The origin of 505nm-peaked PL from Ba3MgSi2O8 Eu2+ Mn2+ phosphor for white-light-emitting diodes

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大学物理西尔斯

1. Atomic Spectra of Hydrogen
1) Atomic Spectra ： emission spectrum：the spectrum formed during the process of radiation of electromagnetic waves.
absorption spectrum： the spectrum formed during the process of absorption of electromagnetic waves with certain wavelengths.
1) Ground-state level : Every atom has a lowest energy level that includes the minimum internal energy state that the atom can have.
2) Excited level :
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➢ Atomic Spectra of Hydrogen
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Q1：There must be something with positive charge in the atom. How does the mass of the atom distribute? Q2：How does the charges of the atom distribute?

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Flame structure of steady and pulsed sooting inversejet diﬀusion ﬂamesChristopher R.Shaddix *,Timothy C.Williams,Linda G.Blevins,Robert W.ScheferCombustion Research Facility,Sandia National Laboratories,Livermore,CA 94550,USAAbstractIn turbulent buoyant ﬁre plumes,local inverse diﬀusion ﬂames of air injected into gaseous fuel or fuel vapors occur,but little is known about the tendency to form soot and produce thermal radiation in these ﬂame structures.To investigate these phenomena,steady and pulsed normal and inverse jet diﬀusion ﬂames of methane/air and ethylene/air have been stabilized on a slot burner,which has advantages over a coan-nular ﬂame geometry for performing ﬂame imaging measurements in sooty ﬂames.OH and PAH laser-in-duced ﬂuorescence (LIF),soot laser-induced incandescence (LII),and soot thermal emission at 850nm have been measured in the lower ﬂame region of steady and pulsed ﬂames.These measurements reveal that the relative positions of these diﬀerent structural features are very similar in the normal and inverse steady ﬂames of each fuel.Also,the OH signals are nearly identical in the normal and inverse ﬂames.The inverse ﬂame PAH signals and soot concentrations are somewhat smaller than for the normal ﬂames,and the near-infrared radiation is approximately 25%lower for the inverse ﬂame.When the central slot is pulsed,the primary buoyant vortex roll-up occurs on the fuel-rich side of inverse ﬂames,resulting in strongly enhanced PAH signals and soot concentrations.The near-infrared radiation also increases in the pulsed ﬂames,but not from the soot within the vortex roll-up region.In general,enhancements in peak signals from soot and near-infrared radiation similar to those in pulsed normal diﬀusion ﬂames are apparent in pulsed inverse diﬀusion ﬂames.PAH signals are clearly greatest in the pulsed inverse ﬂames.Ó2004The Combustion Institute.Published by Elsevier Inc.All rights reserved.Keywords:Soot;PAH;Diﬀusion ﬂame;Laminar;Unsteady1.IntroductionNormal,overventilated diﬀusion ﬂames,with an overall excess of oxidizer surrounding a cen-tral,burning fuel source,naturally constitute the vast majority of laboratory research on laminar jet diﬀusion ﬂames.This type of ﬂame is simple to establish and stabilize,and has ready analogsin a number of practical applications.However,in consideration of the practical problem of soot formation and radiant heat transfer from large,turbulent buoyant ﬁre plumes,the occurrence of turbulent eddies locally injecting a limited amount of air into a large gaseous fuel column appears comparable to the occurrence of gaseous fuel being locally injected into an expanse of air [1,2].Thus,an understanding of the tendency of soot to form and radiate in an inverse diﬀusion ﬂame (where a central oxidizer is injected into an abun-dance of fuel)is important to the overall problem1540-7489/$-see front matter Ó2004The Combustion Institute.Published by Elsevier Inc.All rights reserved.doi:10.1016/j.proci.2004.08.244*Corresponding author.Fax:+19252942276.E-mail address:crshadd@ (C.R.Shaddix).Proceedings of the Combustion Institute 30(2005)1501–1508/locate/prociProceedings of theCombustion Instituteof soot formation and thermal radiation in turbu-lent,buoyantﬁres.Also,with the use of air stag-ing in many burner designs,primary combustion zones often have more of an inverse diﬀusion ﬂame(IDF)structure than a normal diﬀusion ﬂame(NDF)structure[3,4].This can have impor-tant implications for emissions of air toxins,such as polycyclic aromatic hydrocarbons(PAH)and soot,because in the IDF structure these entities are transported away from the reaction zone after they are formed and become part of theﬂame products[5,6].Despite the importance of the IDF geometry in practical applications,few studies of IDFs have been undertaken,and fewer yet have investigated the tendency of soot and PAH to form in IDFs.In particular,no previous study has investigated the eﬀect ofﬂame unsteadiness on PAH and soot for-mation and thermal radiation in IDFs,as is ex-plored here through periodic pulsing of the central air jet.Previous investigations of unsteady NDFs have found there to be signiﬁcant diﬀer-ences in these quantities relative to steadyﬂames [7–9].A slot burner is used in this study,which af-fords several advantages over the coannular burn-ers that have been employed in all previously reported investigations of laminar ser-in-ducedﬂuorescence(LIF)measurements can be performed of theﬂame structure in slot burners without the complication of signal absorption through a curved soot layer.Also,the slot geom-etry allows direct measurements of radiant emis-sion across theﬂame sheet,without the necessity of deconvolving radial projection.A speciﬁc aim of the work reported here was to clarify the relative tendency of soot formation and radiation in IDFs in comparison to NDFs.Arthur and Napier[10],Kent and Wagner[11],and Wu and Essenhigh[12]have all reported signiﬁcantly lower luminosity,and sometimes even blueﬂames, when operating laminar,steady diﬀusionﬂames in an inverse mode.Similarly,Makel and Kennedy [13]have reported peak soot concentrations an or-der of magnitude lower in an IDF in comparison to an NDF.However,there are concerns regard-ingﬂame height and product gas recirculation in these previous investigations.The earliest IDF studies typically used near-stoichiometric overall ﬂowrates of fuel and oxidizer,and simply switched the fuel and airstreams to alternate be-tween normal and inverse jetﬂames[10,11]. Unfortunately,while yieldingﬂames of similar height,this approach gives very low fuel coﬂow velocities in the inverse geometry,leading to prob-lems ofﬂame stability[12]and recirculation of ﬂame products into the fuel stream[11].Later studies,in general,increased fuelﬂowrates in the IDFs,but failed to properly account for combus-tion stoichiometry in setting the airﬂow,resulting in signiﬁcantly shorter IDFs than the NDFs to which they were being compared[13–15].Because soot formation is strongly linked to the character-istic residence time in high-temperature,fuel-rich regions,diﬀerences inﬂame height in laminar, buoyant diﬀusionﬂames have a signiﬁcant impact on soot concentrations.A related goal of the present work was to clar-ify the structural relationships of the soot,PAH, and radiation layers relative to the primary reac-tion zone.In the only previous investigation of these quantities in an IDF,Kang et al.[14]re-ported the PAH proﬁle overlapped the soot layer andﬁlled in on its lean side(above the tip of the reaction zone).The peak luminous emission was also found to reside well to the inside of the soot layer.However,as clearly demonstrated by Wu and Essenhigh[12],the fundamental thermal and chemical structure of an inverse diﬀusion ﬂamesheet is essentially identical to that of a nor-mal diﬀusionﬂamesheet.Therefore,toﬁrst order one would expect the physical structure of an IDF to simply be inverted from the corresponding NDF.Also,any diﬀerences in the local formation rates of PAH and soot would be expected to result from diﬀerences in the temperatures in fuel-rich regions.Diﬀerences in the thermal radiation from soot would be dependent on diﬀerences in the lo-cal soot concentration,its temperature,and in optical property diﬀerences that may arise from diﬀerent degrees of soot carbonization[5,6,16].In inverse jetﬂames,the axis of symmetry lies in the oxidizer stream,rather than the fuel stream, so the temperature decays more quickly in the fuel-rich regions,especially as one moves up in height[11],in comparison to normal jetﬂames. On this basis,one expects PAH and soot forma-tion rates to be lower in IDFs because the key overlap of high temperatures and rich stoichiome-tries occurs over a smaller region[17,18].On the other hand,the steeper temperature gradient on the fuel-side of IDFs enhances the thermophoretic transport of existing soot into richer regions, which might act to oﬀset the temperature eﬀect on soot particle growth.Also,to the extent that soot is transported to somewhat cooler regions in buoyant IDFs,it will experience a lower verti-cal velocity,and hence will have more residence time for surface growth.Globally,soot formation and thermal radiation in NDFs diﬀer from IDFs in that the soot formed low in a NDF is trans-ported towards the high temperature oxidation zone at the top of theﬂame.In contrast,at the top of an IDF the soot is transported away from the reaction zone,along with the combustion products.2.Experimental methodsA Wolfhard–Parker burner was used to sup-port laminar diﬀusionﬂames of methane/air and ethylene/air(see Fig.1).The central,rectangular1502 C.R.Shaddix et al./Proceedings of the Combustion Institute30(2005)1501–1508slot is12mm wide by95mm long.Coﬂow is introduced on each of the long sides of the slot, and nitrogen gas is introduced at each end of the central slot to prevent the formation of end ﬂames.Theﬂame sheets are conﬁned and pro-tected from room air disturbances by a rectangu-lar chimney enclosure with a cross-section of140 mm·191mm.Within the chimney,two curved wire mesh screens form a two-dimensional con-traction to assist in stabilizing theﬂame.UV-qual-ity quartz windows provide optical access from all four walls of the enclosure.For studies of pulsed ﬂame behavior,the centralﬂow is modulated by two100-mm diameter loudspeakers that face each other across the slot.The natural puﬃng fre-quency of the burner is11Hz.Air,fuel,and nitrogenﬂows to the burner were metered using calibrated massﬂow controllers. For the NDF experiments,fuel was fed to the slot, and air formed the coﬂow.For the IDFs,these connections were reversed.Fuel and airﬂow parameters for the investigatedﬂames are shown in Table1.The NDFs extended nearly0.50m above the top of the chimney,for a totalﬂame height of approximately0.70m.Calculations using the Roper correlations forﬂame heights of laminar jet diﬀusionﬂames[19]suggest that essen-tially the sameﬂowrate of air will yield a common ﬂame height for methane and ethylene IDFs. Therefore,a common airﬂowrate of30slpm was used for these two IDFs,and indeed these ﬂames both had a height of80mm,as determined from the location of the dark inner edge of the luminous zone.This choice of airﬂow yielded rel-atively stable IDFs with near-verticalﬂame sheets in the50-mm high interrogation region at the base of theﬂames,similar to the NDFs.The methane and ethylene fuelﬂows in the IDFs were set to the50slpm maximumﬂow capability of the fuel ﬂow handling system,to improveﬂame stability and to minimize product recirculation.For operation of the methane IDF,an outlet screen was necessary to prevent self-ignition of the fuel-rich product gases.Recirculation of soot and tar-laden gases occurred along the sides of the chimney enclosure when operating IDFs of either fuel(consistent with the observations in [11]),resulting in attenuation of the incident laser beam and of the imaged optical signals.To ad-dress these diﬃculties,a chimney extension was incorporated,which conﬁned the product gas recirculation to a region above the stabilizing screens.A frequency-doubled,Nd:YAG-pumped dye laser provided pulsed ultraviolet light for the simultaneous planar excitation of OHÆand PAHﬂuorescence,as detailed in[8].The laser light also excited laser-induced incandescence (LII)emission from the soot particles.The laser sheet was50mm high,with a thickness of250 l m through theﬂame zone.The beam was used to pump the relatively temperature-insensitive Q21(8)line of the(1,0)band of the OHÆA2R+ÀX2P i electronic transition at283.57nm. Theﬂuorescence and LII signals were collected through a45-mm focal length,f/1.8UV lens at-tached to a gated,intensiﬁed charge-coupled de-vice(ICCD)camera.A Schott WG295long-pass ﬁlter eliminated laser reﬂections as well as scatter-ing from soot particles.For theﬂuorescence mea-surements,an additionalﬁlter was used to reduce the signal contribution from soot LII,C2Swan band emission,and naturalﬂame emission.A 450nm UV-quality short-passﬁlter was usedfor Table1Flow conditions for laminar diﬀusionﬂamesFlame Q fuel(slpm)Q air(slpm)V fuel(cm/s)V air(cm/s)slot Re/a CH4NDF15.025022.317.62700.57 C2H4NDF7.525011.217.62600.43 CH4IDF5030.0 3.544.659015.8 C2H4IDF5030.0 3.544.659023.7a global stoichiometry of gases supplied to the burner.C.R.Shaddix et al./Proceedings of the Combustion Institute30(2005)1501–15081503methaneﬂames,whereas a340nm band-passﬁlter was used for ethyleneﬂames.OH and PAH sig-nals were clearly distinguished by tuning the laser oﬀof the OH absorption line.To prevent irising eﬀects with the slow-gating ICCD camera,an intensiﬁer gate width of200ns was used for both theﬂuorescence and LII measurements.For the LII measurements,a200-ns gate delay was imposed on the ICCD and a Schott LG450 long-passﬁlter was used.Even with this long gate delay and long-wavelengthﬁlter,some contribu-tion of PAH LIF was evident,but,in general,it amounted to no more than10%of the soot LII signals.With the use of a UV excitation wave-length and such a long gate delay and gate width, the LII signals should be regarded as a semi-quan-titative measure of the soot concentration,with some sensitivity expected to variations in soot pri-mary particle size[20].The LII signals were cali-brated for soot volume fraction by comparing the LII signal strength at a low height in the stea-dy normal methaneﬂame to a286-nm laser extinction proﬁle(oﬀ-resonant from OH)col-lected with the laser beam aligned to the burner slot.The extinction measurement was interpreted using an assumption of Rayleigh-limit soot absorption(at this location),together with a non-dimensional extinction coeﬃcient of8.8(derived from the dispersion formula in[21]and consistent with the data in[22]).For investigation of the pulsedﬂames,the speakers were synchronized to the frequency of the Nd:YAG laser,thus allowingﬂame excitation frequencies at multiples of the7.5Hzﬂash-lamp frequency.The LIF images were corrected for the mean laser sheet intensity proﬁle determined from200-shot images of Rayleigh scattering from room temperature air.Shot-to-shot variations in laser power at any given position in the laser sheet were found to average less than5%.The laser intensities used for imaging were veriﬁed to be suf-ﬁciently low to produce a linear power depen-dence for both the OH and PAH LIF.The LII signals were veriﬁed to be in a laser power insen-sitive‘‘plateau’’region,so no corrections were necessary for the laser sheet intensity proﬁle.In addition to these laser-based measurements,natu-ralﬂame emission at a wavelength of850nm was measured through a10nm FWHM interference ﬁlter.3.Results and discussionFigure2shows photographs of the steady methane diﬀusionﬂames.With the use of the slotﬂame geometry,the blue CH*chemiluminescence, marking the fuel-rich edge of the reaction zone [23],is clearly seen to lie outside of the yellow-or-ange soot luminosity for the NDF and inside of the luminous zone for the IDF.Figure3shows images of simultaneous OH and PAH LIF in steady normal and inverseﬂames of methane and ethylene.Weak signals from soot LII are also apparent in the ethyleneﬂame images. The expected,inverted structural relationshipof Fig.2.Photographs of the steady CH4/air NDF(left) and IDF(right).Fig.3.Single-shot planar images of OH and PAH LIF in the normal and inverse steady diﬀusionﬂames.Soot LII signals are slightly evident in the ethyleneﬂame images.1504 C.R.Shaddix et al./Proceedings of the Combustion Institute30(2005)1501–1508PAH,soot,and OH in the IDFs,in comparison to the NDFs,is clearly evident.It is also apparent that the signal intensities of PAH and OH for a gi-ven fuel are similar for both the normal and inverseﬂame conﬁgurations.Finally,the high-temperature reaction zone,as indicated by OH, is much closer to the burner centerline for IDFs. This is true despite the signiﬁcantly higher slot ﬂow rates in IDFs and reﬂects the important role of the stoichiometric mixture fraction(which is strongly weighted towards the air side)in deter-mining the location of theﬂame sheet.Both the methane and ethylene steady IDFs were relatively unstable in comparison to the NDFs,resulting in some slight local expansion or contraction of theﬂame in the single-shot laser images.It is believed that this instability is the sameﬂame puﬃng instability that aﬀects buoyant normal jet diﬀusionﬂames[24–26]and that the instability is accentuated for the inverse jetﬂames because of the low(fuel)coﬂow velocity that is used.Normal jet diﬀusionﬂames become increas-ingly unstable as the coﬂow velocity is decreased. The ethylene steady IDF proved to be signiﬁ-cantly less stable than the methane IDF,and ac-tual vortex roll-up of the PAH layer was evident near the top of most images of the‘‘steady’’ethyl-ene IDF(such as in Fig.3).It is hypothesized that this striking diﬀerence in stability for methane and ethylene IDFs,which is not reﬂected in NDFs of methane and ethylene,results from the presence of a higher density coﬂow(fuel)for ethylene. The low-density(i.e.,high-temperature)gas pro-duced in theﬂamesheet has a stronger buoyant potential in the ethylene coﬂow in comparison to methane.Figure4provides a detailed analysis of the spatial relationships between the measured com-ponents of the steadyﬂame structure:OH,PAH, soot,and radiant emission.In thisﬁgure,the transverse location of the peak signal for each of these components is plotted as a function of the height above the burner.Approximately20 images of eachﬂame were interrogated to deter-mine the mean location and amplitude of the peaksignals.Calculated standard deviations of the positions of the peak signals were used to deter-mine the maximumﬂame heights at which the mean quantities could be usefully compared and revealed that analysis of the‘‘steady’’ethylene IDF was limited to heights below35mm.For the NDFs,the peak signal locations are accurate to within0.2mm,whereas for the IDFs the loca-tions are accurate to within0.4mm.As shown in Fig.4,for the methane NDF the positions of OH,PAH,and soot separate with height,reﬂecting the air-entrainmentﬂowﬁeld that brings streamlines from the air-side of the base of theﬂame across theﬂamesheet and then up[27]. The position of the peak radiant emission sepa-rates only slightly from the peak soot concentra-tion(on the high-temperature edge of the soot), at heights above30mm.In the methane IDF, the same relationships in position are seen,except the soot moves further away from the high tem-perature reaction zone with increasing height. This trend may reﬂect an outwardly expanding convectiveﬂowﬁeld in the IDF or the eﬀect of stronger thermophoretic transport of the soot (from a greater transverse temperature gradient). Also,the peak radiant emission separates more clearly from the peak soot location in the methane IDF,as expected for a greater transverse temper-ature gradient.For the steady ethyleneﬂames,the structural entities separate more quickly with height,particularly for the NDF,reﬂectinggreat-C.R.Shaddix et al./Proceedings of the Combustion Institute30(2005)1501–15081505er transverse air entrainment into the ethylene ﬂame to match the stoichiometric requirements for combustion.In the ethylene IDF,the trans-verse positions of the radiant emission,soot,and PAH relative to OH are virtually identical to those in the NDF.Figure5shows the magnitudes of the peak sig-nals as a function of height in the steadyﬂames. The OH signals are essentially identical for the methane normal and inverseﬂames,and they peak at a height of around7mm,as previously found in a smaller Wolfhard–Parker methane NDF[28].The OHﬂuorescence signals in the eth-ylene IDF are almost identical to those in the methaneﬂames,whereas the OH signals from the normal ethyleneﬂame are slightly lower at theﬂame base.Previous measurements of OH ﬂuorescence in coannular methane and ethylene NDFs also showed a small decrease in signals for the ethyleneﬂames,depending on the ethylene ﬂowrate[29].This decrease inﬂuorescence signals was attributed,in part,to a higher OHﬂuores-cence quenching rate in the ethyleneﬂames.For the IDFs,the incident laser beam is attenuated by soot before exciting the OH LIF.This eﬀect probably accounts for the faster falloﬀin OH sig-nals with height that is apparent for the inverse ethyleneﬂame.Therefore,there is no evidence of any signiﬁcant diﬀerences in OH concentrations for the NDFs and IDFs of these fuels,at least near theﬂame base,before radiant energy losses from soot become important.PAH is observed to form at lower heights and has higher signals in the steady ethyleneﬂames in comparison to the methaneﬂames,as has been previously observed for normal co-annular diﬀu-sionﬂames of these fuels[8].In the IDFs,the PAH LIF intensity generally follows the NDF proﬁles in the lowerﬂame region,but at a slightly lower level.For the ethylene NDF,the PAH inten-sity peaks and begins to decrease as the PAH loca-tion approaches theﬂame centerline.The other PAH proﬁles appear to be reaching a peak magni-tude near the top of the interrogation region.The soot concentrations,derived from LII sig-nals,diﬀer by approximately a factor of20for the ethylene and methaneﬂames,as has been previ-ously observed for normal co-annular diﬀusion ﬂames[7,27].The soot concentrations are some-what lower for the IDFs(by25–30%)of both fuels.However,this level of reduction in soot con-centrations in IDFs is not nearly as dramatic as the order of magnitude reduction that some previ-ous IDF studies have suggested[13,15].The soot radiance at850nm,in general,shows the same trends with height in the steadyﬂames as evidenced by the soot concentration data.The near-infrared radiation from each IDF is about 25%lower than from the corresponding NDF, at a given height,except low in the ethyleneﬂame, where the diﬀerence reaches50%.Again,these dif-ferences in the thermal radiation from soot in NDFs versus IDFs are not as great as suggested by previous studies[10–13].Figure6shows images from a pulsed inverse ethylene/air diﬀusionﬂame.Pulsing of the central slot of IDFs was found to result in repeatable,fre-quency-lockedﬂame pulsing behavior for a lower range of pulsing amplitudes than could be used for NDFs.The eﬀect of central-slot pulsing on the fuel-rich portion of theﬂame structure is, however,much greater for IDFs.This is because, for a pulsing frequency near the natural puﬃng frequency(as studied here),the primary buoyant vortex pair forms and propagates outside the ﬂamesheet(as for pulsed NDFs),which in this case is in the fuel-rich region.A comparison of the relative OH and PAHﬂuorescence intensities in Figs.3and6shows that the PAH LIF signals increase considerably in the vortex roll-up region of the pulsedﬂame(the OH signals do not change considerably,as expected).The cycle-mean peak PAH LIF intensity is four times higher thanthe 1506 C.R.Shaddix et al./Proceedings of the Combustion Institute30(2005)1501–1508peak in the steady ﬂame,whereas pulsed normal ethylene ﬂames show a cycle-averaged peak PAH signal that is only 50%higher than that of the steady ﬂame,even when pulsed at a higher amplitude than the IDF.Soot concentrations also strongly increase in the vortex roll-up regions of the pulsed ethylene IDFs and show over a factor of 2enhancement relative to the corresponding steady inverse ﬂame.Peak soot concentrations in the pulsed normal ethylene ﬂames reach similar levels.The 850-nm radiance in the pulsed IDF is,on average,twice that in the corresponding steady ﬂame,with the peak emission occurring adjacent to the OH layer and not in the outer vor-tex.Pulsed normal ethylene ﬂames average a little more than twice the peak 850-nm radiance in comparison to steady NDFs,or approximately 50%more than the pulsed IDFs.The similarity of soot production and near-in-frared radiance observed here for IDFs,both stea-dy and when pulsed,in comparison to NDFs,suggests that existing modeling approaches for soot formation and radiation in buoyant ﬁre plumes (which have not diﬀerentiated between lo-cal inverse versus normal ﬂame geometries)are probably suﬃcient,to reasonable accuracy.When the soot formation and radiation submodels are calibrated against measurements in laminar NDFs,as has been typically done,they would be expected to overpredict soot concentrations and radiation in ﬁre plumes,if,in fact,all of the other relevant chemistry and physics were accu-rately represented.PAH production in unsteady IDFs appears to be enhanced relative to unsteady NDFs,though this ﬁnding needs to be tempered by uncertainties associated with interpretation of broadband ﬂuorescence from a mixture of PAH species [8].4.ConclusionsMeasurements have been performed on both steady and pulsed inverse jet diﬀusion ﬂamesof methane/air and ethylene/air stabilized on a slot burner,aﬀording several advantages over previous studies using coannular burners.Planar measurements of OH and PAH LIF,soot LII,and soot radiance at 850nm have been per-formed in the lower ﬂame region of both normal and inverse ﬂames,revealing broad sim-ilarities in many aspects of the ﬂame structures.PAH signals and soot concentrations are slightly lower in the steady inverse ﬂames,as is the near-infrared soot radiance,possibly as a conse-quence of the steep temperature gradient ex-pected on the fuel-rich side of these ﬂames.Pulsed inverse ﬂames show a strong eﬀect of buoyancy-induced vortex roll-up on the fuel-rich region of the ﬂame,resulting in enhanced PAH signals relative to pulsed normal ﬂames.Soot concentrations are similar in pulsed normal and inverse ﬂames,whereas near-infrared soot radiance is somewhat higher in the pulsed nor-mal ﬂames.Overall,these measurements demon-strate that PAH and soot production and thermal radiation are signiﬁcant in both steady and pulsed inverse diﬀusion ﬂames,and that existing modeling approaches for soot formation and radiation in buoyant ﬁre plumes may ade-quately represent the eﬀects of local inverse ﬂames.AcknowledgmentsThis work has been supported by a Laboratory Directed Research and Development project man-aged by the Engineering Science Research Foun-dation at Sandia National Labs.Bob Harmon and Matt Boisselle of Sandia assisted in labora-tory measurements.Pascale Desgroux of CNRS at the University of Lille,France,assisted with experimental setup and measurements on the nor-mal diﬀusion ﬂames.Sandia is operated by the Sandia Corporation,a Lockheed Martin Com-pany,for the U.S.DOE under contract DE-AC04-94-AL85000.Fig.6.Single-shot planar images of OH and PAH LIF (top)and soot LII (bottom)for evenly spaced phase intervals of an ethylene IDF with the air pulsed at 15Hz.Contributions of the soot LII to the LIF images and of the PAH LIF to the LII images are evident.C.R.Shaddix et al./Proceedings of the Combustion Institute 30(2005)1501–15081507References[1]A.F.Ghoniem,kkis,M.Soteriou,Proc.Combust.Inst.26(1996)1531–1539.[2]X.Zhou,K.H.Luo,J.J.R.Williams,Combust.Flame129(2002)11–29.[3]D.M.Stansel,urendeau, D.W.Senser,Combust.Sci.Tech.104(1995)207–234.[4]J.M.Ballester,C.Dopazo,N.Fueyo,M.Herna´n-dez,P.J.Vidal,Fuel76(1997)435–446.[5]L.G.Blevins,R.A.Fletcher,B.A.Benner Jr.,E.B.Steel,G.W.Mulholland,b.Instit.29 (2002)2325–2333.[6]L.G.Blevins,M.A.Mikofski,G.W.Mulholland,E.F.Moore,R.W.Davis,Combust.Flame,submitted.[7]C.R.Shaddix,K.C.Smyth,Combust.Flame107(1996)418–452.[8]K.C.Smyth,C.R.Shaddix,D.A.Everest,Combust.Flame111(1997)185–207.[9]M.E.Decroix,W.L.Roberts,Combust.Sci.Tech-nol.160(2000)165–189.[10]J.R.Arthur,D.H.Napier,bust.Inst.5(1955)303–316.[11]J.H.Kent,H.Gg.Wagner,Z.Phys.Chem.139(1984)59–68.[12]K.-T.Wu,R.H.Essenhigh,bust.Inst.20(1984)1925–1932.[13]D.B.Makel,I.M.Kennedy,Combust.Sci.Technol.97(1994)303–314.[14]K.T.Kang,J.Y.Hwang,S.H.Chung,W.Lee,Combust.Flame109(1997)266–281.[15]C.R.Kaplan,K.Kailasanath,Combust.Flame124(2001)275–294.[16]C.R.Shaddix,A´.B.Palota´s,C.M.Megaridis,M.Y.Choi,N.Y.C.Yang,Int.J.Heat Mass Transfer, submitted.[17]P.B.Sunderland,R.L.Axelbaum,D.L.Urban,B.H.Chao,S.Liu,Combust.Flame132(2003)25–33. [18]J.H.Kent,D.R.Honnery,in:H.Bockhorn(Ed.),Soot Formation in Combustion.Springer-Verlag, Berlin,1994,p.199.[19]F.G.Roper,Combust.Flame29(1977)219–226.[20]R.J.Santoro,C.R.Shaddix,in:K.Kohse-Ho¨ing-haus,J.Jeﬀries(Eds.),Applied Combustion Diag-nostics,Taylor and Francis,New York,1992,p.252.[21]T.T.Charalampopoulos,H.Chang,Combust.Sci.Tech.59(1988)401–421.[22]H.Chang,T.T.Charalampopoulos,Proc.RoyalSoc.London A430(1990)577–591.[23]R.W.Schefer,Combust.Sci.Tech.126(1997)255–270.[24]J.Buckmaster,N.Peters,b.Instit.21(1986)1829–1836.[25]A.Hamins,J.C.Yang,T.Kashiwagi,b.Instit.24(1992)1695–1702.[26]B.M.Cetegen,T.A.Ahmed,Comb.Flame93(1993)157–184.[27]R.J.Santoro,T.T.Yeh,J.J.Horvath,H.G.Semerj-ian,Combust.Sci.Technol.53(1987)89–115. [28]K.C.Smyth,P.J.H.Tjossem, A.Hamins,J.H.Miller,Comb.Flame79(1990)360–380.[29]R.Puri,M.Moser,R.J.Santoro,K.C.Smyth,b.Instit.24(1992)1015–1022.CommentGus Nathan,University of Adelaide,Australia.The phase correlation between natural radiation at850nm and the soot and OH is very insightful.Please comment more on the lateral resolution of this measurement and the relative phase locking to the other measurements. Also,please comment further on why you chose this wavelength.Reply.The lateral resolution of the imaging of natural (thermal)radiation is limited by the depth-of-ﬁeld of the imaging lens and its acceptance angle,applied over the length of the slotﬂame.For the camera standoﬀand lens used in this investigation(45mm focal length,f1.8,in con-trast to the lens described in the main text),the nominal depth-of-ﬁeld is4mm.A comparison of LII-measured soot volume fraction and natural radiation proﬁles at a height of25mm in the steady,normal ethyleneﬂame shows a FWHM of2.4mm for soot volume fraction and3.0mm for natural radiation.Simulated radiant emis-sion proﬁles from an established database of soot volume fraction and temperature proﬁles in a coannular ethylene ﬂame show essentially identical FWHMs,suggesting that the additional width of the850nm emission proﬁles may be attributed to the inferior spatial resolution of the radi-ation measurement.Applying a2mm wide boxcar smoothing algorithm to the soot volume fraction proﬁle yields a proﬁle width similar to that of the emission proﬁle, but a more rounded shape.For the pulsedﬂame measure-ments,the natural radiation is phase-locked in the same manner as the laser-based measurements,but uses a4l s detection gate width.This corresponds to a phase resolu-tion of a fraction of a degree.The850nm bandwidthﬁlter was chosen for the measurement of the natural radiation because it was the longest wavelength that could be de-tected with suﬃcient sensitivity to permit a detection gate width short enough for good phase locking.A longer wavelength is preferred to shorter ones both to avoid con-tributions from chemiluminescent emissions and to more faithfully track changes in soot radiant emission(which peaks at around1400nm in theseﬂames).1508 C.R.Shaddix et al./Proceedings of the Combustion Institute30(2005)1501–1508。

微生物屏障试验 DIN 58953-6_2010 Test report

Interlaboratory T est …Microbial barrier testing of packa ging materials for medical devices which are tobe ster ili ze d“according to DIN 58953-6:2010Test re portJanuary 2013Author: Daniel ZahnISEGA Forschungs- und Untersuchungsgesellschaft mbHTest report Page 2 / 15Table of contentsSeite1.General information on the Interlaboratory Test (3)1.1 Organization (3)1.2 Occasion and Objective (3)1.3 Time Schedule (3)1.4 Participants (4)2.Sample material (4)2.1 Sample Description and Execution of the Test (4)2.1.1 Materials for the Analysis of the Germ Proofness under Humidityaccording to DIN 58953-6, section 3 (5)2.1.2 Materials for the Analysis of the Germ Proofness with Air Permeanceaccording to DIN 58953-6, section 4 (5)2.2 Sample Preparation and Despatch (5)2.3 Additional Sample and Re-examination (6)3.Results (6)3.1 Preliminary Remark (6)3.2 Note on the Record of Test Results (6)3.3 Comment on the Statistical Evaluation (6)3.4 Outlier tests (7)3.5 Record of Test Results (7)3.5.1 Record of Test Results Sample F1 (8)3.5.2 Record of Test Results Sample F2 (9)3.5.3 Record of Test Results Sample F3 (10)3.5.4 Record of Test Results Sample L1 (11)3.5.5 Record of Test Results Sample L2 (12)3.5.6 Record of Test Results Sample L3 (13)3.5.7 Record of Test Results Sample L4 (14)4.Overview and Summary (15)Test report Page 3 / 15 1. General Information on the Interlaboratory Test1.1 OrganizationOrganizer of the Interlaboratory Test:Sterile Barrier Association (SBA)Mr. David Harding (director.general@)Pennygate House, St WeonardsHerfordshire HR2 8PT / Great BritainRealization of the Interlaboratory Test:Verein zur Förderung der Forschung und Ausbildung fürFaserstoff- und Verpackungschemie e. V. (VFV)vfv@isega.dePostfach 10 11 0963707 Aschaffenburg / GermanyTechnical support:ISEGA Forschungs- u. Untersuchungsgesellschaft mbHDr. Julia Riedlinger / Mr. Daniel Zahn (info@isega.de)Zeppelinstraße 3 – 563741 Aschaffenburg / Germany1.2 Occasion and ObjectiveIn order to demonstrate compliance with the requirements of the ISO 11607-1:2006 …Packaging for terminally sterilized medical devices -- Part 1: Requirements for materials, sterile barrier systems and packaging systems“ validated test methods are to be preferably utilized.For the confirmation of the microbial barrier properties of porous materials demanded in the ISO 11607-1, the DIN 58953-6:2010 …Sterilization – Sterile supply – Part 6: Microbial barrier testing of packaging materials for medical devices which are to be sterilized“ represents a conclusive method which can be performed without the need for extensive equipment.However, since momentarily no validation data on DIN 58953-6 is at hand concerns emerged that the method may lose importance against validated methods in a revision of the ISO 11607-1 or may even not be considered at all.Within the framework of this interlaboratory test, data on the reproducibility of the results obtained by means of the analysis according to DIN 58953-6 shall be gathered.1.3 Time ScheduleSeptember 2010:The Sterile Barrier Association queried ISEGA Forschungs- und Unter-suchungsgesellschaft about the technical support for the interlaboratory test.For the realization, the Verein zur Förderung der Forschung und Ausbildungfür Faserstoff- und Verpackungschemie e. V. (VFV) was won over.November 2010: Preliminary announcement of the interlaboratory test / Seach for interested laboratoriesTest report Page 4 / 15 January toDecember 2011: Search for suitable sample material / Carrying out of numerous pre-trials on various materialsJanuary 2012:Renewed contact or search for additional interested laboratories, respectively February 2012: Sending out of registration forms / preparation of sample materialMarch 2012: Registration deadline / sample despatchMay / June 2012: Results come in / statistical evaluationJuly 2012: Despatch of samples for the re-examinationSeptember 2012: Results of the re-examination come in / statistical evaluationNovember 2012: Results are sent to the participantsDecember 2012/January 2013: Compilation of the test report1.4 ParticipantsFive different German laboratories participated in the interlaboratory test. In one laboratory, the analyses were performed by two testers working independently so that six valid results overall were received which can be taken into consideration in the evaluation.To ensure an anonymous evaluation of the results, each participant was assigned a laboratory number (laboratory 1 to laboratory 6) in random order, which was disclosed only to the laboratory in question. The complete laboratory number breakdown was known solely by the ISEGA staff supporting the proficiency test.2. Sample Material2.1 Sample Description and Execution of the TestUtmost care in the selection of suitable sample material was taken to include different materials used in the manufacture of packaging for terminally sterilized medical devices.With the help of numerous pre-trials the materials were chosen covering a wide range of results from mostly germ-proof samples to germ permeable materials.Test report Page 5 / 15 2.1.1 Materials for the Analysis of Germ Proofness under Humidity according to DIN 58953-6, section 3:The participants were advised to perform the analysis on the samples according to DIN 58953-6, section 3, and to protocol their findings on the provided result sheets.The only deviation from the norm was that in case of the growth of 1 -5 colony-forming units (in the following abbreviated as CFU) per sample, no re-examination 20 test pieces was performed.2.1.2 Materials for the Analysis of Germ Proofness with Air Permeance according to DIN 58953-6, section 4:The participants were advised to perform the analysis on the samples according to DIN 58953-6, section 4, and to protocol their findings on the provided result sheets.2.2 Sample Preparation and DespatchFor the analysis of the germ proofness under humidity, 10 test pieces in the size of 50 x 50 mm were cut out of each sample and heat-sealed into a sterilization pouch with the side to be tested up.Out of the 10 test pieces, 5 were intended for the testing and one each for the two controls according to DIN 58953-6, sections 3.6.2 and 3.6.3. The rest should remain as replacements (e.g. in case of the dropping of a test piece on the floor etc.).For the analysis of the germ proofness with air permeance, 15 circular test pieces with a diameter of 40 mm were punched out of each sample and heat-sealed into a sterilization pouch with the side to be tested up.Test report Page 6 / 15 Out of the 15 test pieces, 10 were intended for the testing and one each for the two controls according to DIN 58953-6, section 4.9. The rest should remain as replacements (e.g. in case of the dropping of a test piece on the floor etc.).The sterilization pouches with the test pieces were steam-sterilized in an autoclave for 15 minutes at 121 °C and stored in an climatic room at 23 °C and 50 % relative humidity until despatch.2.3 Additional Sample and Re-examinationFor the analysis of the germ proofness under humidity another test round was performed in July / August 2012. For this, an additional sample (sample L4) was sent to the laboratories and analysed (see 2.1.2). The results were considered in the evaluation.For validation or confirmation of non-plausible results, occasional samples for re-examination were sent out to the laboratories. The results of these re-examinations (July / August 2012) were not taken into consideration in the evaluation.3. Results3.1 Preliminary RemarkSince the analysis of germ proofness is designed to be a pass / fail – test, the statistical values and precision data were meant only to serve informative purposes.The evaluation of the materials according to DIN58953-6,sections 3.7and 4.7.6by the laboratories should be the most decisive criterion for the evaluation of reproducibility of the interlaboratory test results. Based on this, the classification of a sample as “sufficiently germ-proof” or “not sufficiently germ-proof” is carried out.3.2 Note on the Record of Test Results:The exact counting of individual CFUs is not possible with the required precision if the values turn out to be very high. Thus, an upper limit of 100 CFU per agar plate or per test pieces, respectively, was defined. Individual values above this limit and values which were stated with “> 100” by the laboratories, are listed as 100 CFU per agar plate or per test piece, respectively, in the evaluation.Test report Page 7 / 153.3 Comment on the Statistical EvaluationThe statistical evaluation was done based on the series of standards DIN ISO 5725-1ff.The arithmetic laboratory mean X i and the laboratory standard deviation s i were calculated from the individual measurement values obtained by the laboratories.The overall mean X of the laboratory means as well as the precision data of the method (reproducibility and repeatability) were determined for each sample3.4 Outlier testsThe Mandel's h-statistics test was utilised as outlier test for differences between the laboratory means of the participants.A laboratory was identified as a “statistical outlier” as soon as an exceedance of Mandel's h test statistic at the 1 % significance level was detected.The respective results of the laboratories identified as outliers were not considered in the statistical evaluation.3.5 Record of Test ResultsOn the following pages, the records of the test results for each interlaboratory test sample with the statistical evaluation and the evaluation according to DIN 58953-6 are compiled.Test report Page 8 / 153.5.1 Record of Test Results Sample F1Individual Measurement values:Statistical Evaluation:Comment:Laboratory 4, as an outlier, has not been taken into consideration in the statistical Evaluation.Outlier criterion: Mandel's h-statistics (1 % level of significance)Overall mean X:91.0CFU / agar plateRepeatability standard deviation s r:17.9CFU / agar plateReproducibility standard deviation s R:19.8CFU / agar plateRepeatability r:50.0CFU / agar plateRepeatability coefficient of variation:19.6%Reproducibility R:55.5CFU / agar plateReproducibility coefficient of variation:21.8%Evaluation according to DIN 58953-6, Section 3.7:Lab. 1 - 6:Number of CFU > 5, i.e. the material is classified as not sufficiently germ-proof.Conclusion:All of the participants, even the Laboratory 4 which was identified as an outlier, came to the same results and would classify the sample material as “not sufficiently germ-proof”Test report Page 9 / 153.5.2 Record of Test Results Sample F2Individual Measurement values:Statistical Evaluation:Comment:Laboratory 4, as an outlier, has not been taken into consideration in the statistical Evaluation.Outlier criterion: Mandel's h-statistics (1 % level of significance)Overall mean X:0CFU / agar plateRepeatability standard deviation s r:0CFU / agar plateReproducibility standard deviation s R:0CFU / agar plateRepeatability r:0CFU / agar plateRepeatability coefficient of variation:0%Reproducibility R:0CFU / agar plateReproducibility coefficient of variation:0%Evaluation according to DIN 58953-6, Section 3.7:Lab. 1 – 3:Number of CFU = 0, i.e. the material is classified as sufficiently germ-proofLab. 4:Number of CFU ≤ 5, i.e. a re-examination on 20 test pieces would have to be done Lab. 5 – 6:Number of CFU = 0, i.e. the material is classified as sufficiently germ-proofConclusion:All of the participants, except for the Laboratory 4 which was identified as an outlier, came to the same results and would classify the sample material as “sufficiently germ-proof”.Test report Page 10 / 153.5.3 Record of Test Results Sample F3Individual Measurement values:Statistical Evaluation:Overall mean X:30.1CFU / agar plateRepeatability standard deviation s r:17.2CFU / agar plateReproducibility standard deviation s R:30.9CFU / agar plateRepeatability r:48.2CFU / agar plateRepeatability coefficient of variation:57.1%Reproducibility R:86.5CFU / agar plateReproducibility coefficient of variation:103%Evaluation according to DIN 58953-6, Section 3.7:Lab. 1 - 4:Number of CFU > 5, i.e. the material is classified as not sufficiently germ-proof. Lab. 5:Number of CFU = 0, i.e. the material is classified as sufficiently germ-proof. Lab. 6:Number of CFU > 5, i.e. the material is classified as not sufficiently germ-proof.Conclusion:Five of the six participants came to the same result and would classify the sample as “not sufficiently germ-proof”. Only laboratory 5 would classify the sample material as “sufficiently germ-proof”.Test report Page 11 / 153.5.4 Record of Test Results Sample L1Individual Measurement values:Statistical Evaluation:Overall mean X:0.09CFU / test pieceRepeatability standard deviation s r:0.32CFU / test pieceReproducibility standard deviation s R:0.33CFU / test pieceRepeatability r:0.91CFU / test pieceRepeatability coefficient of variation:357%Reproducibility R:0.93CFU / test pieceReproducibility coefficient of variation:366%Evaluation according to DIN 58953-6, Section 4.7:Lab. 1 - 6:Number of CFU < 15, i.e. the material is classified as sufficiently germ-proof.Conclusion:All participants came to the same result and would classify the sample as “sufficiently germ-proof”.Test report Page 12 / 153.5.5 Record of Test Results Sample L2Individual Measurement values:Statistical Evaluation:Overall mean X:0.73CFU / test pieceRepeatability standard deviation s r: 1.10CFU / test pieceReproducibility standard deviation s R: 1.18CFU / test pieceRepeatability r: 3.07CFU / test pieceRepeatability coefficient of variation:151%Reproducibility R: 3.32CFU / test pieceReproducibility coefficient of variation:163%Evaluation according to DIN 58953-6, Section 4.7:Lab. 1:Number of CFU > 15, i.e. the material is classified as not sufficiently germ-proof. Lab. 2 - 6:Number of CFU < 15, i.e. the material is classified as sufficiently germ-proof.Conclusion:Five of the six participants came to the same result and would classify the sample as “sufficiently germ-proof”. Only laboratory 1 exceeds the limit value slightly by 1 CFU, so that the sample would be classified as “not sufficiently germ-proof”.Test report Page 13 / 153.5.6 Record of Test Results Sample L3Individual Measurement values:Statistical Evaluation:Overall mean X:0.36CFU / test pieceRepeatability standard deviation s r: 1.00CFU / test pieceReproducibility standard deviation s R: 1.06CFU / test pieceRepeatability r: 2.79CFU / test pieceRepeatability coefficient of variation:274%Reproducibility R: 2.98CFU / test pieceReproducibility coefficient of variation:293%Evaluation according to DIN 58953-6, Section 4.7:Lab. 1 - 6:Number of CFU < 15, i.e. the material is classified as sufficiently germ-proof.Conclusion:All participants came to the same result and would classify the sample as “sufficiently germ-proof”.Test report Page 14 / 153.5.7 Record of Test Results Sample L4Individual Measurement values:Statistical Evaluation:Overall mean X:35.1CFU / test pieceRepeatability standard deviation s r:18.8CFU / test pieceReproducibility standard deviation s R:42.6CFU / test pieceRepeatability r:52.7CFU / test pieceRepeatability coefficient of variation:53.7%Reproducibility R:119CFU / test pieceReproducibility coefficient of variation:122%Evaluation according to DIN 58953-6, Section 4.7:Lab. 1 - 3:Number of CFU > 15, i.e. the material is classified as not sufficiently germ-proof. Lab. 4:Number of CFU < 15, i.e. the material is classified as sufficiently germ-proof. Lab. 5 - 6:Number of CFU > 15, i.e. the material is classified as not sufficiently germ-proof.Conclusion:Five of the six participants came to the same result and would classify the sample as“not sufficiently germ-proof”.Test report Page 15 / 15 4. Overview and SummarySummary:In case of four of the overall seven tested materials, a 100 % consensus was reached regarding the evaluation as“sufficiently germ-proof”and“not sufficiently germ-proof”according to DIN 58 953-6.As for the other three tested materials, there were always 5 concurrent participants out of 6 (83 %). In each case, only one laboratory would have evaluated the sample differently.It is noteworthy that the materials about the evaluation of which a 100 % consensus was reached were the smooth sterilization papers. The differences with one deviating laboratory each occurred with the slightly less homogeneous materials, such as with the creped paper and the nonwoven materials.。

marked manuscript

Quality evaluation of Flos Lonicerae through a simultaneous determination of seven saponins by HPLC with ELSDXing-Yun Chai1, Song-Lin Li2, Ping Li1*1Key Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China2Institute of Nanjing Military Command for Drug Control, Nanjing, 210002, People’s Republic of China*Corresponding author: Ping LiKey Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China.E-mail address: lipingli@Tel.: +86-25-8324-2299; 8539-1244; 135********Fax: +86-25-8532-2747AbstractA new HPLC coupled with evaporative light scattering detection (ELSD) method has been developed for the simultaneous quantitative determination of seven major saponins, namely macranthoidinB (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)in Flos Lonicerae, a commonly used traditional Chinese medicine (TCM) herb.Simultaneous separation of these seven saponins was achieved on a C18 analytical column with a mixed mobile phase consisting of acetonitrile(A)-water(B)(29:71 v/v) acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min on linear gradient, and then keep isocratic elution with 54%B from 25 to 30min.The drift tube temperature of ELSD was set at 106℃, and with the nitrogen flow-rate of 2.6 l/min. All calibration curves showed good linear regression (r2 0.9922) within test ranges. This method showed good reproducibility for the quantification of these seven saponins in Flos Lonicerae with intra- and inter-day variations of less than 3.0% and 6.0% respectively. The validated method was successfully applied to quantify seven saponins in five sources of Flos Lonicerae, which provides a new basis of overall assessment on quality of Flos Lonicerae.Keywords: HPLC-ELSD; Flos Lonicerae; Saponins; Quantification1. IntroductionFlos Lonicerae (Jinyinhua in Chinese), the dried buds of several species of the genus Lonicera (Caprifoliaceae), is a commonly used traditional Chinese medicine (TCM) herb. It has been used for centuries in TCM practice for the treatment of sores, carbuncles, furuncles, swelling and affections caused by exopathogenic wind-heat or epidemic febrile diseases at the early stage [1]. Though four species of Lonicera are documented as the sources of Flos Lonicerae in China Pharmacopeia (2000 edition), i.e. L. japonica, L. hypoglauca,L. daystyla and L. confusa, other species such as L. similes and L. macranthoides have also been used on the same purpose in some local areas in China [2]. So it is an important issue to comprehensively evaluate the different sources of Flos Lonicerae, so as to ensure the clinical efficacy of this Chinese herbal drug.Chemical and pharmacological investigations on Flos Lonicerae resulted in discovering several kinds of bioactive components, i.e. chlorogenic acid and its analogues, flavonoids, iridoid glucosides and triterpenoid saponins [3]. Previously, chlorogenic acid has been used as the chemical marker for the quality evaluation of Flos Lonicerae,owing to its antipyretic and antibiotic property as well as its high content in the herb. But this compound is not a characteristic component of Flos Lonicerae, as it has also been used as the chemical marker for other Chinese herbal drugs such as Flos Chrysanthemi and so on[4-5]. Moreover, chlorogenic acid alone could not be responsible for the overall pharmacological activities of Flos Lonicerae[6].On the other hand, many studies revealed that triterpenoidal saponins of Flos Lonicerae possess protection effects on hepatic injury caused by Acetaminophen, Cd, and CCl4, and conspicuous depressant effects on swelling of ear croton oil [7-11]. Therefore, saponins should also be considered as one of the markers for quality control of Flos Lonicerae. Consequently, determinations of all types of components such as chlorogenic acid, flavonoids, iridoid glucosides and triterpenoidal saponins in Flos Lonicerae could be a better strategy for the comprehensive quality evaluation of Flos Lonicerae.Recently an HPLC-ELSD method has been established in our laboratory for qualitative and quantitative determination of iridoid glucosides in Flos Lonicerae [12]. But no method was reported for the determination of triterpenoidal saponins in Flos Lonicera. As a series studies on the comprehensive evaluation of Flos Lonicera, we report here, for the first time, the development of an HPLC-ELSD method for simultaneous determination of seven triterpenoidal saponins in the Chinese herbal drug Flos Lonicerae, i.e.macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) (Fig. 1).2. Experimental2.1. Samples, chemicals and reagentsFive samples of Lonicera species,L. japonica from Mi county, HeNan province (LJ1999-07), L. hypoglauca from Jiujang county, JiangXi province (LH2001-06), L. similes from Fei county, ShanDong province (LS2001-07), L. confuse from Xupu county, HuNan province (LC2001-07), and L. macranthoides from Longhu county, HuNan province (LM2000-06) respectively, were collected in China. All samples were authenticated by Dr. Ping Li, professor of department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. The voucher specimens were deposited in the department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. Seven saponin reference compounds: macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) were isolated previously from the dried buds of L. confusa by repeated silica gel, sephadex LH-20 and Rp-18 silica gel column chromatography, their structures were elucidated by comparison of their spectral data (UV, IR, MS, 1H- NMR and 13C-NMR) with references [13-15]. The purity of these saponins were determined to be more than 98% by normalization of the peak areas detected by HPLC with ELSD, and showed very stable in methanol solution.HPLC-grade acetonitrile from Merck (Darmstadt, Germany), the deionized water from Robust (Guangzhou, China), were purchased. The other solvents, purchased from Nanjing Chemical Factory (Nanjing, China) were of analytical grade.2.2. Apparatus and chromatographic conditionsAglient1100 series HPLC apparatus was used. Chromatography was carried out on an Aglient Zorbax SB-C18 column（250 4.6mm, 5.0µm）at a column temperature of 25℃.A Rheodyne 7125i sampling valve (Cotati, USA) equipped with a sample loop of 20µl was used for sample injection. The analog signal from Alltech ELSD 2000 (Alltech, Deerfield, IL, USA)was transmitted to a HP Chemstation for processing through an Agilent 35900E (Agilent Technologies, USA).The optimum resolution was obtained by using a linear gradient elution. The mobile phase was composed of acetonitrile(A) and water(B) which acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min in linear gradient, and back to the isocratic elution of 54%B from 25 to 30 min.The drift tube temperature for ELSD was set at 106℃and the nitrogen flow-rate was of 2.6 l/min. The chromatographic peaks were identified by comparing their retention time with that of each reference compound tried under the same chromatographic conditions with a series of mobile phases. In addition, spiking samples with the reference compounds further confirmed the identities of the peaks.2.3. Calibration curvesMethanol stock solutions containing seven analytes were prepared and diluted to appropriate concentration for the construction of calibration curves. Six concentrationof the seven analytes’ solution were injected in triplicate, and then the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The results were demonstrated in Table1.2.4. Limits of detection and quantificationMethanol stock solution containing seven reference compounds were diluted to a series of appropriate concentrations with methanol, and an aliquot of the diluted solutions were injected into HPLC for analysis.The limits of detection (LOD) and quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD and LOQ for each compound were shown in Table1.2.5. Precision and accuracyIntra- and inter-day variations were chosen to determine the precision of the developed assay. Approximate 2.0g of the pulverized samples of L. macranthoides were weighted, extracted and analyzed as described in 2.6 Sample preparation section. For intra-day variability test, the samples were analyzed in triplicate for three times within one day, while for inter-day variability test, the samples were examined in triplicate for consecutive three days. Variations were expressed by the relative standard deviations. The results were given in Table 2.Recovery test was used to evaluate the accuracy of this method. Accurate amounts of seven saponins were added to approximate 1.0g of L. macranthoides,and then extracted and analyzed as described in 2.6 Sample preparation section. The average recoveries were counted by the formula: recovery (%) = (amount found –original amount)/ amount spiked ×100%, and RSD (%) = (SD/mean) ×100%. The results were given in Table 3.2.6. Sample preparationSamples of Flos Lonicerae were dried at 50℃until constant weight. Approximate 2.0g of the pulverized samples, accurately weighed, was extracted with 60% ethanol in a flask for 4h. The ethanol was evaporated to dryness with a rotary evaporator. Residue was dissolved in water, followed by defatting with 60ml of petroleum ether for 2 times, and then the water solution was evaporated, residue was dissolved with methanol into a 25ml flask. One ml of the methanol solution was drawn and transferred to a 5ml flask, diluted to the mark with methanol. The resultant solution was at last filtrated through a 0.45µm syringe filter (Type Millex-HA, Millipore, USA) and 20µl of the filtrate was injected to HPLC system. The contents of the analytes were determined from the corresponding calibration curves.3. Results and discussionsThe temperature of drift tube and the gas flow-rate are two most important adjustable parameters for ELSD, they play a prominent role to an analyte response. In ourprevious work [12], the temperature of drift tube was optimized at 90°C for the determination of iridoids. As the polarity of saponins are higher than that of iridoids, more water was used in the mobile phase for the separation of saponins, therefore the temperature for saponins determination was optimized systematically from 95°C to 110°C, the flow-rate from 2.2 to 3.0 l/min. Dipsacoside B was selected as the testing saponin for optimizing ELSD conditions, as it was contained in all samples. Eventually, the drift tube temperature of 106℃and a gas flow of 2.6 l/min were optimized to detect the analytes. And these two exact experimental parameters should be strictly controlled in the analytical procedure [16].All calibration curves showed good linear regression (r2 0.9922) within test ranges. Validation studies of this method proved that this assay has good reproducibility. As shown in Table 2, the overall intra- and inter-day variations are less than 6% for all seven analytes. As demonstrated in Table 3, the developed analytical method has good accuracy with the overall recovery of high than 96% for the analytes concerned. The limit of detection (S/N=3) and the limit of quantification (S/N=10) are less than 0.26μg and 0.88μg respectively (Table1), indicating that this HPLC-ELSD method is precise, accurate and se nsitive enough for the quantitative evaluation of major non- chromaphoric saponins in Flos Lonicerae.It has been reported that there are two major types of saponins in Flos Lonicerae, i.e. saponins with hederagenin as aglycone and saponins with oleanolic acid as the aglycone [17]. But hederagenin type saponins of the herb were reported to have distinct activities of liver protection and anti-inflammatory [7-11]. So we adoptedseven hederagenin type saponins as representative markers to establish a quality control method.The newly established HPLC-ELSD method was applied to analyze seven analytes in five plant sources of Flos Lonicerae, i.e. L. japonica,L. hypoglauca,L. confusa,L. similes and L. macranthoides(Table 4). It was found that there were remarkable differences of seven saponins contents between different plant sources of Flos Lonicerae. All seven saponins analyzed could be detected in L. confusa and L. hypoglauca, while only dipsacoside B was detected in L. japonica. Among all seven saponins interested, only dipsacoside B was found in all five plant species of Flos Lonicerae analyzed, and this compound was determined as the major saponin with content of 53.7 mg/g in L. hypoglauca. On the other hand, macranthoidin B was found to be the major saponin with the content higher than 41.0mg/g in L. macranthoides,L. confusa, and L. similis, while the contents of other analytes were much lower.In our previous study [12], overall HPLC profiles of iridoid glucosides was used to qualitatively and quantitatively distinguish different origins of Flos Lonicerae. As shown in Fig.2, the chromatogram profiles of L. confusa, L. japonica and L. similes seem to be similar, resulting in the difficulty of clarifying the origins of Flos Lonicerae solely by HPLC profiles of saponins, in addition to the clear difference of the HPLC profiles of saponins from L. macranthoides and L. hypoglauca.Therefore, in addition to the conventional morphological and histological identification methods, the contents and the HPLC profiles of saponins and iridoids could also be used as accessory chemical evidence toclarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.4. ConclusionsThis is the first report on validation of an analytical method for qualification and quantification of saponins in Flos Lonicerae. This newly established HPLC-ELSD method can be used to simultaneously quantify seven saponins, i.e. macranthoidin B, macranthoidin A, dipsacoside B, hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester, macranthoside B, macranthoside A, and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside in Flos Lonicerae. Together with the HPLC profiles of iridoids, the HPLC-ELSD profiles of saponins could also be used as an accessory chemical evidence to clarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.AcknowledgementsThis project is financially supported by Fund for Distinguished Chinese Young Scholars of the National Science Foundation of China (30325046) and the National High Tech Program（2003AA2Z2010）.[1]Ministry of Public Health of the People’s Republic of China, Pharmacopoeia ofthe People’s Republic of China, V ol.1, 2000, p. 177.[2]W. Shi, R.B. Shi, Y.R. Lu, Chin. Pharm. J., 34(1999) 724.[3]J.B. Xing, P. Li, D.L. Wen, Chin. Med. Mater., 26(2001) 457.[4]Y.Q. Zhang, L.C. Xu, L.P. Wang, J. Chin. Med. Mater., 21(1996) 204.[5] D. Zhang, Z.W. Li, Y. Jiang, J. Pharm. Anal., 16(1996) 83.[6]T.Z. Wang, Y.M. Li, Huaxiyaoxue Zazhi, 15(2000) 292.[7]J.ZH. Shi, G.T. Liu. Acta Pharm. Sin., 30(1995) 311.[8]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 209.[9]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 213.[10]J.ZH. Shi, L. Wan, X.F. Chen.ZhongYao YaoLi Yu LinChuang, 6 (1990) 33.[11]J. Liu, L. Xia, X.F. Chen. Acta Pharmacol. Sin., 9 (1988) 395[12]H.J. Li, P. Li, W.C. Ye, J. Chromatogr. A 1008(2003) 167-72.[13]Q. Mao, D. Cao, X.SH. Jia. Acta Pharm. Sin., 28(1993) 273.[14]H. Kizu, S. Hirabayashi, M. Suzuki, et al. Chem. Pharm. Bull., 33(1985) 3473.[15]S. Saito, S. Sumita, N. Tamura, et al. Chem Pharm Bull., 38(1990) 411.[16]Alltech ELSD 2000 Operating Manual, Alltech, 2001, p. 16. In Chinese.[17]J.B. Xing, P. Li, Chin. Med. Mater., 22(1999) 366.Fig. 1 Chemical structures of seven saponins from Lonicera confusa macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)Fig. 2Representative HPLC chromatograms of mixed standards and methanol extracts of Flos Lonicerae.Column: Agilent Zorbax SB-C18 column（250 4.6mm, 5.0µm）, temperature of 25℃; Detector: ELSD, drift tube temperature 106℃, nitrogen flow-rate 2.6 l/min.A: Mixed standards, B: L. confusa, C: L. japonica, D: L. macranthoides, E: L. hypoglauca, F: L. similes.Table 1 Calibration curves for seven saponinsAnalytes Calibration curve ar2Test range(μg)LOD(μg)LOQ(μg)1 y=6711.9x－377.6 0.9940 0.56–22.01 0.26 0.882 y=7812.6x－411.9 0.9922 0.54–21.63 0.26 0.843 y=6798.5x－299.0 0.9958 0.46–18.42 0.22 0.724 y=12805x－487.9 0.9961 0.38–15.66 0.10 0.345 y=4143.8x－88.62 0.9989 0.42–16.82 0.18 0.246 y=3946.8x－94.4 0.9977 0.40–16.02 0.16 0.207 y=4287.8x－95.2 0.9982 0.42–16.46 0.12 0.22a y: Peak area; x: concentration (mg/ml)Table 2 Reproducibility of the assayAnalyteIntra-day variability Inter-day variability Content (mg/g) Mean RSD (%) Content (mg/g) Mean RSD (%)1 46.1646.2846.2246.22 0.1346.2245.3647.4226.33 2.232 5.385.385.165.31 2.405.285.345.045.22 3.043 4.374.304.184.28 2.244.284.464.024.255.204 nd1)-- -- nd -- --5 1.761.801.821.79 1.701.801.681.841.77 4.706 1.281.241.221.252.451.241.341.201.26 5.727 tr2)-- -- tr -- -- 1): not detected; 2): trace. RSD (%) = (SD/Mean) ×100%Table 3 Recovery of the seven analytesAnalyteOriginal(mg) Spiked(mg)Found(mg)Recovery(%)Mean(%)RSD(%)1 23.0823.1423.1119.7122.8628.1042.7346.1351.0199.7100.699.399.8 0.722.692.672.582.082.913.164.735.515.7698.197.6100.698.8 1.632.172.152.091.732.182.623.884.404.6598.8103.297.799.9 2.94nd1)1.011.050.980.981.101.0297.0104.8104.1102.0 4.250.880.900.910.700.871.081.561.752.0197.197.7101.898.9 2.660.640.620.610.450.610.751.081.211.3397.796.796.096.8 0.97tr2)1.021.101.081.031.111.07100.9102.799.1100.9 1.81): not detected; 2): trace.a Recovery (%) = (Amount found –Original amount)/ Amount spiked ×100%, RSD (%) = (SD/Mean) ×100%Table 4 Contents of seven saponins in Lonicera spp.Content (mg/g)1 2 3 4 5 6 7 L. confusa45.65±0.32 5.13±0.08 4.45±0.11tr1) 2.04±0.04tr 1.81±0.03 L. japonica nd2)nd 3.44±0.09nd nd nd nd L. macranthoides46.22±0.06 5.31±0.13 4.28±0.10 tr 1.79±0.03 1.25±0.03 tr L. hypoglauca11.17±0.07 nq3)53.78±1.18nd 1.72±0.02 2.23±0.06 2.52±0.04 L. similes41.22±0.25 4.57±0.07 3.79±0.09nd 1.75±0.02tr nd 1): trace; 2): not detected.. 3) not quantified owing to the suspicious purity of the peak.。

Methods for Obtaining and Analyzing Whole Chloroplast Genome Sequences

Yokobori,S.,Watanabe,Y.,and Oshima,T.(2003).Mitochondrial genome of Ciona savignyi (Urochordata,Ascidiacea,Enterogona):Comparison of gene arrangement and tRNA genes with Halocynthia roretzi mitochondrial genome.J.Mol.Evol.57,574–587. Yost,H.J.,Phillips,C.R.,Boore,J.L.,Bertman,J.,Whalen,B.,and Danilchik,M.V.(1995).Relocation of mitochondrial RNA to the prospective dorsal midline during Xenopus embryogenesis.Dev.Biol.170,83–90.Zardoya,R.,and Meyer,A.(2001).On the origin of and phylogenetic relationships among living A98(13),7380–7383.[20]Methods for Obtaining and Analyzing WholeChloroplast Genome SequencesBy R obert K.J ansen,L inda A.R aubeson,J effrey L.B oore,C laude W.de P amphilis,T imothy W.C humley,R osemarie C.H aberle,S tacia K.W yman,A ndrew J.A lverson,R hiannon P eery,S allie J.H erman,H.M atthew F ourcade,J ennifer V.K uehl, J oel R.M c N eal,J ames L eebens-M ack,and L iying C ui AbstractDuring the past decade,there has been a rapid increase in our under-standing of plastid genome organization and evolution due to the availabil-ity of many new completely sequenced genomes.There are45complete genomes published and ongoing projects are likely to increase this sam-pling to nearly200genomes during the next5years.Several groups of researchers including ours have been developing new techniques for gathering and analyzing entire plastid genome sequences and details of these developments are summarized in this chapter.The most important developments that enhance our ability to generate whole chloroplast ge-nome sequences involve the generation of pure fractions of chloroplast genomes by whole genome ampliﬁcation using rolling circle ampliﬁcation, cloning genomes into Fosmid or bacterial artiﬁcial chromosome(BAC) vectors,and the development of an organellar annotation program(Dual Organellar GenoMe Annotator[DOGMA]).In addition to providing de-tails of these methods,we provide an overview of methods for analyzing complete plastid genome sequences for repeats and gene content,as well as approaches for using gene order and sequence data for phylogeny recon-struction.This explosive increase in the number of sequenced plastid genomes and improved computational tools will provide many insights into the evolution of these genomes and much new data for relationships at deep nodes in plants and other photosynthetic organisms.Copyright2005,Elsevier Inc.All rights reserved. METHODS IN ENZYMOLOGY,VOL.3950076-6879/05$35.00IntroductionHistorical Overview of Chloroplast GenomicsThe study of chloroplast genomes dates back to the1950s when plant biologistsﬁrst discovered that chloroplasts contain their own DNA(see Sugiura(2003)for a review).Early work used electron microscopy,cloning, comparative restriction site mapping,and gene mapping to characterize genome structure–gene order and organization(Palmer,1991;Sugiura, 1992).Such comparisons yielded numerous phylogenetic studies based on restriction site polymorphisms and gene order changes(Downie and Palmer,1992;Jansen et al.,1998;Olmstead and Palmer,1994).The publi-cation of complete plastid sequences for Nicotiana(Shinozaki et al.,1986) and Marchantia(Ohyama et al.,1986)provided theﬁrst opportunity for nucleotide-level whole genome comparisons(Morton,1994;Wolfe et al., 1987).Currently the list of completely sequenced plastid genomes has increased to45and includes a wide diversity of taxonomic groups.The number of sequenced chloroplast genomes is growing rapidly:19of these 45genomes(Table I)have appeared in the last two years.In spite of the availability of so many complete genome sequences,our understanding of chloroplast genome evolution is still limited because this remains a very small sampling of plastid-containing species and because previous sequenc-ing efforts were not designed to address phylogenetic or molecular evolu-tionary issues.A number of groups(e.g.,algae and various lineages of land plants,including bryophytes,ferns and fern allies,gymnosperms,and cer-tain angiosperm groups,especially monocots other than the cereal grasses) remain poorly sampled.However,several groups of scientists are now focusing their sequencing efforts atﬁlling these gaps,and the number of completely sequenced chloroplast genomes will continue to increase dra-matically in the next few years (for details of three such projects see http:// megasun.bch.umontreal.ca/ogmp/projects/sumprog.html, http://www.jgi. /programs/comparative/second_levels/chloroplasts/jansen_project_ home/chlorosite.html, and /TreeofLife/).Brief Overview of Chloroplast Genome Structure and Evolution Plastid genomes vary in size from35to217kilobases(kb),but the vast majority from photosynthetic organisms are between115and165kb (Table I).The45completely sequenced genomes(Table I)encode from63 (Toxiplasma)to209(Porphyra)genes with most containing110–130genes. Most of these genes code for proteins,mostly involved in photosynthesis or gene expression,with the remainder being transfer RNA or ribosomalTABLE IA lphabetical L ist of45C omplete P lastid G enome S equences as of F ebruary17,2005aSpecies NCBI classiﬁcation AccessionnumberYearcompletedGenomesize(bp)Adiantum capillus-veneris Embryophyta AY1788642003150,568 Amborella trichopoda Embryophyta AJ5061562003162,686 Anthoceros formosae Embryophyta AB0861792003161,162 Arabidopsis thaliana Embryophyta AP0004231999154,478 Atropa belladonna Embryophyta AJ3165822003156,687 Calycanthus fertilis var.ferax Embryophyta AJ4284132003153,337 Chaetosphaeridium globosum Streptophyta AF4942782002131,183 Chlamydomonas reinhardtii Chlorophyta BK0005542004203,828 Chlorella vulgaris Chlorophyta AB0016841997150,613Cyanidioschyzon merolae Rhodophyta AB002583/AY2861232003/2004149,987149,705Cyanidium caldarium Rhodophyta AF0221861999164,921 Cyanophora paradoxa Glaucocystophyceae U308211995135,599 Eimeria tenella b Alveolata AY217738200334,750 Epifagus virginiana c Embryophyta M81884199370,028 Euglena gracilis Euglenozoa X708101993143,171 Euglena longa Euglenozoa AJ294725200173,345 Gracilaria tenuistipitata Rhodophyta AY6739962004183,883 Guillardia theta Cryptophyta AF0414681998121,524 Huperzia lucidula Embryophyta AY6605662005154,373 Lotus corniculatus Embryophyta AP0029832001150,519 Marchantia polymorpha Embryophyta X044651986121,024 Medicago truncatula Embryophyta AC0935442001124,033 Mesostigma viride Chlorophyta AF1661142000118,360 Nephroselmis olivacea Chlorophyta AF1373791999200,799 Nicotiana tabacum Embryophyta Z000441986155,939 Nymphaea alba Embryophyta AJ6272512004159,930 Odontella sinensis Stramenopiles Z677531996119,704 Oenothera elata Embryophyta AJ2710792000163,935 Oryza nivara Embryophyta AP0067282004134,494Oryza sativa Embryophyta X15901/AY522329/AY5223311989/2004/2004134,525/134,496/134,551Ponax schinsing Embryophyta AY5821392004156,318 Physcomitrella patens Embryophyta AP0056722003122,890 Pinus koraiensis Embryophyta AY2284682003116,866 Pinus thunbergii Embryophyta D175101996119,707 Porphyra purpurea Rhodophyta U388041996191,028 Psilotum nudum Embryophyta AP0046382002138,829 Saccharum hybrid Embryophyta AE0099472004141,182 Saccharum ofﬁcinarum Embryophyta AP0067142004141,182 Spinacia oleracea Embryophyta AJ4008482000150,725 Toxoplasma gondii b Alveolata U87145199934,996 Triticum aestivum Embryophyta AB0422402001134,545 Zea mays Embryophyta X865631995140,384RNA genes.Although the number of genes may be similar between even distantly related lineages,the exact gene complement may be quite differ-ent.Although gene content is largely consistent within land plants,Martin et al.(2002)found only44protein-coding genes to be common among15 chloroplast genomes representing all major lineages of photosynthetic organisms.A few genes have evidently been gained during plastid genome evolution,but the vast majority of gene content changes represent gene losses,some of which have been lost independently in different lineages (Martin et al.,2002;Maul et al.,2002).In all plastid genomes,most genes are part of polycistronic transcription units,suggestive of bacterial operons (Fig.1)(Mullet et al.,1992;Palmer,1991).Plastid operons often have multiple promoters that enable a subset of genes to be transcribed within the operon(Kuroda and Maliga,2002;Miyagi et al.,1998).Both group I and group II types of self-splicing introns are found in cpDNAs;the majority are group II(Palmer,1991).A unique intron type(known as a ‘‘twintron’’)that contains an intron within an intron is found in Euglena (Copertino and Hallick,1991)and possibly other organisms(Maier et al., 1995).Although intron content is quite variable among algal genomes,it is highly conserved among land plant cpDNAs.Most land plant(and some algal)genomes have a quadripartite organi-two sections of unique DNA,which are referred to as the‘‘large’’and ‘‘small single copy regions’’(LSC and SSC,respectively).The gene content and organization of the chloroplast genome change by several mechanisms. Transposition has been suggested as a mechanism of genomic change inin Trachelium in(Cosner et al.,1997)and in the et al.,1989),but few deﬁnitive have been Only one clear case ofpositional gain has been documented in Chlamydomonas(Fan et al., where a transposable element that is no longer active has beenized.The frequency of the other types of rearrangements,including gene and intron gains and losses,expansion,and contraction of the IR,and inversions,varies from group to group.Most genomes have very few gene order changes,at least in comparison to close relatives.However,several lineages have cpDNAs that are highly rearranged.The most notableamples are in the algae(e.g.,Chlamydomonas)(Maul et al.,2002),a See http://megasun.bch.umontreal.ca/ogmp/projects/other/cp_list.html, http://www.ncbi.:80/genomes/static/euk_o.html, and http://www.rs.noda.tus.ac.jp/$kunisawa/ order/front.html for access to these genomic sequences. All listed genomes are chloroplasts except as noted.b Plastid genome remnant,nonphotosynthetic protist.c Plastid genome,nonphotosyntheticﬂowering plant.(e.g.,Pinus )(Wakasugi et al .,1994),and(e.g.,Campanulaceae [Cosner et al .,1997],],Geraniaceae [Palmer et al .,1987],and Lobeliaceae [Knox and Palmer,F ig .1.Gene map of tobacco chloroplast genome (from Raubeson and Jansen [2005]).The inner circle shows the four major regions of the genome:the two copies of the inverted repeat (IRA and IRB)and the large and small single-copy regions (LSC and SSC).The outer circle represents the tobacco genome with the transcribed regions shown as boxes proportional to gene size.Genes inside the circle are transcribed in a clockwise direction,and genes outside of the circle are transcribed counterclockwise.The IR by the increased width of the circle representing the tobacco genome.Genes with are marked with asterisks (*).the gene boxes and gene names operons known to occur in Genes coding for products that function in protein synthesis are dark gray;genes products that function in photosynthesis are stippled;and genes coding for products with various other functions are lighter gray.cpDNAs of algae(and Stern,2002)and land plants(Raubeson and Jansen,2005).changes in plastid genomes have proven useful for resolving relationships within a number of plant groups(Raubeson and).OverviewThis chapter focuses on the methods used to gather and analyze plastid genomic sequences.This includes methods for(1)isolating chloroplasts and puriﬁed cpDNA,(2)amplifying,cloning,and sequencing cpDNA,(3)as-sembling drafts andﬁnishing genomes,(4)annotating chloroplast genomes, and(5)analyzing genome sequence and structure.Most of areequally applicable to the plastid genomes of plants, except for the initial isolation steps,which typically involve of a large insert genomic library.In our treatment of genomic analysis,we focus on evolutionary issues,and even then we will not be able to be comprehen-sive.In addition to reviewing methods that others have used,this chapter provides some more detailed protocols used by our group in an ongoing project for which we are sequencing60plastid genomes from seed plants. Whole Chloroplast Genome SequencingChloroplast genomes had been sequenced by cloning cpDNA intoplasmid vectors,selecting cpDNA-containing clones,and then sequencing the clones using both plasmid and chloroplast-speciﬁc primers.This process is very labor intensive and involves of highly puriﬁed cpDNA,which can be quite difﬁcult for many Now,faster and more cost-effective approaches have been There are four basic ap-proaches to sequencing entire chloroplast genomes:(1)isolation of pure cpDNA,followed by random shearing,shotgun cloning,and sequencing;(2)ampliﬁcation using long polymerase chain reaction(PCR)of large segments of the genome,followed by cloning,and then sequencing of the products using chloroplast-speciﬁc primers;(3)ampliﬁcation of the entire genome using rolling circle ampliﬁcation(RCA)followed by shearing of the RCA product and shotgun cloning and sequencing of the fragments; and(4)construction of bacterial artiﬁcial chromosome(BAC)or Fosmid libraries from total DNA preparations,preferably ones that are enriched for cpDNA,followed by shearing,cloning,and sequencing.Weﬁrst outline our general genomic sequencing methods and then go on to describe the unique parts of each of the four approaches,with anemphasis on those used by ourDraft sequences of chloroplast genomes from our group are being produced at the DOE Joint Genome Institute(JGI)in Walnut Creek, California.This facility is a very high-throughput operation that relies on robotics for many of the steps in the process.Details of JGI protocols can be found at /Internal/protocols/prots_production. html,but a general description is given here.Our approach is to shear the DNA,select3-kb fragments,and clone these fragmentsinto vectors.coli are then transformed withbinant and large plates from which areand placed into384-well plates containing theate growth medium.Picking of colonies from the library is random,so the percentage of wells in the plates that contain cpDNA clones will be proportional to the percentage of cpDNA(as opposed to nuclear or mitochondrial‘‘contaminant’’)in the DNA sample used to create the library.The inserts are sequenced from the384-well plates using forward and reverse plasmid primers,yielding about500–800bp of sequence from each end of the insert.Sequencing proceeds until the depth of coverage, from many overlapping sequence reads,enables the assembly of the reads into one contiguous genomic sequence.In this approach,most steps are performed robotically minimizing human effort compared to earlier meth-ods.The tradeoff is that unlike directed approaches such as chromosome walking with custom primers,the genome must be sequenced to a depth of 6–10Âcoverage to ensure accurate characterization of the entire genome. Isolation of Chloroplast DNAIf pure cpDNA can be obtained in sufﬁcient quantity,it can serve as the template for the sequencing approach just described.Many methods have been developed for isolating puriﬁed cpDNA from plants(Palmer,1986). Most of these methods involve three basic steps:separation of plastids from other organelles,lysis of the chloroplasts,and puriﬁcation of DNA.The most commonly applied methods use sucrose or Percoll gradients(Palmer, 1986),DNAse I treatment(Kolodner and Tewari,1979),or high salt buffers(Bookjans et al.,1984)to isolate puriﬁed cpDNA(or more realisti-cally,a total DNA preparation enriched for cpDNA).The use of sucrose gradients is most generally applicable at least in land plants and a detailed protocol is provided in Table II.Basically,sucrose step gradients are used to obtain chloroplasts that are then lysed and the DNA is recovered from the lysate.We include several modiﬁcations of the basic method that have been used by our group to improve the quality and quantity of cpDNA. Consistent problems are encountered with two aspects of cpDNA isola-tions,using this method or any other:(1)collecting a sufﬁcient quantityTABLE III solation of C hloroplasts or cp DNA by S ucrose S tep-G radient C entrifugation(seeP almer(1986)and S andbrink ET AL.(1989))1.Before extraction,place plants in the dark for1–2days to reduce chloroplast starch levels.Approximately!100g of leaf tissue is required to get sufﬁcient quantities of cpDNA. If the chloroplast isolation is being prepared for rolling circle ampliﬁcation(RCA),at least 10g of leaf tissue is generally necessary.The quality of the plant tissue is probably the most important criterion for a successful isolation.Leaves that are fresher and younger are far superior to older senescing leaves.2.Wash healthy green leaves in tap water if visibly dirty and cut into small pieces ($2–10cm2in surface area).3.Place10–100g of cut leaves in400ml of ice-cold isolation buffer.Steps3–5are done in a cold room at4 or on ice.We have found that the isolation buffer in Sandbrink et al. (1989)often yields a much purer chloroplast pellet(see recipes at end of protocol).This buffer contains higher concentrations of salts and2-mercaptoethanol.4.Homogenize in a prechilled blender forﬁve5-s bursts at high speed.5.Filter through four layers of cheesecloth and squeeze remaining liquid through the cloth.Thenﬁlter through one layer of Miracloth(Calbiochem,catalog no.475855)without squeezing.6.Divideﬁltrate into multiple centrifuge bottles and centrifuge at1000g for15min at 4 .Pour off supernatant.7.Resuspend pellet7ml of ice-cold wash buffer using a soft paintbrush and by vigorous swirling.8.Gently load the resuspended pellet onto a step gradient consisting of18ml of52% sucrose,overlayered with7ml of30%sucrose.The overlay should be added with sufﬁcient mixing to create a diffuse interface.It is best to pour the sucrose gradients1–2days before the extraction and allow them to sit at4 to allow for mixing of the interface.To enhance the purity of your cpDNA isolation,it is best to use more sucrose gradients,each with material from a smaller amount of tissue so the nuclei can better penetrate the chloroplast band.At least six sucrose gradients are recommended for up to200g of starting material.When preparing chloroplasts(rather than cpDNA),we will use three gradients for just20g of tissue. We also have experimented with modifying the percentage of sucrose in the step gradients. We have found that the optimal percentage varies from one taxon to the next.For example, 52/30%gradients work well for most angiosperms,Ginkgo,and conifers,but we found that a 44–48%sucrose in the bottom layer yielded DNA with a much higher proportion of cpDNA for cycads.9.Centrifuge the step gradients at25,000rpm for30–60min at4 in an SW-27 (Beckman)or AH-627(Sorvall)swinging bucket rotor.10.Remove the chloroplast band from the30–52%interface using a wide-bore pipette, dilute with3–10volumes of wash buffer,and centrifuge at1500g for15min at4 .We have found that the use of the Sandbrink wash buffer often improves the purity of the cpDNA. Multiple cycles of washing,pelleting,and resuspending of the chloroplasts often renders much purer cpDNA.11.Resuspend the chloroplast pellet in wash buffer to aﬁnal volume of2ml.Depending on the size of theﬁnal pellet,it may be necessary to resuspend the pellet in a larger volume and then divide resuspended pellet into separate tubes with no more than3ml per tube.If you are planning to use the chloroplasts for RCA,this is the point at which you proceed to the RCA protocol in Table III.(continued)TABLE II(continued)12.Add one-tenth volume of a10mg/ml solution of self-digested(2h at37 )Pronase (Calbiochem,catalog no.537088)and incubate for2min at room temperature.13.Gently add one-ﬁfth volume of1Âlysis buffer and mix in by slowly inverting the tube several times over a period of10–15min at room temperature.We experimented with higher concentrations of lysis buffer(a5Âlysis buffer vs.the normal1Âbuffer)and with doing the lysis at higher temperatures for longer periods(37 for15–60min).In general,we found that the5Âlysis buffer incubated at37 gave much higher yields of cpDNA.We also tried several alternative lysis buffers that used cetyltrimethylammonium bromide(CTAB)(Milligan et al., 1989)or sodium dodecyl sulfate(SDS)(Triboush et al.,1998),but in general we did not have much success with these buffers.14.Centrifuge for10min at room temperature in a clinical centrifuge to remove residual starch and cell-wall debris from the chloroplast lysate.Transfer lysate to a new tube.This step is optional.15.Add1.0g of technical-grade cesium chloride(CsCl)per1ml of lysate and add ethidium bromide(EtBr)to aﬁnal concentration of200mg/ml.Fill remaining volume of ultracentrifuge tubes with a premixed solution of1g CsCl per1ml of TE buffer.16.Centrifuge the small CsCl/EtBr gradients(5ml)in a vertical rotor for5–8h at 65,000rpm at20 .17.Remove the band from gradient,and if necessary,reband in a second gradient or move on to step18.High-molecular-weight chloroplast DNA will be very viscous and easily removed‘‘en masse’’from near the center of the gradient.18.Remove EtBr by at least three extractions with isopropanol saturated with NaCl and H2O and dialyze against at least three changes of2liters of dialysis buffer over a period of1–2 days.19.Check purity of cpDNA by doing restriction digests and agarose gel electrophoresis.20.Store the chloroplast DNA at4 for short-term and atÀ20 for long-term use.Digests of cpDNA produce well-deﬁned bands,whereas nuclear DNA produces so many bands that it appears as a smear on the gel.Standard isolation buffer Sandbrink isolation buffer 0.35M sorbitol 1.25M NaCl50m M tris-HCl,pH8.050m M Tris–HCl,pH8.05m M EDTA5m M EDTA0.1%BSA(w/v,Sigma A-4503)1%BSA(w/v,Sigma A-4503)1.5m M2-mercaptoethanol10m M2-mercaptoethanol5%poly pyrrolidone(PVP-40) Standard wash buffer Sandbrink wash buffer0.35M sorbitol10m M Tris–HCl,pH8.050m M Tris–HCl,pH8.05m M EDTA25m M EDTA10m M2-mercaptoethanol100 g/ml proteinase K52%sucrose solution30%sucrose solution52%Sucrose(w/v)30%Sucrose(w/v)50m M Tris pH8.050m M Tris pH8.025m M EDTA25m M EDTA(continued)TABLE II(continued)Standard isolation buffer Sandbrink isolation buffer 1Âlysis buffer5Âlysis buffer5%sodium sarcosinate(w/v)20%sodium sarcosinate(w/v) 50m M Tris pH8.050m M Tris pH8.025m M EDTA25m M EDTADialysis buffer10m M Tris,pH8.010m M NaCl0.1m M EDTAof chloroplasts while eliminating nuclear contamination and(2)lysing the chloroplasts and releasing the membrane-bound cpDNA.Nuclear DNA tends to adhere to the outer chloroplast membrane,leading to theﬁrst challenge.Regarding the second challenge,chloroplasts can be surprisingly difﬁcult to lyse.If harsh enough detergents are used to lyse the chloroplasts abruptly,then the DNA is degraded.Because the DNA is bound to the thylakoid membranes,the membranes must be solubilized to release the DNA,but if the chloroplast is lysed too gently,the DNA remains bound to the membrane and is lost.Our modiﬁcations to the basic procedure help reduce these problems but do not totally overcome them.Two other approaches to cpDNA isolation are the DNAse I(Kolodner and Tewari,1979),which is used as a modiﬁcation of the sucrose gradient technique, and the high salt (Bookjans et al., 1984) methods (see http:// /programs/comparative/second_levels/chloroplasts/jansen_ project_home/cpDNA_protocols.html for protocols). In the DNAse I method,the chloroplast pellet in step7(Table I)is treated with DNAse I to destroy nuclear DNA.This treatment also will destroy any cpDNA that is not protected within intact plastids.Thus,although the purity of cpDNA is very high,the yield is much lower and much more leaf material is needed to obtain sufﬁcient cpDNA.In our experience,this method yields very pure cpDNA when it works,but it has only worked for two species of the many that we have attempted(Lactuca sativa[Fig.2]and Ginkgo biloba).Even in those cases,sufﬁcient quantities of cpDNA for shearing and shotgun cloning were not always recovered.The second alternative method employs a high NaCl(1.25M)concentration in the isolation and wash buffers,and it does not involve any step-gradient centrifugation. The high salt concentration is supposed to signiﬁcantly reduce nuclear contamination.According to Bookjans et al.(1984),the undissociatedchromatin or nuclear DNA tends to stick to chloroplast membranesbecause of electrostatic interactions.The high salt concentration di-minishes these electrostatic interactions,yielding a DNA prep that is en-riched in cpDNA.We have had only limited success with this approach;one isolation by this method yielded cpDNA of suf ﬁcient purity andquantity to proceed to genomic sequencing (Ranunculus macranthus ,Fig.2).However,the use of high-salt wash buffers in combination withthe sucrose gradient technique has proven quite valuable for decreasingnuclear DNA contamination in chloroplast preps.The methods just described can also be used (stopping prior to lysis)to collect chloroplasts for use in whole genome ampli ﬁcations (describedlater in this chapter).Other workers are experimenting with the use of aﬂuorescence-activated cell sorter (FACS)to separate chloroplasts frommitochondria and nuclei (D.Mandoli,personal communication,2004).This method may be particularly valuable when limited tissue is available.Once puri ﬁed chloroplasts have been obtained from the FACS,they can befurther processed using one of the methods described below.Anotheradvantage of the FACS approach is that it may also provide puri ﬁedfractions of both mitochondria and nuclei in addition tochloroplasts.F ig .2.Gel photo showing chloroplast DNA isolations for Lactuca (Asteraceae)usingDNAse I method and Ranunuculus using the NaCl method (see the section ‘‘Isolation ofChloroplast DNA ’’). Lanes 1 and 2 and 4 and 5 were digested with KpnI and HaeII,respectively;lane 3is a lambda DNA digest used as a size marker.358comparing macromolecules [20]Whole Genome AmpliﬁcationIf puriﬁed chloroplasts can be obtained,they can serve as a template from which to produce abundant cpDNA via RCA,a powerful approach for performing whole genome ampliﬁcation.This process involves an iso-thermal ampliﬁcation using bacteriophage Phi29polymerase,which is capable of performing strand-displacement DNA synthesis for more than 70kb without disassociating from the template(Dean et al.,2002).This feature,combined with the stability of this polymerase and its low error rate,makes this enzyme a powerful tool for template preparation.RCA involves the use of random hexamer primers that are exonuclease resistant, necessary because the DNA polymerase has a30–50exonuclease proofread-ing activity.Most applications of RCA have been directed toward per-forming human genome ampliﬁcation and a kit for this purpose(Repli-G) is available from Qiagen.Our group has been using this kit routinely for amplifying entire chloroplast genomes,and we have modiﬁed the Repli-G protocol to improve cpDNA ampliﬁcation(see Table III for protocol).We have had considerable success with the RCA approach for a wide diversity of seed plants.Figure3shows restriction digests of RCA products for two taxa that had sufﬁcient quality and quantity of cpDNA to proceed with genome sequencing.One possible further modiﬁcation of this protocol would be to develop genome-speciﬁc primers for chloroplast or mitochon-drial genomes,which would enable the ampliﬁcation of the chloroplast and mitochondrial genomes from total DNA isolations.Although the low tem-perature of the RCA reaction limits the speciﬁcity of annealing for these primers,experiments are in progress,focusing on buffer modiﬁcations that show promise for increasing the speciﬁcity of the ampliﬁcation.Long PCR and SequencingA third approach for obtaining DNA template from which to generatewhole chloroplast genome sequences involves PCRof largefragments of the genome using conserved chloroplast This ap-proach has been employed to sequence three basal genomes (Goremykin et al.,2003a,b,2004).Goremykin et al.conserved primers by aligning sequences from seven seed-plant genomes(Arabidopsis, Nicotiana,Oenothera,Oryza,Pinus,Spinacia,and Zea).These primers then were used to amplify long fragments ranging in size from4to20kb and covering the entire chloroplast genome.The long PCR products were then sheared into smaller pieces,shotgun cloned,and sequenced.Although this approach worked well for Goremykin’s group,it does have several disad-vantages:(1)The primer combinations may not work for seed-plant gen-omes that have experienced gene order changes or substantial sequence [20]analyzing chloroplast genome sequences359。

电荷补偿剂增强的 Ca2.96 Eu0.04（PO4）2红色荧光粉

电荷补偿剂增强的 Ca2.96 Eu0.04（PO4）2红色荧光粉张志伟;王晓娟;任艳军【摘要】Novel Li + -, Na + -, K + -, and Si4 + -doped Ca2. 96 Eu0. 04 (PO4 ) 2 red phosphors were syn-thesized using a conventional solid-state reaction route. The effects of codoping of charge compensa-tory ions on the phase and luminescent properties of Ca2. 96 Eu0. 04 (PO4 ) 2 red phosphors were investi-gated by X-ray diffraction and photoluminescence spectra. The excitation spectra include both broad band (200 - 310 nm) and sharp peaks (310 - 500 nm). From emission spectra, it is observed that these phosphors exhibit two dominating bands situated at 593 and 616 nm, originating from the 5 D0→7 F1 and 5 D0→7 F2 transition of the Eu3 + ion, respectively. The luminescence of Ca2. 96 Eu0. 04-(PO4 ) 2 can be enhanced by the incorporation of Li + , Na + , K + , and Si4 + . Furthermore, the charge compensation mechanism was discussed. The charge compensatory additives have little influ-ence on the decay times and CIE of all the phosphors. The CIE chromaticity coordinates of these phosphors all locate in the red region. It implies that Ca2. 96 Eu0. 04 (PO4 ) 2 is a good candidate as a red-emitting phosphor pumped by near-ultraviolet ( NUV) InGaN chip for fabricating white light-emitting diodes (wLEDs).%采用高温固相法合成了 Li +、Na +、K +和 Si4+作为电荷补偿剂的Ca2.96 Eu0.04(PO4)2白光 LED 用红色荧光粉。

核磁常见溶剂峰

NMR Chemical Shifts of Common Laboratory Solvents as Trace Impurities Hugo E.Gottlieb,*Vadim Kotlyar,andAbraham Nudelman*Department of Chemistry,Bar-Ilan University,Ramat-Gan52900,IsraelReceived June27,1997In the course of the routine use of NMR as an aid for organic chemistry,a day-to-day problem is the identifica-tion of signals deriving from common contaminants (water,solvents,stabilizers,oils)in less-than-analyti-cally-pure samples.This data may be available in the literature,but the time involved in searching for it may be considerable.Another issue is the concentration dependence of chemical shifts(especially1H);results obtained two or three decades ago usually refer to much more concentrated samples,and run at lower magnetic fields,than today’s practice.We therefore decided to collect1H and13C chemical shifts of what are,in our experience,the most popular “extra peaks”in a variety of commonly used NMR solvents,in the hope that this will be of assistance to the practicing chemist.Experimental SectionNMR spectra were taken in a Bruker DPX-300instrument (300.1and75.5MHz for1H and13C,respectively).Unless otherwise indicated,all were run at room temperature(24(1°C).For the experiments in the last section of this paper,probe temperatures were measured with a calibrated Eurotherm840/T digital thermometer,connected to a thermocouple which was introduced into an NMR tube filled with mineral oil to ap-proximately the same level as a typical sample.At each temperature,the D2O samples were left to equilibrate for at least 10min before the data were collected.In order to avoid having to obtain hundreds of spectra,we prepared seven stock solutions containing approximately equal amounts of several of our entries,chosen in such a way as to prevent intermolecular interactions and possible ambiguities in assignment.Solution1:acetone,tert-butyl methyl ether,di-methylformamide,ethanol,toluene.Solution2:benzene,di-methyl sulfoxide,ethyl acetate,methanol.Solution3:acetic acid,chloroform,diethyl ether,2-propanol,tetrahydrofuran. Solution4:acetonitrile,dichloromethane,dioxane,n-hexane, HMPA.Solution5:1,2-dichloroethane,ethyl methyl ketone, n-pentane,pyridine.Solution6:tert-butyl alcohol,BHT,cyclo-hexane,1,2-dimethoxyethane,nitromethane,silicone grease, triethylamine.Solution7:diglyme,dimethylacetamide,ethyl-ene glycol,“grease”(engine oil).For D2O.Solution1:acetone, tert-butyl methyl ether,dimethylformamide,ethanol,2-propanol. Solution2:dimethyl sulfoxide,ethyl acetate,ethylene glycol, methanol.Solution3:acetonitrile,diglyme,dioxane,HMPA, pyridine.Solution4:1,2-dimethoxyethane,dimethylacetamide, ethyl methyl ketone,triethylamine.Solution5:acetic acid,tert-butyl alcohol,diethyl ether,tetrahydrofuran.In D2O and CD3OD nitromethane was run separately,as the protons exchanged with deuterium in presence of triethylamine.ResultsProton Spectra(Table1).A sample of0.6mL of the solvent,containing1µL of TMS,1was first run on its own.From this spectrum we determined the chemical shifts of the solvent residual peak2and the water peak. It should be noted that the latter is quite temperature-dependent(vide infra).Also,any potential hydrogen-bond acceptor will tend to shift the water signal down-field;this is particularly true for nonpolar solvents.In contrast,in e.g.DMSO the water is already strongly hydrogen-bonded to the solvent,and solutes have only a negligible effect on its chemical shift.This is also true for D2O;the chemical shift of the residual HDO is very temperature-dependent(vide infra)but,maybe counter-intuitively,remarkably solute(and pH)independent. We then added3µL of one of our stock solutions to the NMR tube.The chemical shifts were read and are presented in Table 1.Except where indicated,the coupling constants,and therefore the peak shapes,are essentially solvent-independent and are presented only once.For D2O as a solvent,the accepted reference peak(δ)0)is the methyl signal of the sodium salt of3-(trimeth-ylsilyl)propanesulfonic acid;one crystal of this was added to each NMR tube.This material has several disadvan-tages,however:it is not volatile,so it cannot be readily eliminated if the sample has to be recovered.In addition, unless one purchases it in the relatively expensive deuterated form,it adds three more signals to the spectrum(methylenes1,2,and3appear at2.91,1.76, and0.63ppm,respectively).We suggest that the re-sidual HDO peak be used as a secondary reference;we find that if the effects of temperature are taken into account(vide infra),this is very reproducible.For D2O, we used a different set of stock solutions,since many of the less polar substrates are not significantly water-soluble(see Table1).We also ran sodium acetate and sodium formate(chemical shifts: 1.90and8.44ppm, respectively).Carbon Spectra(Table2).To each tube,50µL of the stock solution and3µL of TMS1were added.The solvent chemical shifts3were obtained from the spectra containing the solutes,and the ranges of chemical shifts(1)For recommendations on the publication of NMR data,see: IUPAC Commission on Molecular Structure and Spectroscopy.Pure Appl.Chem.1972,29,627;1976,45,217.(2)I.e.,the signal of the proton for the isotopomer with one less deuterium than the perdeuterated material,e.g.,C H Cl3in CDCl3or C6D5H in C6D6.Except for CHCl3,the splitting due to J HD is typically observed(to a good approximation,it is1/6.5of the value of the corresponding J HH).For CHD2groups(deuterated acetone,DMSO, acetonitrile),this signal is a1:2:3:2:1quintet with a splitting of ca.2 Hz.(3)In contrast to what was said in note2,in the13C spectra the solvent signal is due to the perdeuterated isotopomer,and the one-bond couplings to deuterium are always observable(ca.20-30Hz). Figure1.Chemical shift of H DO as a function of tempera-ture..Chem.1997,62,7512-7515S0022-3263(97)01176-6CCC:$14.00©1997American Chemical Societyshow their degree of variability.Occasionally,in order to distinguish between peaks whose assignment was ambiguous,a further1-2µL of a specific substrate were added and the spectra run again.Table1.1H NMR Dataproton mult CDCl3(CD3)2CO(CD3)2SO C6D6CD3CN CD3OD D2O solvent residual peak7.26 2.05 2.507.16 1.94 3.31 4.79 H2O s 1.56 2.84a 3.33a0.40 2.13 4.87acetic acid CH3s 2.10 1.96 1.91 1.55 1.96 1.99 2.08 acetone CH3s 2.17 2.09 2.09 1.55 2.08 2.15 2.22 acetonitrile CH3s 2.10 2.05 2.07 1.55 1.96 2.03 2.06 benzene CH s7.367.367.377.157.377.33tert-butyl alcohol CH3s 1.28 1.18 1.11 1.05 1.16 1.40 1.24 OH c s 4.19 1.55 2.18tert-butyl methyl ether CCH3s 1.19 1.13 1.11 1.07 1.14 1.15 1.21 OCH3s 3.22 3.13 3.08 3.04 3.13 3.20 3.22 BHT b ArH s 6.98 6.96 6.877.05 6.97 6.92OH c s 5.01 6.65 4.79 5.20ArCH3s 2.27 2.22 2.18 2.24 2.22 2.21ArC(CH3)3s 1.43 1.41 1.36 1.38 1.39 1.40chloroform CH s7.268.028.32 6.157.587.90 cyclohexane CH2s 1.43 1.43 1.40 1.40 1.44 1.451,2-dichloroethane CH2s 3.73 3.87 3.90 2.90 3.81 3.78 dichloromethane CH2s 5.30 5.63 5.76 4.27 5.44 5.49diethyl ether CH3t,7 1.21 1.11 1.09 1.11 1.12 1.18 1.17 CH2q,7 3.48 3.41 3.38 3.26 3.42 3.49 3.56 diglyme CH2m 3.65 3.56 3.51 3.46 3.53 3.61 3.67 CH2m 3.57 3.47 3.38 3.34 3.45 3.58 3.61OCH3s 3.39 3.28 3.24 3.11 3.29 3.35 3.37 1,2-dimethoxyethane CH3s 3.40 3.28 3.24 3.12 3.28 3.35 3.37 CH2s 3.55 3.46 3.43 3.33 3.45 3.52 3.60 dimethylacetamide CH3CO s 2.09 1.97 1.96 1.60 1.97 2.07 2.08 NCH3s 3.02 3.00 2.94 2.57 2.96 3.31 3.06NCH3s 2.94 2.83 2.78 2.05 2.83 2.92 2.90 dimethylformamide CH s8.027.967.957.637.927.977.92 CH3s 2.96 2.94 2.89 2.36 2.89 2.99 3.01CH3s 2.88 2.78 2.73 1.86 2.77 2.86 2.85 dimethyl sulfoxide CH3s 2.62 2.52 2.54 1.68 2.50 2.65 2.71 dioxane CH2s 3.71 3.59 3.57 3.35 3.60 3.66 3.75 ethanol CH3t,7 1.25 1.12 1.060.96 1.12 1.19 1.17 CH2q,7d 3.72 3.57 3.44 3.34 3.54 3.60 3.65OH s c,d 1.32 3.39 4.63 2.47ethyl acetate CH3CO s 2.05 1.97 1.99 1.65 1.97 2.01 2.07C H2CH3q,7 4.12 4.05 4.03 3.89 4.06 4.09 4.14CH2C H3t,7 1.26 1.20 1.170.92 1.20 1.24 1.24 ethyl methyl ketone CH3CO s 2.14 2.07 2.07 1.58 2.06 2.12 2.19C H2CH3q,7 2.46 2.45 2.43 1.81 2.43 2.50 3.18CH2C H3t,7 1.060.960.910.850.96 1.01 1.26 ethylene glycol CH s e 3.76 3.28 3.34 3.41 3.51 3.59 3.65“grease”f CH3m0.860.870.920.860.88CH2br s 1.26 1.29 1.36 1.27 1.29n-hexane CH3t0.880.880.860.890.890.90CH2m 1.26 1.28 1.25 1.24 1.28 1.29HMPA g CH3d,9.5 2.65 2.59 2.53 2.40 2.57 2.64 2.61 methanol CH3s h 3.49 3.31 3.16 3.07 3.28 3.34 3.34 OH s c,h 1.09 3.12 4.01 2.16nitromethane CH3s 4.33 4.43 4.42 2.94 4.31 4.34 4.40 n-pentane CH3t,70.880.880.860.870.890.90CH2m 1.27 1.27 1.27 1.23 1.29 1.292-propanol CH3d,6 1.22 1.10 1.040.95 1.09 1.50 1.17 CH sep,6 4.04 3.90 3.78 3.67 3.87 3.92 4.02 pyridine CH(2)m8.628.588.588.538.578.538.52 CH(3)m7.297.357.39 6.667.337.447.45CH(4)m7.687.767.79 6.987.737.857.87 silicone grease i CH3s0.070.130.290.080.10 tetrahydrofuran CH2m 1.85 1.79 1.76 1.40 1.80 1.87 1.88 CH2O m 3.76 3.63 3.60 3.57 3.64 3.71 3.74 toluene CH3s 2.36 2.32 2.30 2.11 2.33 2.32CH(o/p)m7.177.1-7.27.187.027.1-7.37.16CH(m)m7.257.1-7.27.257.137.1-7.37.16 triethylamine CH3t,7 1.030.960.930.960.96 1.050.99 CH2q,7 2.53 2.45 2.43 2.40 2.45 2.58 2.57a In these solvents the intermolecular rate of exchange is slow enough that a peak due to HDO is usually also observed;it appears at2.81and3.30ppm in acetone and DMSO,respectively.In the former solvent,it is often seen as a1:1:1triplet,with2J H,D)1Hz. b2,6-Dimethyl-4-tert-butylphenol.c The signals from exchangeable protons were not always identified.d In some cases(see note a),the coupling interaction between the CH2and the OH protons may be observed(J)5Hz).e In CD3CN,the OH proton was seen as a multiplet atδ2.69,and extra coupling was also apparent on the methylene peak.f Long-chain,linear aliphatic hydrocarbons.Their solubility in DMSO was too low to give visible peaks.g Hexamethylphosphoramide.h In some cases(see notes a,d),the coupling interaction between the CH3and the OH protons may be observed(J)5.5Hz).i Poly(dimethylsiloxane).Its solubility in DMSO was too low to give visible peaks.Notes .Chem.,Vol.62,No.21,19977513.Chem.,Vol.62,No.21,1997NotesTable2.13C NMR Data aCDCl3(CD3)2CO(CD3)2SO C6D6CD3CN CD3OD D2O solvent signals77.16(0.0629.84(0.0139.52(0.06128.06(0.02 1.32(0.0249.00(0.01206.26(0.13118.26(0.02acetic acid CO175.99172.31171.93175.82173.21175.11177.21 CH320.8120.5120.9520.3720.7320.5621.03 acetone CO207.07205.87206.31204.43207.43209.67215.94 CH330.9230.6030.5630.1430.9130.6730.89 acetonitrile CN116.43117.60117.91116.02118.26118.06119.68 CH3 1.89 1.12 1.030.20 1.790.85 1.47 benzene CH128.37129.15128.30128.62129.32129.34tert-butyl alcohol C69.1568.1366.8868.1968.7469.4070.36 CH331.2530.7230.3830.4730.6830.9130.29 tert-butyl methyl ether OCH349.4549.3548.7049.1949.5249.6649.37 C72.8772.8172.0472.4073.1774.3275.62C C H326.9927.2426.7927.0927.2827.2226.60 BHT C(1)151.55152.51151.47152.05152.42152.85C(2)135.87138.19139.12136.08138.13139.09CH(3)125.55129.05127.97128.52129.61129.49C(4)128.27126.03124.85125.83126.38126.11CH3Ar21.2021.3120.9721.4021.2321.38C H3C30.3331.6131.2531.3431.5031.15C34.2535.0034.3334.3535.0535.36chloroform CH77.3679.1979.1677.7979.1779.44cyclohexane CH226.9427.5126.3327.2327.6327.961,2-dichloroethane CH243.5045.2545.0243.5945.5445.11 dichloromethane CH253.5254.9554.8453.4655.3254.78diethyl ether CH315.2015.7815.1215.4615.6315.4614.77 CH265.9166.1262.0565.9466.3266.8866.42 diglyme CH359.0158.7757.9858.6658.9059.0658.67 CH270.5171.0369.5470.8770.9971.3370.05CH271.9072.6371.2572.3572.6372.9271.63 1,2-dimethoxyethane CH359.0858.4558.0158.6858.8959.0658.67 CH271.8472.4717.0772.2172.4772.7271.49 dimethylacetamide CH321.5321.5121.2921.1621.7621.3221.09 CO171.07170.61169.54169.95171.31173.32174.57NCH335.2834.8937.3834.6735.1735.5035.03NCH338.1337.9234.4237.0338.2638.4338.76 dimethylformamide CH162.62162.79162.29162.13163.31164.73165.53 CH336.5036.1535.7335.2536.5736.8937.54CH331.4531.0330.7330.7231.3231.6132.03 dimethyl sulfoxide CH340.7641.2340.4540.0341.3140.4539.39 dioxane CH267.1467.6066.3667.1667.7268.1167.19 ethanol CH318.4118.8918.5118.7218.8018.4017.47 CH258.2857.7256.0757.8657.9658.2658.05 ethyl acetate C H3CO21.0420.8320.6820.5621.1620.8821.15 CO171.36170.96170.31170.44171.68172.89175.26CH260.4960.5659.7460.2160.9861.5062.32CH314.1914.5014.4014.1914.5414.4913.92 ethyl methyl ketone C H3CO29.4929.3029.2628.5629.6029.3929.49 CO209.56208.30208.72206.55209.88212.16218.43C H2CH336.8936.7535.8336.3637.0937.3437.27CH2C H37.868.037.617.918.148.097.87 ethylene glycol CH263.7964.2662.7664.3464.2264.3063.17“grease”CH229.7630.7329.2030.2130.8631.29n-hexane CH314.1414.3413.8814.3214.4314.45CH2(2)22.7023.2822.0523.0423.4023.68CH2(3)31.6432.3030.9531.9632.3632.73HMPA b CH336.8737.0436.4236.8837.1037.0036.46 methanol CH350.4149.7748.5949.9749.9049.8649.50c nitromethane CH362.5063.2163.2861.1663.6663.0863.22 n-pentane CH314.0814.2913.2814.2514.3714.39CH2(2)22.3822.9821.7022.7223.0823.38CH2(3)34.1634.8333.4834.4534.8935.302-propanol CH325.1425.6725.4325.1825.5525.2724.38 CH64.5063.8564.9264.2364.3064.7164.88 pyridine CH(2)149.90150.67149.58150.27150.76150.07149.18 CH(3)123.75124.57123.84123.58127.76125.53125.12CH(4)135.96136.56136.05135.28136.89138.35138.27 silicone grease CH3 1.04 1.40 1.38 2.10 tetrahydrofuran CH225.6226.1525.1425.7226.2726.4825.67 CH2O67.9768.0767.0367.8068.3368.8368.68 toluene CH321.4621.4620.9921.1021.5021.50C(i)137.89138.48137.35137.91138.90138.85CH(o)129.07129.76128.88129.33129.94129.91CH(m)128.26129.03128.18128.56129.23129.20CH(p)125.33126.12125.29125.68126.28126.29triethylamine CH311.6112.4911.7412.3512.3811.099.07 CH246.2547.0745.7446.7747.1046.9647.19a See footnotes for Table1.b2J PC)3Hz.c Reference material;see text.For D2O solutions there is no accepted reference for carbon chemical shifts.We suggest the addition of a drop of methanol,and the position of its signal to be defined as49.50ppm;on this basis,the entries in Table2were recorded.The chemical shifts thus obtained are,on the whole,very similar to those for the other solvents. Alternatively,we suggest the use of dioxane when the methanol peak is expected to fall in a crowded area of the spectrum.We also report the chemical shifts of sodium formate(171.67ppm),sodium acetate(182.02and 23.97ppm),sodium carbonate(168.88ppm),sodium bicarbonate(161.08ppm),and sodium3-(trimethylsilyl)-propanesulfonate[54.90,19.66,15.56(methylenes1,2, and3,respectively),and-2.04ppm(methyls)],in D2O. Temperature Dependence of HDO Chemical Shifts.We recorded the1H spectrum of a sample of D2O, containing a crystal of sodium3-(trimethylsilyl)propane-sulfonate as reference,as a function of temperature.The data are shown in Figure1.The solid line connecting the experimental points corresponds to the equation which reproduces the measured values to better than1 ppb.For the0-50o C range,the simplergives values correct to10ppb.For both equations,T is the temperature in°C.Acknowledgment.Generous support for this work by the Minerva Foundation and the Otto Mayerhoff Center for the Study of Drug-Receptor Interactions at Bar-Ilan University is gratefully acknowledged.JO971176Vδ)5.060-0.0122T+(2.11×10-5)T2(1)δ)5.051-0.0111T(2)Notes .Chem.,Vol.62,No.21,19977515。

病毒学

6
The Role of Animal Viruses in Understanding the Basic Outlines of Eukaryotic Gene Regulation: The first transcriptional enhancer element (acts in an orientation- and distance-independent fashion) was described in the SV40 genome, as was a distance- and orientation-dependent promoter element observed with the same virus. The transcription factors that bind to the promoter, SP-1, or to the enhancer element, such as AP-1, AP-2, and which are essential to promote transcription along with the basal factors, were first described with SV40. Almost everything we know about the steps of messenger RNA (mRNA) processing began with observations made with viruses. For examples, RNA splicing of new transcripts was first described with the adenoviruses. The signal for polyadenylation in the mRNA was first found using SV40.

生物大分子功能化上转换纳米探针的制备及其成像研究

论文摘要掺杂镧系离子(Ln3+)的上转换纳米粒子(UCNPs)因为具有丰富的电子能级、高能带发射、长荧光寿命、出色的光稳定性和从紫外(UV)到红外(IR)的多个发射带等优势而受到了广泛关注，已经在无背景生物传感和生物成像、光触发药物传输、太阳能电池和超分辨生物成像等领域获得了广泛的应用。

然而，UCNPs本身不能识别目标分析物，无法对分析物进行高选择性的传感。

所以，在本论文中，我们选择了不同的生物大分子作为UCNPs的能量受体，同时具备对目标分析物的识别能力，设计了基于UCNPs的新型近红外(NIR)荧光探针，利用上转换荧光(UCL)实现生物成像研究。

具体内容如下：第一章绪论本章首先介绍了上转换荧光(UCL)、上转换纳米晶体的性质，并对其合成方法进行了概述。

重点对基于上转换纳米材料的表面功能化、能量受体的选择，以及在生物传感与成像领域的应用进行了阐述。

最后对本论文研究内容以及意义进行了论述。

第二章藻蓝蛋白修饰上转换纳米探针用于炎症过程中髓过氧化物酶的检测和生物成像研究在本章中，我们合成了PEI-UCNPs，构建了一种基于UCNPs和藻蓝蛋白(PC)共价结合的新型荧光纳米探针(PC-UCNPs)，并将该探针用于髓过氧化物酶(MPO)的检测与生物成像研究。

我们通过酰胺化反应将PC偶联到UCNPs的表面，UCL 被PC吸收后淬灭。

由于PC存在特定的识别位点，该纳米探针可用于灵敏评估MPO的生物活性。

利用该探针的光学优势，PC-UCNPs已成功用于活细胞和小鼠急性炎症过程中MPO的生物成像。

第三章缩醛化葡聚糖修饰上转换纳米探针用于细胞自噬过程中溶酶体pH的传感与成像研究在本章中，我们开发了一种基于UCNPs的新型NIR溶酶体pH探针(Ac-DEX@UCNPs)。

首先，我们合成了核壳结构的UCNPs，并对葡聚糖(DEX)进行修饰得到了缩醛化葡聚糖(Ac-DEX)。

Ac-DEX与UCNPs通过配体交换策略结合，基于聚集诱导淬灭效应(ACQ)，Ac-DEX@UCNPs的UCL淬灭。

SelectiveSynthesisofSingle-Crystalline_Rhombic_Dodecahedral,_Octahedral,_and_Cubic_Gold_Nanocrystals

Selective Synthesis of Single-Crystalline Rhombic Dodecahedral,Octahedral,and Cubic Gold NanocrystalsWenxin Niu,†,‡Shanliang Zheng,§Dawei Wang,†,‡Xiaoqing Liu,†,‡Haijuan Li,†,‡Shuang Han,†,‡Jiuan Chen,†,‡Zhiyong Tang,*,§and Guobao Xu*,†State Key Laboratory of Electroanalytical Chemistry,Changchun Institute of Applied Chemistry,and Graduate Uni V ersity of the Chinese Academy of Sciences,Chinese Academy of Sciences,Changchun 130022,China,and National Center for Nanoscience and Technology,Beijing 100190,ChinaReceived June 6,2008;E-mail:zytang@.;guobaoxu@Abstract:This paper reports a versatile seed-mediated growth method for selectively synthesizing single-crystalline rhombic dodecahedral,octahedral,and cubic gold nanocrystals.In the seed-mediated growth method,cetylpyridinium chloride (CPC)and CPC-capped single-crystalline gold nanocrystals 41.3nm in size are used as the surfactant and seeds,respectively.The CPC-capped gold seeds can avoid twinning during the growth process,which enables us to study the correlations between the growth conditions and the shapes of the gold nanocrystals.Surface-energy and kinetic considerations are taken into account to understand the formation mechanisms of the single-crystalline gold nanocrystals with varying shapes.CPC surfactants are found to alter the surface energies of gold facets in the order {100}>{110}>{111}under the growth conditions in this study,whereas the growth kinetics leads to the formation of thermodynamically less favored shapes that are not bounded by the most stable facets.The competition between AuCl 4-reduction and the CPC capping process on the {111}and {110}facets of gold nanocrystals plays an important role in the formation of the rhombic dodecahedral (RD)and octahedral gold nanocrystals.Octahedral nanocrystals are formed when the capping of CPC on {111}facets dominates,while RD nanocrystals are formed when the reduction of AuCl 4-on {111}facets dominates.Cubic gold nanocrystals are formed by the introduction of bromide ions in the presence of CPC.The cooperative work of cetylpyridinium and bromide ions can stabilize the gold {100}facet under the growth condition in this study,thereby leading to the formation of cubic gold nanocrystals.1.IntroductionThe properties of noble-metal nanocrystals are dictated by their sizes and shapes.1-3Therefore,size-and shape-controlled synthesis of noble-metal nanocrystals is essential for uncovering and understanding their intrinsic properties,which will conse-quently enable us to exploit them for catalytic,plasmonic,sensing,and spectroscopic applications.1-6Over the past decade,great progress has been made in crystallographic control over the nucleation and growth of gold nanocrystals.Most efforts thus far have been focused on the synthesis of octahedral and cubic gold nanocrystals bounded by {111}and {100}facets.7-10Synthesis of gold nanocrystals bounded by {110}facets has seldom been reported because the surface energy of the bare {110}facets is higher than those of bare {111}and {100}facets.11In principle,the shape of single-crystalline gold crystals bounded completely by equivalent {110}facets is supposed to be rhombic dodecahedral (RD),12,13i.e.,such a crystal would be a convex polyhedron with 12rhombic faces that has eight vertices where three faces meet at their obtuse angles and six vertices where four faces meet at their acute angles (Figure 1).14RD gold crystals have been observed in minerals,12and RD gold nano-and microcrystals have also been observed in chemical syntheses.15,16However,the rational synthesis of uniform RD gold nanocrystals in high yield has not been reported to date and is still a challenge that requires exquisite crystal-growth control.Although several parameters have been revealed to greatly affect the growth mode of metal nanocrystals,the exact mechanisms for the shape-controlled synthesis of metal nanoc-†Changchun Institute of Applied Chemistry.‡Graduate University of the Chinese Academy of Sciences.§National Center for Nanoscience and Technology.(1)Murphy,C.J.;Sau,T.K.;Gole,A.M.;Orendorff,C.J.;Gao,J.;Gou,L.;Hunyadi,S.E.;Li,T.J.Phys.Chem.B 2005,109,13857.(2)Wiley,B.;Sun,Y.;Xia,Y.Acc.Chem.Res.2007,40,1067.(3)Tao,A.R.;Habas,S.;Yang,P.Small 2008,4,310.(4)Narayanan,R.;El-Sayed,M.A.J.Am.Chem.Soc.2004,126,7194.(5)Liz-Marzan,ngmuir 2006,22,32.(6)Rosi,N.L.;Mirkin,C.A.Chem.Re V .2005,105,1547.(7)Kim,F.;Connor,S.;Song,H.;Kuykendall,T.;Yang,P.Angew.Chem.,Int.Ed.2004,43,3673.(8)Sau,T.K.;Murphy,C.J.J.Am.Chem.Soc.2004,126,8648.(9)Seo,D.;Park,J.C.;Song,H.J.Am.Chem.Soc.2006,128,14863.(10)Li,C.;Shuford,K.L.;Park,Q.-H.;Cai,W.;Li,Y.;Lee,E.J.;Cho,O.C.Angew.Chem.,Int.Ed.2007,46,3264.(11)Wang,Z.L.J.Phys.Chem.B 2000,104,1153.(12)Taber,S.Am.Mineral.1948,33,482.(13)Francis,C.A.Rocks Miner.2004,79,24.(14)Weisstein,E.W.CRC Concise Encyclopedia of Mathematics ,2nd ed.;CRC Press:Boca Raton,FL,2003;p 2546.(15)Liu,X.;Wu,N.;Wunsch,B.H.,Jr.;Stellacci,F.Small 2006,2,1046.(16)Chen,Y.;Gu,X.;Nie,C.-G.;Jiang,Z.-Y.;Xie,Z.-X.;Lin,C.-J.Chem.Commun.2005,4181.Published on Web 12/22/200810.1021/ja804115r CCC:$40.75 2009American Chemical SocietyJ.AM.CHEM.SOC.2009,131,697–7039697rystals are still not well-understood.3Most of the synthetic methods remain empirical,and understanding their growth mechanisms is still a challenging task.The seed-mediated growth method separates the nucleation and growth stages of nanocrystals,which provides better control over the size,size distribution,and shape evolution of the nanoparticles.1,17A typical seed-mediated growth process involves the preparation of small metal nanoparticles and their subsequent growth in reaction solutions containing metal salts,reducing agents,and surfactants.During the growth procedure,the crystal structures of the metal nanocrystals grown from small seeds are ﬂuctuant.18The small seeds may grow into single-crystalline,twinned,or multitwinned structures,19leading to the formation of polydis-perse nanostructures.The shape evolution of metal nanocrystals from seeds with different crystal structures is quite different,20,21which blurs our understanding of the mechanisms of the growth process.To preserve the single-crystalline nature of the seeds during the seed-mediated growth process,one has to judiciously select appropriate adsorbents 20,22or manipulate the growth kinetics.8Therefore,development of a general strategy that can selectively produce single-crystalline metal nanocrystals without the need for elaborate selection of appropriate adsorbents or manipulation of the growth kinetics could enable us to study the detailed correlations between growth conditions and the shapes of the single-crystalline nanocrystals.In this study,we developed a versatile seed-mediated growth method to selectively synthesize single-crystalline RD,octahe-dral,and cubic gold nanocrystals through manipulation of the growth kinetics and selection of appropriate adsorbates.Single-crystalline gold nanocrystals with diameters of 41.3nm and capped by cetylpyridinium chloride (CPC)were prepared and explored as the seeds in the seed-mediated growth method.The single-crystalline nature and relatively large sizes of the CPC-capped seeds can ﬁx the structure of the ﬁnal nanocrystals as single-crystalline,enabling us to carefully study the parametersthat affect the formation of the gold nanocrystals with varying shapes.Several important parameters that affect the shape evolutions of the gold nanocrystals are revealed,and the growth mechanisms are explained in terms of surface energy and growth kinetics.2.Experimental Section2.1.Materials.Chloroauric acid tetrahydrate (HAuCl 4·4H 2O),sodium borohydride (NaBH 4),and cetyltrimethylammonium bro-mide (CTAB)were obtained from Sinopharm Chemical Reagent Co.,Ltd.L -Ascorbic acid,silver nitrate (AgNO 3),and potassium bromide (KBr)were obtained from Beijing Chemical Reagent Company.CPC was obtained from Shanghai Sangon Company.All of the chemicals were of analytical grade and used without further puriﬁcation.Doubly distilled water was used throughout the experiments.2.2.Synthesis of Gold Nanorods. 2.2.1.Preparation of ∼1.5nm CTAB-Capped Gold Seeds.A 125µL aliquot of 10mM HAuCl 4solution was added to 5mL of 100mM CTAB solution at 30°C.After this combination was gently mixed,0.3mL of 10mM ice-cold NaBH 4solution was added all at once,followed by rapid inversion mixing for 2min.22,23The CTAB-capped gold seed solution was stored at 30°C for future use.2.2.2.Synthesis of Gold Nanorods.In sequence,2mL of 10mM HAuCl 4solution,240µL of 10mM AgNO 3solution,320µL of freshly prepared 100mM ascorbic acid solution,and 48µL of the ∼1.5nm CTAB-capped gold seed solution were added to 40mL of 100mM CTAB solution at 30°C.The solution was thoroughly mixed after each addition.22,23Finally,the gold nanorod solution was left undisturbed and aged for 2h for further use.2.3.Synthesis of the CPC-Capped Gold Seeds from Gold Nanorods.2.3.1.Secondary Overgrowth of Gold Nanorods.A 30mL aliquot of the as-synthesized gold nanorod solution was centrifuged (12000rpm,10min)and redispersed in water.Subsequently,the solution was centrifuged (12000rpm,10min)again and redispersed in 30mL of 10mM CTAB solution at 40°stly,1.5mL of 10mM HAuCl 4solution and 0.3mL of 100mM ascorbic acid solution were added in sequence and mixed thoroughly.The mixture was allowed to react at 40°C for 1h.2.3.2.Synthesis of the CPC-Capped Gold Seeds.The CPC-capped gold seeds were prepared by transformation of the over-grown gold nanorods to near-spherical nanoparticles.24Brieﬂy,the overgrown gold nanorod solution was centrifuged (12000rpm,10min)and redispersed in 30mL of 10mM CTAB solution.Next,at 40°C,0.6mL of 10mM HAuCl 4solution was added.After the solution was gently mixed,it was left undisturbed and aged for 12h.The solution was then washed three times with 100mM CPC solution by centrifugation (12000rpm,10min)and dissolution and ﬁnally dispersed in 30mL of 100mM CPC solution.We designated these nanoparticles as the CPC-capped seeds.2.4.Seed-Mediated Growth of Three Types of Gold Nanocrystals.In a typical synthesis of the RD gold nanocrystals,100µL of 10mM HAuCl 4solution,200µL of freshly prepared 100mM ascorbic acid solution,and 200µL of the CPC-capped seed solution were added to 5mL of 10mM CPC solution at 30°C in sequence.The solution was thoroughly mixed after each addition.The reaction was stopped after 2h by centrifugation (12000rpm,10min).The gold nanocrystal solution was washed twice with water and concentrated for characterization by electron microscopy.In a typical synthesis of the octahedral gold nanocrystals,100µL of 10mM HAuCl 4solution,13µL of freshly prepared 100mM ascorbic acid solution,and 200µL of the CPC-capped seed solution were added to 5mL of 100mM CPC solution at 30°C in(17)Murphy,C.J.;Gole,A.M.;Hunyadi,S.E.;Orendorff,C.J.Inorg.Chem.2006,45,7544.(18)Elechiguerra,J.L.;Reyes-Gasga,J.;Yacaman,J.M.J.Mater.Chem.2006,16,3906.(19)Wu,H.-Y.;Huang,W.-L.;Huang,M.H.Cryst.Growth Des.2007,7,831.(20)Liu,M.;Guyot-Sionnest,G.J.Phys.Chem.B 2005,109,22192.(21)Lim,B.;Xiong,Y.;Xia,Y.Angew.Chem.,Int.Ed.2007,46,9279.(22)Nikoobakht,B.;El-Sayed,M.A.Chem.Mater.2003,15,1957.(23)Sau,T.K.;Murphy,ngmuir 2004,20,6414.(24)Rodriguez-Fernandez,J.;Perez-Juste,J.;Mulvaney,P.;Liz-Marzan,L.M.J.Phys.Chem.B 2005,109,14257.Figure 1.Geometrical models of rhombic dodecahedra:(A)overall view;(B)view perpendicular to one of the rhombic faces;(C)view centered on one of the vertices where three faces meet at their obtuse angles;(D)view centered on one of the vertices where four faces meet at their acute angles.698J.AM.CHEM.SOC.9VOL.131,NO.2,2009A R T I C L E S Niu et al.sequence.The solution was thoroughly mixed after each addition. The reaction was stopped after2h by centrifugation(12000rpm, 10min).In a typical synthesis of the cubic gold nanocrystals,500µL of 100mM KBr solution,100µL of10mM HAuCl4solution,13µL of freshly prepared100mM ascorbic acid solution,and200µL of the CPC-capped seed solution were added to5mL of100mM CPC solution at30°C in sequence.The solution was thoroughly mixed after each addition.The reaction was stopped after2h by centrifugation(12000rpm,10min).2.5.Instrumentation.Scanning electron microscopy(SEM) images were taken using an FEI XL30ESEM FEG scanning electron microscope operating at25kV.Transmission electron microscopy(TEM)and selected-area electron diffraction(SAED) studies were performed on a FEI Tecnai G220S-TWIN transmis-sion electron microscope operated at200kV.A drop of the concentrated nanocrystal solution was deposited on ITO glass or a TEM grid for SEM or TEM measurements,respectively.During natural evaporation of water,part of the nanocrystals migrated to the edge of the drop and formed a solid ring.SEM observations revealed that ordered assemblies mainly existed at the coffee-ring region,while random assemblies mainly existed at the center. UV-vis extinction spectra were taken at room temperature on a CARY500Scan UV-vis-near-IR spectrophotometer using a quartz cuvette with an optical path of1cm.X-ray diffraction(XRD) measurements were obtained with a Rigaku D/MAX-2500instru-ment(Cu K R1radiation)operated at50kV and250mA over a range of30-90°by step scanning with a step size of0.02°.3.Results and Discussion3.1.Preparation and Characterization of the CPC-Capped Gold Seeds.Gold nanorods were overgrown and then oxidized by Au(III)-CTAB complexes to prepare the CPC-capped gold seeds.The oxidation occurred preferentially at the ends of the overgrown gold nanorods and eventually led to the formation of near-spherical nanoparticles.24These near-spherical nano-particles wereﬁnally dispersed in100mM CPC solution and designated as the CPC-capped gold seeds.As shown in Figure 2,the average width,length,and aspect ratio of the gold nanorods are27.2nm,58.8nm,and2.2,respectively,and the average width,length,and aspect ratio of the overgrown gold nanorods are32.4nm,63.7nm,and2.0,respectively;the CPC-capped gold seeds have an average diameter of41.3nm and a size distribution of5.4%.The detailed morphology and struc-tures of the CPC-capped seeds were studied through TEM. Figure3A shows that the shapes of the CPC-capped gold seeds are slightly ellipsoidal rather than perfectly spherical.Therefore, the average diameter of the seeds is bigger than the average width of the overgrown nanorods from which they originate. The SAED pattern and high-resolution TEM(HRTEM)image of a single CPC-capped seed demonstrate that the CPC-capped seeds are single-crystalline(Figure3B,C).The process of preparing the CPC-capped seeds was also studied by UV-vis spectroscopy.The UV-vis extinction spectra of the gold nanorods,overgrown gold nanorods,and CPC-capped seeds are shown in Figure2D.The two peaks in the UV-vis extinction spectra of the gold nanorods and overgrown gold nanorods can be attributed to the transverse and longitudinal plasmon modes of gold nanorods.22The blue shift in the longitudinal plasmon mode of the overgrown nanorods conﬁrms a decrease in their aspect ratios.25The UV-vis extinction spectrum of the CPC-capped seeds has only one peak[curve(c)in Figure2D],which conﬁrms their near-spherical morphology.3.2.Synthesis of Single-Crystalline RD,Octahedral,and Cubic Gold Nanocrystals.3.2.1.Synthesis and Characterization of RD Gold Nanocrystals.Synthesis of RD gold nanocrystals was performed at30°C.Typically,100µL of10mM HAuCl4 solution,200µL of100mM ascorbic acid solution,and200µL of the CPC-capped seed solution were added to5mL of10 mM CPC solution in sequence.The reaction was stopped after 2h by centrifugation.Figure4A,B shows SEM images of orderly assembled and randomly distributed RD gold nanoc-rystals,respectively.In the ordered assembly of RD gold nanocrystals,the RD gold nanocrystals are in the orientation shown in Figure1C.From the randomly distributed RD gold nanocrystals,the geometrical shapes of these gold nanocrystals can be viewed from different directions and identiﬁed as RD, i.e.,bounded by12equivalent rhombic faces(Figure S1in the Supporting Information).14Figure4C shows the TEM image of the RD gold nanocrystals.Most of the RD nanocrystals tend to lieﬂat and exhibit elongated hexagonal shapes.For aﬂat-(25)Tsung,C.-K.;Kou,X.;Shi,Q.;Zhang,J.;Yeung,M.H.;Wang,J.;Stucky,G.D.J.Am.Chem.Soc.2006,128,5352.Figure2.SEM images of(A)the gold nanorods,(B)the overgrown goldnanorods,and(C)the CPC-capped gold seeds(scale bars:200nm).(D)UV-vis extinction spectra of(a)the gold nanorods,(b)the overgrown goldnanorods,and(c)the CPC-capped gold seeds(each spectrum is normalizedagainst the intensity of its strongest peak).Figure3.(A)TEM image of the CPC-capped gold seeds.(B)TEM imageand corresponding SAED pattern of a single CPC-capped seed.(C)HRTEMimage of the CPC-capped seed in(B).J.AM.CHEM.SOC.9VOL.131,NO.2,2009699 Dodecahedral,Octahedral,and Cubic Gold Nanocrystals A R T I C L E Slying RD gold nanocrystal,the top and bottom {110}faces of the RD gold nanocrystal are parallel to the substrate.Directing the electron beam perpendicular to the upper face of the ﬂat-lying RD nanocrystal produces the typical SAED pattern of a gold crystal along the [011]zone axis,12,13,26as shown in the inset of Figure 4D.The HRTEM and corresponding SAED images of gold nanocrystals viewed from different directions further conﬁrm their RD shapes (Figure S2in the Supporting Information).It should be pointed out that the XRD pattern of the RD gold nanocrystals (Figure S3in the Supporting Informa-tion)does not have an abnormally intense (220)peak because SEM observations (Figure 4A,B)reveal that not all of the RD nanocrystals lie ﬂat on the substrate.The TEM image in Figure 4D also reveals that the corners of the RD gold nanocrystal are slightly stretched-out,which indicates that the faces of the RD nanocrystals are not perfectly ﬂat.In order to clearly distinguish the surface morphologies of the RD nanocrystals,larger RD nanocrystals were also synthe-sized by reducing the added volume of the seed solution to 25µL.As shown in Figure 5,the faces of the RD nanocrystals are concave with relatively ﬂat center parts parallel to the ideal {110}facets.Therfore,although the gold nanocrystals in Figures 4and 5take an RD overall shape,their faces are not perfect {110}facets,and the center part of each facet is supposed to be a {110}facet.3.2.2.Synthesis and Characterization of Octahedral Gold Nanocrystals.Octahedral gold nanocrystals were synthesizedunder conditions similar to those for the synthesis of the RD gold nanocrystals in Figure 4,except that the concentration of CPC was increased from 10to 100mM and the volume of 100mM ascorbic acid solution was decreased from 200to 13µL.SEM and TEM images of the octahedral gold nanocrystals are presented in Figure 6A,B,respectively.The SAED study indicates that the octahedral gold nanocrystal is a piece of single crystal bounded by {111}facets (Figure 6C,D).9,103.2.3.Synthesis and Characterization of Cubic Gold Nano-crystals.Cubic gold nanocrystals were produced when 0.5mLof 100mM KBr solution was introduced while the other conditions were kept the same as those in the synthesis of the octahedral gold nanocrystals.Figure 7A,B shows the SEM and TEM images of the cubic gold nanocrystals,respectively,and Figure 7C,D shows the TEM image and corresponding SAED pattern of a single cubic gold nanocrystal.The square spot array of the SAED pattern indicates that the cubic gold nanocrystal is a single crystal bounded by {100}facets.7,9Cubic gold nanocrystals are also produced when only 0.05mL of 100mM KBr solution is introduced,as shown in Figure S4in the Supporting Information.3.2.4.Average Edge Lengths,Size Distributions,Yields,and Corresponding Optical Properties of the Gold Nano-crystals.Table 1summarizes the average sizes,size distributions,and yields of the gold nanocrystals presented in Figures 4,6,and 7.All three types of gold nanocrystals are prepared in high yields with good pared with the CPC-capped seeds,the ﬁnal nanocrystals have improved monodis-(26)Wang,Z.L.Reﬂection Electron Microscopy and Spectroscopy forSurface Analysis ;Cambridge University Press:Cambridge,U.K.,1996;Appendix D.Figure 4.SEM images of (A)orderly assembled and (B)randomly distributed RD gold nanocrystals (scale bars:200nm).(C)TEM image of the RD gold nanocrystals (scale bar:200nm).(D)TEM image and corresponding SAED pattern of a single ﬂat-lying RD gold nanocrystal recorded along the [011]zone axis (scale bar:50nm).Figure 5.SEM images of larger RD gold nanocrystals at differentmagniﬁcations [scale bars:(A)1µm,(B)500nm,(C)200nm].(D)RD geometrical models with typical orientations corresponding to RD gold nanocrystals at the same positions in (C).Figure 6.(A)SEM image of octahedral gold nanocrystals (scale bar:200nm).(B)TEM image of octahedral gold nanocrystals (scale bar:100nm).(C)TEM image and (D)corresponding SAED pattern of a single ﬂat-lyingoctahedral gold nanocrystal recorded along the [1j 11]zone axis (scale bar:20nm).700J.AM.CHEM.SOC.9VOL.131,NO.2,2009A R T I C L E S Niu et al.persity.In seed-mediated growth procedures,the improvement in the monodispersity is attributed to the “self-focusing”tendency that small particles grow faster than larger ones,causing the ﬁnal sizes of the nanocrystals to become uniform.27,28Such observations are consistent with previous reports that the monodispersity of the grown nanoparticles is better than that of the seeds.29-31It is well-known that noble metal nanoparticles exhibit size-and shape-dependent optical properties arising from surface plasmon resonances.32As shown in Figure 8,the UV -vis extinction spectra of the RD,octahedral,and cubic gold nanocrystals show peaks at 582,568,and 551nm,respectively.The differences in the UV -vis extinction spectra may result from the differences in the sizes,shapes,and outstretched or truncated corners of the gold nanocrystals.3.3.Growth Mechanisms of the Gold Nanocrystals.In a seed-mediated growth procedure,several parameters are simulta-neously responsible for the ﬁnal shapes of the gold nanocrys-tals.17Hence several control experiments are conducted to elucidate the growth mechanisms of the gold nanocrystals.In the nucleation stage,the size and crystal structure of seeds are proved to be important for the formation of single-crystalline structures of the gold nanocrystals.In the growth stage,surfactants,growth kinetics,and adsorbates are found to determine the ﬁnal shapes of the resultant single-crystalline gold nanocrystals.3.3.1.The Importance of the Size and Crystal Structure of the Seeds.To investigate how the size and crystal structureof the seeds affect the growth of gold nanocrystals,another two types of seeds commonly used in the seed-mediated growth method were adopted here (under the same growth conditions)in addition to the CPC-capped gold seeds.These two types of seeds were ∼3nm citrate-capped twinned gold seeds and ∼1.5nm CTAB-capped single-crystalline gold seeds.20Among the three types of seeds,the ∼1.5nm CTAB-capped single-crystalline seed solution contains bromide ions,and bromide ions from this seed solution may affect the growth of gold nanocrystals.33Therefore,we intentionally adopted the growth conditions with the addition of KBr to counteract the inﬂuence of the presence or absence of bromide ions in different seed solutions.SEM images of the gold nanocrystals synthesized with the different types of seeds are shown in Figure 9,and their corresponding shape distributions are summarized in Table S1in the Supporting Information.When the CPC-capped single-crystalline gold seeds were used,cubic gold nanocrystals were produced in high yield (95.2%).In contrast,only 4.0%of the products were cubic nanocrystals when the ∼3nm twinned nanoparticles were used,and 26.1%of the products were cubic nanocrystals when ∼1.5nm single-crystalline nanoparticles were used.These results provide evidence that both the single-crystalline nature and the relatively large sizes of the CPC-capped seeds play important roles in the growth of single-crystalline gold nanocrystals with high yields.It is believed that the crystal structure of the seeds ﬂuctuates at very small sizes,whereas their structure will be ﬁxed as single-crystalline or multitwinned as the size of the crystals increases.18,34Because of their relatively large sizes,the CPC-capped single-crystalline seeds can avoid twinning during the growth process,enabling us to study the parameters that affect the growth of the gold nanocrystals.It should also be noted that the present procedure for preparing the CPC-capped seeds is somewhat tedious.Ongoing efforts in our group are being directed toward ﬁnding simpler methods for synthesis of high-quality single-crystalline gold seeds with relatively large sizes.(27)Reiss,H.J.Chem.Phys.1951,19,482.(28)Peng,X.G.;Wickham,J.;Alivisatos,A.P.J.Am.Chem.Soc.1998,120,5343.(29)Brown,K.R.;Walter,D.G.;Natan,M.J.Chem.Mater.2000,12,306.(30)Henglein,ngmuir 1999,15,6738.(31)Jana,N.R.;Gearheart,L.;Murphy,ngmuir 2001,17,6782.(32)Xia,Y.;Halas,N.J.MRS Bull.2005,30,338.(33)Ha,T.H.;Koo,H.J.;Chung,B.H.J.Phys.Chem.C 2007,111,1123.(34)Im,S.H.;Lee,Y.T.;Wiley,B.;Xia,Y.Angew.Chem.,Int.Ed.2005,44,2154.Figure 7.(A)SEM image of cubic gold nanocrystals (scale bar:500nm).(B)TEM image of cubic gold nanocrystals (scale bar:100nm).(C)TEM image and (D)corresponding SAED pattern of a single ﬂat-lying cubic gold nanocrystal recorded along the [001]zone axis (scale bar:20nm).Table 1.Average Edge Lengths (L av ),Size Distribution Standard Deviations (σ),and Yields of the RD,Octahedral,and Cubic Gold NanocrystalsshapeL av (nm)σ(%)yield (%)ﬁgure no.RD53.4 5.3∼1004octahedral 59.8 4.197.26cubic 55.7 4.496.17Figure 8.UV -vis extinction spectra of (A)the RD gold nanocrystals inFigure 4,(B)the octahedral gold nanocrystals in Figure 6,and (C)the cubic gold nanocrystals in Figure 7(each spectrum is normalized against the intensity of its strongest peak).J.AM.CHEM.SOC.9VOL.131,NO.2,2009701Dodecahedral,Octahedral,and Cubic Gold Nanocrystals A R T I C L E S3.3.2.Surface Energies of Different Gold Crystallographic Facets in the Presence of CPC.Surface energies associated withdifferent gold crystallographic facets usually increase in the order γ{111}<γ{100}<γ{110}.11,35In a solution-phase synthesis,adsorbates (including surfactants,polymers,small molecules,and atomic adsorbates)can interact selectively with different metal crystal facets and alter their surface energies.3It has been reported that gold nanorods have {110}side facets.20,36These {110}facets disappear during overgrowth in the presence of CTAB or poly(vinyl pyrrolidone).37,38In contrast,CPC can selectively stabilize the {110}facets of gold,and thus,RD gold nanocrystals can exist as ﬁnal products.The preparation of octahedral gold nanocrystals indicates that CPC can also stabilize the {111}facets of gold.Through a comparison between the surface structures of the larger RD and octahedral gold nanoc-rystals shown in Figures 5and 10,respectively,we found that the faces of the octahedral gold nanocrystals are ﬂat and smooth but that the faces of the RD gold nanocrystals are not well-deﬁned {110}facets,which suggests that CPC stabilizes {110}facets relatively poorly.Moreover,octahedral gold nanocrystals bounded by {111}facets are obtained at relatively high CPC concentrations,suggesting that the enhanced capping of CPC promotes the formation of {111}facets of gold.These observa-tions suggest that the gold {111}facets should be more stable than gold {110}facets in the presence of CPC under the growth conditions in this study.Since gold nanocrystals bounded by {100}facets are not observed in the presence of CPC,we suppose that gold {100}facets tend to disappear during the growth procedure,and it should be the most unstable low-index facet of gold in the presence of CPC.Therefore,it is reasonable to assume that CPC alters the surface energies of gold facets,ordering them as γ{111}<γ{110}<γ{100}under the growth conditions in this study.The selective interactions of CPC with different gold facets are responsible for the alteration.3.3.3.Growth Kinetics of the RD and Octahedral Gold Nanocrystals.The above surface-energy considerations suggestthat the RD gold nanocrystal is not the thermodynamically most favored shape.The formation of thermodynamically less favored shapes of nanocrystals are generally governed by growth kinetics.39-41In the case of the selective synthesis of RD and octahedral gold nanocrystals,growth kinetics and the capping effect of CPC must be taken into account simultaneously in order to understand their growth mechanisms.Several control experiments were conducted to investigate the formation mechanisms of RD and octahedral gold nanoc-rystals.RD gold nanocrystals could still be prepared when the concentration of CPC was increased to 100mM and the volume of 100mM ascorbic acid solution was between 200and 25µL,while other conditions were the same as those in the synthesis of the RD gold nanocrystals in Figure 4.The corresponding SEM images of the RD nanocrystals are shown in Figure 11A,B.When the volume of ascorbic acid solution was further reduced to 13µL,octahedral gold nanocrystals were produced,as shown in Figure 6.These results indicate that the competition between AuCl 4-reduction and the CPC capping process on the {111}and {110}surfaces plays an important role.Octahedral gold nanocrystals bounded by {111}facets were obtained at relatively high CPC concentrations,suggesting that the capping of CPC promotes the formation of {111}facets of gold.At relatively high ascorbic acid concentrations,RD nanocrystals were(35)Xiong,Y.;Wiley,B.;Xia,Y.Angew.Chem.,Int.Ed.2007,46,7157.(36)Wang,Z.L.;Mohamed,M.B.;Link,S.;El-Sayed,M.A.Surf.Sci.1999,440,L809.(37)Xiang,Y.;Wu,X.;Liu,D.;Feng,L.;Zhang,K.;Chu,W.;Zhou,W.;Xie,S.J.Phys.Chem.C 2008,112,3203.(38)Carbo-Argibay,E.;Rodriguez-Gonzalez,B.;Paciﬁco,J.;Pastoriza-Santos,I.;Perez-Juste,J.;Liz-Marzan,L.M.Angew.Chem.,Int.Ed.2007,46,8983.(39)Petroski,J.M.;Wang,Z.L.;Green,T.C.;El-Sayed,M.A.J.Phys.Chem.B 1998,102,3316.(40)Xiong,Y.;Cai,H.;Wiley,B.J.;Wang,J.;Kim,M.J.;Xia,Y.J.Am.Chem.Soc.2007,129,3665.(41)Tao,A.;Sinsermsuksakul,P.;Yang,P.Angew.Chem.,Int.Ed.2006,45,4597.Figure 9.SEM images of gold nanocrystals synthesized with different types of seeds:(A)41.3nm CPC-capped single-crystalline gold seeds;(B)∼3nmcitrate-capped twinned gold seeds;(C)∼1.5nm CTAB-capped single-crystalline gold seeds (all scale bars:500nm).The gold nanocrystals were synthesized by adding 50µL of 100mM KBr solution,100µL of 10mM HAuCl 4solution,15µL of 100mM ascorbic acid solution,and the corresponding amount of seed solution (200µL for the CPC-capped seed solution,1µL for the ∼3nm citrate-capped twinned seed solution,and 0.02µL for the ∼1.5nm CTAB-capped single-crystalline seed solution)to 5mL of 100mM CPC solution at 30°C.Figure 10.SEM image of larger octahedral gold nanocrystals synthesizedwith 25µL of the CPC-capped seed solution added (scale bar:200nm).702J.AM.CHEM.SOC.9VOL.131,NO.2,2009A R T I C L E S Niu et al.。

超高效液相串联质谱法测定牛奶中8种氟喹诺酮类兽药残留

分析检测超高效液相串联质谱法测定牛奶中8种氟喹诺酮类兽药残留张瑞婷（北京市产品质量监督检验研究院，北京 101300）摘要：建立了超高效液相串联质谱法同时测定牛奶中8种氟喹诺酮类兽药残留的分析方法。

牛奶样品用提取剂提取后，用固相萃取柱净化，选用C18色谱柱梯度洗脱分离。

通过串联质谱在电喷雾电离-正离子、多反应监测模式下进行定性和定量分析。

结果表明，8种兽药在0.5～500.0 μg·mL-1呈良好的线性关系，R2＞0.996，在10 μg·kg-1、50 μg·kg-1、100 μg·kg-1 3个浓度添加水平下，回收率为81.3%～98.8%，RSD为2.3%～9.8%。

该方法灵敏、快速、可靠，适用于牛奶中多种兽药残留的快速筛查。

关键词：超高效液相串联质谱法；氟喹诺酮类；牛奶Determination of 8 Fluoroquinolones Veterinary Drug Residues in Milk by Ultra Performance Liquid ChromatographyTandem Mass SpectrometryZHANG Ruiting(Beijing Institute of Product Quality Supervision and Inspection, Beijing 101300, China) Abstract: A method for the simultaneous determination of eight ﬂuoroquinolone veterinary drug residues in milk by ultra performance liquid chromatography tandem mass spectrometry was established. After the milk sample was extracted with the extractant, it was purified by a solid phase extraction column and separated by gradient elution on a C18 chromatographic column. Qualitative and quantitative analyzes were then performed by tandem mass spectrometry in electrospray ionization-positive ion, multiple reaction monitoring mode. The results showed that the eight veterinary drugs showed a good linear relationship in the range of 0.5～500.0 μg·mL-1, R2>0.996. The recoveries ranged from 81.3% to 98.8% at the spiked levels of 10 μg·kg-1, 50 μg·kg-1, 100 μg·kg-1 with the RSD of 2.3% to 9.8%. The method is sensitive, rapid and reliable, and is suitable for rapid screening of various veterinary drug residues in milk.Keywords: ultra performance liquid chromatography tandem mass spectrometry; ﬂuoroquinolones; milk氟喹诺酮类药物为白色或淡黄色晶型粉末，属于广谱抗生素，常被用于预防和治疗奶牛疾病[1]。

Yang Fan-JMS-ME online

13Journal of Materials Science:Materials in ElectronicsISSN 0957-4522J Mater Sci: Mater ElectronDOI 10.1007/s10854-014-2405-1A novel scheme to obtain tunablefluorescent colors based on electrospun composite nanofibersFan Yang, Qianli Ma, Xiangting Dong,Wensheng Yu, Jinxian Wang & Guixia LiuYour article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further depositthe accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publicationor later and provided acknowledgement is given to the original source of publicationand a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at ”.13A novel scheme to obtain tunable ﬂuorescent colors based on electrospun composite nanoﬁbersFan Yang ·Qianli Ma ·Xiangting Dong ·Wensheng Yu ·Jinxian Wang ·Guixia LiuReceived:5September 2014/Accepted:17October 2014©Springer Science+Business Media New York 2014Abstract Europium complexes Eu(BA)3phen (BA =benzoic acid,phen =1,10-phenanthroline)and Terbium complexes Tb(BA)3phen are incorporated into polyvinyl pyrrolidone (PVP)and electrospun into [Eu(BA)3phen +Tb (BA)3phen]/PVP composite nanoﬁbers.The morphologies and properties of the ﬁnal products are investigated via scanning electron microscopy,energy dispersive spec-trometry,X-ray powder diffractometry,ﬂuorescence spectroscopy,Fourier-transform infrared spectroscopy and thermogravimetry.The diameter of the composite nanoﬁ-bers is 431.55± 3.14nm.The obtained composite nanoﬁbers are amorphous in structure and stable below 220°C.By adjusting the ratio of Eu(BA)3phen to Tb (BA)3phen complexes,the ﬂuorescence color of composite nanoﬁbers can be tuned from red to yellow,yellow green,and green under the excitation of 276-nm single-wavelength ultraviolet light.Besides,the ﬂuorescence lifetime of Eu 3+ions in composite nanoﬁbers is extended gradually,while that of Tb 3+ions is decreased slowly with increase of Tb (BA)3phen content.The novel color-tunable composite nanoﬁbers have potential applications in the ﬁelds of optical devices,color displays and sensor systems.1IntroductionRecently,tremendous efforts have been dedicated to mul-ticolor and color-tunable luminescent materials because of their extensive applications in various areas such as panel display,imaging,illumination and etc [1–3].Currently,the most common color-tunable luminescent materials were prepared by introducing inorganic phosphors [4],semi-conductor quantum dots [5],functional organic materials [6]and rare earth (RE)organic complexes [7],etc.In most cases,the multicolor tuning of materials can be achieved through co-doping the host nanocrystals with Ln 3+ions.For instance,Wang et al.[8]successfully prepared Ca 5(PO 4)3F:Tb 3+,Eu 3+microrods which had potential applications in ﬁeld emission display devices.Multicolor emitting NaGdF 4:Yb 3+/Ln 3+(Ln =Er,Ho,Tm)phosphor powders were also successfully synthesized via a hydro-thermal method by Ren et al.[9].It is also feasible by using a mixture of different emissive dyes or RE complexes to achieve color-tunable materials.At present,some prepara-tions of color-tunable materials by using RE complexes can be found in the references.Wang et al.[10]prepared color-tunable and white-light emitting lanthanide complexes,and demonstrated the energy transfer mechanism was involved.Liu et al.[11]fabricated nano/micro-scaled Eu 3+and Tb 3+co-doped La(1,3,5-BTC)(H 2O)6coordination polymer.Europium and Terbium complexes have many potential applications in laser [12],phosphor [13],chemosensors and bioimaging probes [14],owing to the antenna effect of ligands and the f–f electron transition of Eu 3+ions and Tb 3+ions.However,pure RE complexes have serious limitations in practical applications because of their poor mechanical properties and physicochemical stabilities.In order to over-come these defects,doping of RE complexes into a polymer matrix has attracted much attention.The polymer-basedF.Yang ·Q.Ma ·X.Dong (&)·W.Yu ·J.Wang (&)·G.LiuKey Laboratory of Applied Chemistry and Nanotechnology at Universities of Jilin Province,Changchun University of Science and Technology,Changchun 130022,China e-mail:dongxiangting888@ J.Wange-mail:wjx87@123J Mater Sci:Mater ElectronDOI 10.1007/s10854-014-2405-1composites present excellentﬂuorescent properties,besides the enhanced mechanical and thermal properties[15–19].Electrospinning is an outstanding technique to process viscous solutions or melts into continuous compositeﬁbers or belts with1D nanostructure[20–22].It can manufacture compositeﬁbers with diameters ranging from micrometer to submicron or nanometer.The incorporation of optical active materials,such as quantum dots,organic dyes,or RE com-plexes,into electrospun composite nanoﬁbers is an attractive method to investigate their optical properties and to manu-facture optical devices on the nanometer scale[23–29].Yu et al.[30]incorporated terbium complexes Tb(acac)2AA (acac:acetyl acetone,AA:acrylic acid)into polystyrene(PS) matrix and electrospun into compositeﬁbers,and the lumi-nescence properties of the Tb(acac)2AA/PS compositeﬁbers were studied as well.However,few report concerning the fabrication of color-tunable composite nanoﬁbers can befound in literatures.Therefore,the construct of the color-tunable luminescent composite nanoﬁbers via electrospin-ning technique is a meaningful subject of study.Herein,we suggest a simple fabrication method for emissive composite nanoﬁbers composed of polyvinyl pyrrolidone(PVP)as a matrix polymer and two different RE complexes,including Eu(BA)3phen and Tb(BA)3phen, asﬂuorescence materials.The emission color of the obtained composite nanoﬁbers is tuned by adjusting the mass ratio of Eu(BA)3phen to Tb(BA)3phen.The mor-phology andﬂuorescence property of the products were systematically investigated.The as-prepared color-tunable composite nanoﬁbers have potential applications in the ﬁelds of optical devices,color displays and sensor systems. 2Experimental sections2.1ChemicalsBenzoic acid(BA),phenanthroline(phen),polyvinyl pyr-rolidone(PVP,M w≈90,000),N,N-dimethylformamide (DMF)were bought from Tianjin Guangfu Technology Development Co.,Ltd.Terbium oxide(Tb4O7)(99.9%) was purchased from Aladdin Chemistry Co.,Ltd.Europium oxide(Eu2O3)(99.99%),nitric acid(HNO3),ammonia (NH3·H2O)and anhydrous ethanol were purchased from Sinopharm Chemical Reagent Co.,Ltd.All the reagents were of analytical grade and directly used as received without further puriﬁcation.2.2Synthesis of Eu(BA)3phen and Tb(BA)3phencomplexesEu(BA)3phen powders were synthesized according to the traditional method as described in Ref.[31].1.7600g of Eu2O3was dissolved in10mL of concentrated nitric acid and then crystallized by evaporation of excess nitric acid and water by heating,and Eu(NO3)3was acquired.Eu (NO3)3ethanol solution was prepared by adding10mL of anhydrous ethanol into the above Eu(NO3)3.3.6640g of BA and1.8020g of phen were dissolved in100mL of ethanol.The Eu(NO3)3solution was then added into the solution of BA and phen under magnetic agitation for3h at 60°C.The precipitates were collected byﬁltration and washed for three times using anhydrous ethanol,and then dried in an electric oven for12h at60°C.The synthetic method of Tb(BA)3phen complexes was similar to the above method,except that the using dosages of Tb4O7,BA and phen were1.8693,1.8320and0.9910g,respectively.2.3Fabrication of[Eu(BA)3phen+Tb(BA)3phen]/PVP composite nanoﬁbers via electrospinningA series of[Eu(BA)3phen+Tb(BA)3phen]/PVP spinning solutions with different mass ratios of Eu(BA)3phen to Tb (BA)3phen were prepared.The preparation process was as follows:certain amounts of Eu(BA)3phen and Tb (BA)3phen powders were added in6.0g of DMF,and then 1.5g of PVP was slowly added into the above solution with magnetic stirring for12h at room temperature.During the electrospinning process,a traditional elec-trospinning apparatus was used to prepare the[Eu (BA)3phen+Tb(BA)3phen]/PVP composite nanoﬁbers. Spinning solution was loaded into a plastic syringe with a plastic spinneret.A piece ofﬂat iron net was put about 16cm away from the tip of the plastic nozzle as a nanof-ibers collector.A positive direct current(DC)voltage of 14kV was applied between the spinneret and the collector to generate stable,continuous PVP-based composite nanoﬁbers at20–25°C,and the relative humidity was45–50%.In this process,PVP-based composite nanoﬁbers Table1Compositions of[Eu(BA)3phen+Tb(BA)3phen]/PVP composite nanoﬁbersCompositionsSamples Eu(BA)3phen Tb(BA)3phen/g PVP/g S a10.15000 1.5000 S a20.15000.0150 1.5000 S a30.15000.0450 1.5000 S a40.15000.0750 1.5000 S a50.15000.1050 1.5000 S b100.1500 1.5000 S b20.01500.1500 1.5000 S b30.04500.1500 1.5000 S b40.07500.1500 1.5000 S b50.10500.1500 1.5000J Mater Sci:Mater Electron123were formed along with the volatilization of DMF.Thus the composite nanoﬁbers consisted of Eu(BA)3phen,Tb (BA)3phen and PVP were successfully prepared.The obtained composite nanoﬁbers were marked as S a1–S b5, and their compositions were summarized in Table1.2.4CharacterizationThe morphology and size of[Eu(BA)3phen+Tb (BA)3phen]/PVP composite nanoﬁbers was observed by a ﬁeld emission scanning electron microscope(FESEM,XL-30).The elements analysis for nanoﬁbers was determined by energy dispersive spectrometer(EDS,Oxford ISIS300) attached to the FESEM.The as-prepared[Eu (BA)3phen+Tb(BA)3phen]/PVP composite nanoﬁbers were identiﬁed by an X-ray powder diffractometer(XRD, Bruker,D8FOCUS)with Cu Kɑradiation.The operation voltage and current were kept at40kV and20mA, respectively.Theﬂuorescent properties of the samples were investigated by a Hitachiﬂuorescence spectropho-tometer F-7000.All the measures were performed at room temperature.A thermogravimetric analyzer(TG,TA Q600)was used to measure the thermal stability of the composite nanoﬁbers under air atmosphere,and the heating rate is kept at10°C/min.Fourier-transform infrared spectra (FT-IR)were measured by a Shimadzu model8400s FT-IR spectrophotometer with KBr pellet technique.All deter-minations were performed at room temperature except for thermal analysis.3Results and discussion3.1Characterizations of morphology and structure Figure1a shows the scanning electron microscopy(SEM) images of[10%Eu(BA)3phen+1%Tb(BA)3phen]/PVP composite nanoﬁbers(S a2).One can see that the as-prepared nanoﬁbers are considerably smooth.In order to investi-gate the diameter distribution of this sample,Image-Pro Plus 6.0software is used to measure diameters of100nanoﬁbers from SEM images,and the results are analyzed with statis-tics,and then the histogram of diameters distribution of the composite nanoﬁbers is drawn by using Origin8.5software. According to the above analysis result,it is found that the diameters of composite nanoﬁbers are431.55±3.14nm under the conﬁdence level of95%(see Fig.1b).The energy dispersive spectrum(EDS)shown in Fig.1c reveals that the nanoﬁbers are composed of C,O,N,Eu and Tb elements. The Au peak in the spectrum originated from gold conduc-tiveﬁlm plated on the surface of the sample for SEM observation(Coated by Eiko IB-5iron coater).The phase compositions of[10%Eu(BA)3phen+1%Tb(BA)3phen]/PVP composite nanoﬁbers were characterized by means of XRD analysis,as shown in Fig.1d.Obviously,A broad diffraction peak of the amorphous PVP(2θ=22°)could be observed,and no other diffraction peaks are identiﬁed, indicating that the obtained composite nanoﬁbers are amorphous in structure.From the above SEM,EDS and XRD analysis,it is demonstrated that[Eu(BA)3phen+Tb (BA)3phen]/PVP composite nanoﬁbers have been success-fully fabricated.3.2Infrared spectra analysisFigure2shows the FT-IR spectrum of PVP,Eu(BA)3phen complexes,Tb(BA)3phen complexes and[10%Eu (BA)3phen+1%Tb(BA)3phen]/PVP composite nanoﬁbers.A prominent peak at1,650cm−1is attributed to the C=O groups in PVP,and the peaks at1,444and1,294cm−1cor-respond to the bending vibration of–CH and the stretching vibration of–CN,respectively.The broad band centered at 3,420cm−1(ʋO–H)is attributed to coordinated waters among which hydrogen bonds exist,and the broad band located at 2,960cm−1is assigned to C–H stretching vibration.The FT-IR spectra of Eu(BA)3phen complexes and Tb(BA)3phen complexes are similar,as seen from Fig.2.The peaks at 1,608and1,396cm−1can be assigned to the antisymmetric and symmetric stretching vibration of COO−group in Eu (BA)3phen complexes and Tb(BA)3phen complexes.One sharp peak is observed at1,517cm−1,which is attributed to C=N group in Eu(BA)3phen and Tb(BA)3phen complexes.It is also observed that Eu3+–O2−or Tb3+–O2−stretching vibration peaks locate at430cm−1.The FT-IR spectrum of [10%Eu(BA)3phen+1%Tb(BA)3phen]/PVP composite nanoﬁbers is similar to that of pure PVP.The reason is that Eu(BA)3phen and Tb(BA)3phen complexes are co-doped in PVP matrix and the amount of Eu(BA)3phen and Tb (BA)3phen complexes are little.3.3Thermal stability analysisThe TG curve for[10%Eu(BA)3phen+1%Tb (BA)3phen]/PVP composite nanoﬁbers is shown in Fig.3. The composite nanoﬁbers lose about1%of their initial weight when the temperature arises from60to220°C,due to the volatilization of residual solvents and surface absorbed water.With the continuous increasing in tem-perature,Eu(BA)3phen and Tb(BA)3phen complexes start to decompose,and the composite nanoﬁbers lose12.3%of their initial weight when the temperature reaches up to 390°C.At this temperature,PVP begins to decompose,and the decomposition process isﬁnished at495°C.No weight loss is detected when temperature is above495°C,and the total weight loss percentage is99.8%for the composite nanoﬁbers.J Mater Sci:Mater Electron1233.4Fluorescent performanceFluorescent properties of the [Eu(BA)3phen +Tb (BA)3phen]/PVP composite nanoﬁbers with different mass ratios of Eu(BA)3phen to Tb(BA)3phen were investigated.In order to perform the contrast experiments,the masspercentage of Eu(BA)3phen to PVP was ﬁxed as 10%from samples S a1to S a5,and the Tb(BA)3phen to PVP also was ﬁxed as 10%from S b1to S b5.Figure 4gives the excitation and emission spectra of Eu(BA)3phen/PVP composite nanoﬁbers (S a1).The left section in Fig.4indicates the12345678910c Au Tb Tb Eu Au I n t e n s i t y (a .u .)Binding Energy (KeV)CO N Au Eu Tb aSEM images (a ),histogram of diameter distribution (b ),EDS phen]/PVP compositenanoﬁbers29603420129414441650PVPJ Mater Sci:Mater Electron123excitation spectrum of the sample monitored at616nm.Broad excitation band extending from200to380nm is observed.The peak at276nm is ascribed to theπ→π* electron transition of the ligands.As shown in the right section of Fig.4,characteristic emission peaks of the Eu3+ ions are observed under the excitation of276-nm ultraviolet light.They are assigned to the energy levels transitions of the5D0→7F0(580nm),5D0→7F1(592nm),and 5D0→7F2(616nm)of Eu3+,respectively.The5D0→7F2 hypersensitive transition at616nm is the predominant emission.The excitation and emission spectra of Tb (BA)3phen/PVP nanoﬁbers(S b1)are revealed in Fig.5.As shown the left section in Fig.5,a broad excitation band extending from200to380nm was observed when moni-toring wavelength was545nm,and the excitation peak also locates at276nm.Through comparing the excitation spectra of S a1and S b1,it can be seen that the excitation spectra of the two samples are similar in shape.Moreover,the excitation peaks of the two samples both locate at276nm,indicating that both the Eu(BA)3phen and Tb(BA)3phen complexes can be simultaneously and most effectively excited using 276-nm single-wavelength ultraviolet light.The right sec-tion of Fig.5reveals that the characteristic emission peaks of the Tb3+ions and the corresponding energy levels tran-sitions are5D4→7F6(490nm),5D4→7F5(545nm), 5D4→7F4(584nm),and5D4→7F3(622nm),and the 5D4→7F6hypersensitive transition at545nm is the pre-dominant emission peak.It is obvious that S a1and S b1 respectively emit red and green light under the excitation of 276-nm single-wavelength ultraviolet light.Figure6a shows emission spectra of[Eu(BA)3phen+ x%Tb(BA)3phen]/PVP composite nanoﬁbers with different mass ratios of Eu(BA)3phen to Tb(BA)3phen under the excitation of276-nm single-wavelength ultraviolet light. The mass of Eu(BA)3phen complexes isﬁxed as10%in samples S a1-S a5,and the mass ratios of Eu(BA)3phen to Tb (BA)3phen are10:0,10:1,10:3,10:5and10:7,respectively. The mass percentage of Tb(BA)3phen complexes to PVP is denoted as x%(x=0,1,3,5,7).It can be seen that the predominant peaks at490,545,592,616nm are all observed,and these emission peaks are attributed to the 5D4→7F6(490nm)and5D4→7F5(545nm)energy levels transitions of Tb3+ions,and the5D0→7F1(592nm), 5D0→7F2(616nm)energy levels transitions of Eu3+ions, respectively.In order to further discuss the variation trend, the intensities of predominant emission peaks at545nm and 616nm versus different mass of Tb(BA)3phen are plotted in Fig.6b.Obviously,with the increase of Tb(BA)3phen content,the intensity of predominant emission peak at 545nm of Tb3+ions enhances gradually,whereas the intensity of predominant emission peak at616nm of Eu3+ ions is decreased.The possible reason is that the energy of substrates absorbed from ultraviolet light is constant,and consequently,the energy that the substrates transferred to Eu3+ions is reduced when Tb3+ions concentration is increased,thus resulting in feeblerﬂuorescence peaks at 616nm.Meanwhile,the energy that the substrates trans-ferred to Tb3+ions is increased and theﬂuorescence peaks at545nm becomes stronger.For comparison,Tb(BA)3phen contentﬁxed as10%in samples S b1-S b5is also studied,and the mass ratios of Eu(BA)3phen to Tb(BA)3phen are0:10, 1:10,3:10,5:10and7:10,respectively.The mass percentage of Eu(BA)3phen complexes to PVP is determined as y% (y=0,1,3,5,7).As Fig.7a and b shown,the peaks at490, 545,592,616nm are observed as well.Besides,the red ﬂuorescence predominant emission at616nm of Eu3+ions enhances gradually with the increase of Eu(BA)3phen con-tent,while the greenﬂuorescence predominant emission at 545nm of Tb3+ions is wakened.J Mater Sci:Mater Electron123Theﬂuorescence decay curves of Eu3+ions and Tb3+ ions in[10%Eu(BA)3phen+x%Tb(BA)3phen]/PVP composite nanoﬁbers are shown in Fig.8,and they are used to calculate the lifetime and to investigate theﬂuorescence dynamics of these samples.Figure8a and b respectively give theﬂuorescence decay curves of samples excited by 276nm single-wavelength ultraviolet light and monitored at616and545nm.It is found that the curves follow the single-exponential decay:I t¼I0expðÀt=sÞWhere I t is the intensity at time t,I0is the intensity at t=0 andτis the decay lifetime,and the obtained average lifetime values(τ/ms)of Eu3+ions and Tb3+ions are shown in Fig.8.Obviously,theﬂuorescence lifetime of Eu3+ions in[10%Eu(BA)3phen+x%Tb(BA)3phen]/PVP composite nanoﬁbers is extended gradually,while that of Tb3+ions is decreased slowly with increase of Tb (BA)3phen content.Similar conclusions about polymers doped by RE complexes can be found in literatures[32, 33].On one hand,The relative content of Eu(BA)3phen complexes in the composite nanoﬁbers is reduced with introducing more Tb(BA)3phen.Thus the distance among Eu3+in Eu(BA)3phen molecular clusters and/or nanopar-ticles in the nanoﬁbers is increased,resulting in that the energy transfer among Eu3+to Eu3+is reduced and theJ Mater Sci:Mater Electron 123ﬂuorescence lifetime of Eu3+is prolonged.On the other hand,for Tb3+ions,some aggregates are formed in the polymer matrix when increasing the Tb(BA)3phen content, and the exciton migration between the Tb(BA)3phen mol-ecules shortened theﬂuorescence lifetime of Tb3+ions.In Fig.9,it reveals the same conclusions that the life time of Tb3+ions extends gradually while the life time of Eu3+ ions diminishes with the increase of Eu(BA)3phen complexes.Generally,ﬂuorescence color can be represented by the Commission Internationale de L’Eclairage(CIE)1931 chromaticity coordinates.Figure10depicts the CIE(X,Y, Z)coordinate diagram of[Eu(BA)3phen+Tb(BA)3phen]/ PVP composite nanoﬁbers under the excitation of276-nm single-wavelength ultraviolet light.The CIE chromaticity coordinates of sample S a1are determined to be(0.616, 0.325,0.059),corresponding to red color.Moreover,the CIE chromaticity coordinates of samples S a2and S a3are determined to be(0.476,0.436,0.088),(0.397,0.513, 0.089),which are placed in yellow region.In addition,the samples S a4,S a5and S b5locate at(0.364,0.538,0.098), (0.349,0.551,0.100),(0.257,0.604,0.139),respectively, which performed yellow green st,the CIE chro-maticity coordinates of samples S b3and S b1are determined to be(0.286,0.581,0.132)and(0.327,0.561,0.113),which belong to green color.It is gratify to see that theﬂuores-cence color could be tuned by adjusting the mass ratios of Eu(BA)3phen complexes to Tb(BA)3phen complexes in a wide color range of red–yellow–green under the excitation of276-nm single-wavelength ultraviolet light.TheJ Mater Sci:Mater Electron123corresponding images were obtained by taking pictures,when Hitachi ﬂuorescence spectrophotometer F-7000was exciting the products under the excitation of 276-nm sin-gle-wavelength ultraviolet light.The novel ﬂuorescent composite nanoﬁbers are expected to make a contribution in the ﬁeld of color display.4ConclusionsTo sum up,uniform [Eu(BA)3phen +Tb(BA)3phen]/PVP composite nanoﬁbers were successfully prepared by elec-trospinning.The SEM analysis shows the diameter of the composite nanoﬁbers is 431.55±3.14nm.The ﬂuores-cence intensity of Eu 3+is increased with introducing more Eu(BA)3phen into the composite nanoﬁbers,but the ﬂuo-rescence intensity of Tb(BA)3phen is simultaneously found to decrease monotonically,and and vice versa.Further-more,by adjusting the mass ratio of Eu(BA)3phen complexes and Tb(BA)3phen complexes,the ﬂuorescence color of [Eu(BA)3phen +Tb(BA)3phen]/PVP composite nanoﬁbers could be tuned easily from red to yellow,yellow green,and green under the excitation of 276-nm single-wavelength ultraviolet light.Besides,the design concept and preparation method of the composite nanoﬁbers are of universal signiﬁcance for the fabrication of other color-tunable composite nanoﬁbers.The novel ﬂuorescentcomposite nanoﬁbers have potential applications in many ﬁelds.Acknowledgments This work was ﬁnancially supported by the National Natural Science Foundation of China (NSFC 50972020,51072026),Specialized Research Fund for the Doctoral Program of Higher Education (20102216110002,20112216120003),the Science and Technology Development Planning Project of Jilin Province (Grant Nos.20130101001JC,20070402,20060504),the Research Project of Science and Technology of Department of Education of Jilin Province “11th 5-years plan”(Grant Nos.2010JYT01),Key Research Project of Science and Technology of Ministry of Education of China (Grant No.207026).References1.G.Zhu,Z.P.Ci,Q.Wang,Y.Wen,S.C.Han,Y.R.Shi,S.Y.Xin,Y.H.Wang,J.Mater.Chem.C 1,4490–4496(2013)2.D.M.Yang,G.G.Li,X.J.Kang,Z.Y.Cheng,P.A.Ma,C.Peng,H.Z.Lian,C.X.Li,L.Jun,Nanoscale 4,3450–3459(2012)3.J.F.Sun,Z.P.Lian,G.Q.Shen,D.Z.Shen,RSC Adv.3,18395–18405(2013)4.M.M.Jiao,Y.C.Jia,W.Lu¨,W.Z.Lv,Q.Zhao,B.Q.Shao,H.P.You,Dalton 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A novelﬂuorescent“turn-on”chemosensor for nanomolar detection ofFe(III)from aqueous solution and its application in living cells imagingJitendra Nandre a,Samadhan Patil a,Vijay Patil a,Fabiao Yu b,Lingxin Chen b,n,Suban Sahoo c,Timothy Prior d,Carl Redshaw d,Pramod Mahulikar a,n,Umesh Patil a,na School of Chemical Sciences,North Maharashtra University,P.B.No.80,Jalgaon425001,MS,Indiab Key Laboratory of Coastal Zone Environmental Processes and Ecological Remediation,Yantai Institute of Coastal Zone Research,Chinese Academy ofSciences,Yantai264003,Chinac Department of Applied Chemistry,S.V.National Institute Technology,Surat395007,Gujrat,Indiad Department of Chemistry,University of Hull,Cottingham Road,Hull HU67RX,UKa r t i c l e i n f oArticle history:Received11April2014Received in revised form5June2014Accepted10June2014Available online14June2014Keywords:Fluorescent“turn-on”sensorBenzo-thiazolo-pyrimidineFe3þICTPETLive cells imaging.a b s t r a c tAn electronically active and spectral sensitiveﬂuorescent“turn-on”chemosensor(BTP-1)based on thebenzo-thiazolo-pyrimidine unit was designed and synthesized for the highly selective and sensitivedetection of Fe3þfrom aqueous medium.With Fe3þ,the sensor BTP-1showed a remarkableﬂuorescence enhancement at554nm(λex¼314nm)due to the inhibition of photo-induced electrontransfer.The sensor formed a host-guest complex in1:1stoichiometry with the detection limit down to0.74nM.Further,the sensor was successfully utilized for the qualitative and quantitative intracellulardetection of Fe3þin two liver cell lines i.e.,HepG2cells(human hepatocellular liver carcinoma cell line)and HL-7701cells(human normal liver cell line)by a confocal imaging technique.&2014Elsevier B.V.All rights reserved.1.IntroductionThe development of highly selective and sensitive chemosen-sors for bioactive metal ions has gained enormous importance(Au-Yeung et al.,2013;Callan et al.,2005;Chen et al.,2012;Duttaand Das,2012;Formica et al.,2012;Kim et al.,2012;Sahoo et al.,2012),as metal ions are well known to be involved in a variety offundamental biological processes,which are essential for main-taining the life of organisms and remain sustainable underenvironmental conditions.As an important physiologically rele-vant metal ion,Fe3þexhibits an obligatory role in many biochem-ical processes at the cellular level.Numerous enzymes use Fe3þasa catalyst for electron transfer,oxygen metabolism,and RNA andDNA synthesis(Cairo and Pietrangelo,2000;Crabtree,1994).However,both its deﬁciency(hypoferremia)and excess(hyperfer-remia)can induce a variety of diseases.As a result of theseconcerns,intense research efforts have been focused on thedevelopment of highly sensitive and selective receptors for thequalitative and quantitative detection of Fe3þ.However,becauseof the paramagnetic nature of the Fe3þion,recognition of Fe3þbyﬂuorescence response is mostly signaled by aﬂuorescencequenching mechanism(Fan et al.,2006;Li et al.,2009;Lohaniet al.,2009).Also,the design of a“turn-on”ﬂuorescent chemo-sensor for the Fe3þion remains a challenging task for researchersworking in theﬁeld of chemosensing due to the need to overcomethe usualﬂuorescence quenching nature of Fe3þ.The lack ofsuitable“turn-on”ﬂuorescent iron indicators is even more obviouswhen judged in terms of applications in bioimaging,althoughsigniﬁcant progress has been made onﬂuorescent molecularsensors for intracellular imaging of biologically important metalions.It is worth noting that many reportedﬂuorescent“turn-on”chemosensors for the Fe3þion utilize the rhodamine moiety dueto its advantageous photophysical properties(Sahoo et al.,2012).However,some rhodamine dyes are harmful if swallowed byhumans or animals,and cause irritation of the skin,eyes andrespiratory tract(Jain et al.,2007;Rochat et al.,1978).In addition,among the various sensing methods,sensors based on a naked-eyeresponse have many advantages because of their ability to providea simple,sensitive,selective,precise and economical method forthe detection of the target analyte without the use of sophisticatedinstrumentation.On the basis of previously reported data of Fe3þchemosensors,herein,we focused on the development of a simple“turn-on”ﬂuorescent chemosensor for Fe3þions from purelyContents lists available at ScienceDirectjournal homepage:/locate/biosBiosensors and Bioelectronics/10.1016/j.bios.2014.06.0170956-5663/&2014Elsevier B.V.All rightsreserved.n Corresponding authors.Tel.:þ2572257432;fax:þ2572258403.E-mail addresses:lxchen@(L.Chen),mahulikarpp@(P.Mahulikar),udpatil.nmu@(U.Patil).Biosensors and Bioelectronics61(2014)612–617aqueous medium.To achieve this goal,we have designed and synthesized a novel benzo-thiazolo-pyrimidine basedﬂuorescent sensor(BTP-1)for the selective recognition of Fe3þ.The present sensor BTP-1is nontoxic and successfully used to study its in vitro glycosidase inhibitory activity(Patil et al.,2012).2.Material and methods2.1.Materials and instrumentationsAll the starting reagents and metal perchlorates were pur-chased either from S.D.Fine chemicals or Sigma-Aldrich depend-ing on their availability.All the reagents were used as received.All the solvents were of spectroscopic grade and were used without further treatment.The purity of the compounds and the progress of reactions were determined and monitored by means of analy-tical thin layer chromatography(TLC).Pre-coated silica gel60F254(Merck)on alumina plates(7Â3cm2)was used and visualized by using either an iodine chamber or a short UV–Visible lamp. Melting points were recorded on the Celsius scale by the open capillary method and were uncorrected.IR spectra were recorded on a Perkin-Elmer Spectrum One FT-IR spectrometer as potassium bromide pellets and nujol mulls,unless otherwise mentioned.IR bands are expressed in frequency(cmÀ1).NMR spectra were recorded in CDCl3on a Varian(Mercury Vx)SWBB Multinuclear probe spectrometer,operating at300MHz and75MHz for1H NMR and13C NMR,respectively and shifts are given in ppm downﬁeld from TMS as an internal standard.UV–vis spectra were recorded on a U-3900spectrophotometer(Perkin-Elmer Co.,USA) with a quartz cuvette(path length¼1cm).Fluorescence spectra were recorded on a Fluoromax-4spectroﬂuorometer(HORIBA Jobin Yvon Co.,France).2.2.Spectroscopic studyThe receptor BTP-1was not soluble in water and therefore the stock solutions of BTP-1(1.0Â10À3M)was prepared in CH3CN. All cations(1.0Â10À2M)solutions were prepared in water. These solutions were used for all spectroscopic studies after appropriate dilution.For the spectroscopic(UV–vis andﬂuores-cence)titrations,the required amount of the diluted receptor BTP-1(2mL,2Â10À5M,in CH3CN)was taken directly into the cuvette and the spectra were recorded after each successive addition of cations(0–180m L,1Â10À3M,in H2O)by using a micropipette.putational methodsAll theoretical calculations were carried out using the Gaussian 09W computer program and the Gaussview5.0.9graphical inter-face(Frisch et al.,2009).Optimization of BTP-1and its complex with Fe3þwas carried out without symmetry constraints by applying the B3LYP/6-31G(d,p)method in the gas phase followed by the harmonic vibrational frequency which was calculated using the same methods to ascertain the presence of a local minimum. The basis set LANL2DZ was used for Fe3þatom.2.4.Living cells imagingThe solution of BTP-1(DMSO, 1.0mM)was prepared and maintained in a refrigerator at4°C.FeCl3is as the iron-supple-mented source.The confocalﬂuorescent images were acquired on an Olympus laser-scanning microscope with an objective lens (Â40).Excitation of the probe was carried out using a Spectra Physics InSightDeepSee ultrafast laser at700nm and emission was collected between500and600nm.Prior to imaging,the medium was removed.Cell imaging was carried out after washing cells with RPMI-1640for three times.Cell Culture:HepG2cells(Human hepatocellular liver carci-noma cell line)and HL-7701cells(human normal liver cell line) were purchased from the Committee on type Culture Collection of Chinese Academy of Sciences.Cells were seeded at a density of1Â106cells/mL for confocal imaging in RPMI1640medium supplemented with20%fetal bovine serum(FBS),NaHCO3(2g/ L),and1%antibiotics(penicillin/streptomycin,100U/ml).Cultures were maintained at37°C under a humidiﬁed atmosphere contain-ing5%CO2.The cells were sub-cultured by scraping and seeding on15mm petri-dishes according to the instructions from the manufacturer.2.5.Synthesis of BTP-1Synthesis of BTP-1was achieved by using a mild base through ring transformation of suitably functionalized4-(methylthio)-2-oxo-6-naphthyl-2H-pyran-3-carbonitriles(2)with2-amino-ben-zothiazole in DMF using DBU as the base(Scheme1)(Patil et al., 2012).The precursors,4-(methylthio)-2-oxo-6-naphthyl-2H-pyran-3-carbonitriles(2)was prepared by stirring an equimolar mixture of ethyl2-cyano-3.3-bis(methylthio)acrylate(1),substi-tuted acetophenone and KOH in DMF(Tominaga et al.,1984).Synthesis of ethyl2-cyano-3.3-bis(methylthio)acrylate(1):In a 250mL beaker,a mixture of water(10mL)and DMF(30mL)and KOH(9.87g,176mmol)were cooled to0°C.Ethyl2-cyanoacetate (10g,88mmol)was added dropwise to this cold solution for 30min,and stirred for10–15min.Then carbon disulﬁde(6.70g, 88mmol)was added dropwise for20min at0toÀ5°C.The mixture was stirred for60min at room temperature,and then reaction mixture was again cooled to0°C and dimethyl sulfate (22.20g,176mmol)was added dropwise for30min.The reaction mixture was allowed to remain at room temperature for12h and then it was poured into crushed ice cold water(400mL)and kept at room temperature with vigorous stirring for10–15min.The obtained yellowish solid wasﬁltered,washed with cold water and dried.The crude product was recrystallized from methanol.Synthesis of6-naphthyl-3-cyano-4-methylthio-2H-pyran-2-ones (2):In a100mL round bottomﬂask,a mixture of2-acetyl naphthalene(1.7g,0.01mol)and ethyl2-cyano-3.3-bis (methylthio)acrylate(2.17g,0.01mol,1),powdered KOH(1.12g, 0.02mol)and50mL of dry DMF was stirred at room temperature for5–6h.Progress of the reaction was monitored by TLC(ethyl acetate:hexane,3:7).After completion of the reaction,the reaction mixture was poured onto crushed ice(500mL of ice-water)with vigorous stirring and then it was stirred at room temperature for 4–5h.The yellow precipitate formed wasﬁltered,washed with NCMeS SMeOOC2H5CH3O OCNSCH3NSH2NNBTP-1rtNSNC12Scheme1.Synthesis of BTP-1.J.Nandre et al./Biosensors and Bioelectronics61(2014)612–617613cold water and dried.The crude product was recrystallized from methanol.Synthesis of BTP-1:A mixture of2-aminobenzothiazole (1mmol)and DBU(2mmol)and DMF(15mL)was stirred under nitrogenﬂux for10–15min at room temperature.Then4-(methylthio)-2-oxo-6-naphthalen-2H-pyran-3-carbonitrile(2, 1mmol)was added under nitrogenﬂux with constant stirring. The progress of the reaction was monitored by TLC(ethyl acetate: hexane,2:8).After completion of the reaction(about4–12h),the reaction mixture was poured onto crushed ice with vigorous stirring about30min.The reaction mixture was allowed to remain at room temperature for about20min to settle down the solid, which was the isolated byﬁltration.The obtained crude product was dissolved in hot MeOH(70mL)andﬁltered.The collected insoluble solid was again dissolved in to hot chloroform(40mL),ﬁltered rapidly(impurity remains insoluble in hot chloroform while product is soluble)and cooled.Chloroform was removed to afford the pure product.Mol.Formula:C22H13N3S;Mol.Weight:351.42g;Physical Nature:Yellow solid;IR(cmÀ1) [KBr]:2923,2178,1599,1526,1267;Mass[ESI,70Ev]m/z(%): 353(30),352(100),291(45),153(25);1H NMR(300MHz,CDCl3,δppm):5.15(s,1H,CH¼C),7.43(s,1H,PhH),7.45–7.55(m,4H, PhH),7.69–7.71(dd,J¼1.3Hz,J¼7.5Hz,1H,PhH),7.86–8.06(m, 5H,PhH),8.58(s,1H,PhH);13C NMR(75MHz,CDCl3,δppm): 64.2,104.4,115.7,119.5,122.9,123.3,125.1,126.4,126.5,127.0,127.2, 127.6,128.2,128.4,128.9,132.2,133.1,134.2,135.5,150.1,151.9, 160.4;HRMS(ESI):m/z:Calculated for C22H14N3S1:[MþH]þ352.0920,Found:352.0903.Results and discussion3.1.Synthesis of BTP-1Synthesis of BTP-1was achieved by using a mild base DBU through ring transformation of a suitably functionalized4-(methylthio)-2-oxo-6-naphthyl-2H-pyran-3-carbonitriles(2)with 2-aminobenzothiazole in DMF(Scheme1).The structure of BTP-1 was characterized by IR,1H-NMR and13C-NMR spectroscopy and HRMS(Figs.S1,S2,S3,S4and S5†).Finally,suitable crystal of BTP-1for a single crystal X-ray diffraction‡study was obtained from chloroform,and the molecular structure is shown in Fig.S6†.3.2.Naked-eye selectivity study of BTP-1The recognition properties of BTP-1(2mL,2Â10À5M,in CH3CN)toward different metal ions(20m L,1Â10À2M,in H2O) were studied experimentally by naked-eye,UV–Visible,andﬂuor-escence methods.In the naked-eye experiments(Fig.S7†),no obvious visual color changes of BTP-1were observed in the presence of the tested metal ions.However,under UV light,sensor BTP-1showed a selective“turn-on”ﬂuorescence upon addition of Fe3þover other tested metal ions(Agþ,Ca2þ,Cd2þ,Co2þ,Csþ, Cu2þ,Fe2þ,Hg2þ,Kþ,Liþ,Mg2þ,Mn2þ,Ni2þ,Pd2þand Zn2þ). The observedﬂuorescence intensity reveals that the receptor BTP-1shows higher recognition ability for Fe3þ.Interestingly,this ﬂuorescence“turn-on”response becomes reversibly“turn-off”after the addition of an aq.solution of EDTA.Encouraged by the Fe3þselective and reversible response shown by BTP-1,the quantitative and qualitative metal ions sensing behavior of BTP-1was determined further by spectrophotometric methods.3.3.UV–Visible absorption study of BTP-1Sensor BTP-1exhibited two absorption bands:one at313nm and a weak narrow band at271nm.On addition of5equivalents of Fe3þions(20m L,1Â10À2M,in H2O)to the solution of BTP-1 (2mL,2Â10À5M,in CH3CN),signiﬁcant spectral changes were observed(Figs.1and S8†).A hypochromic shift was observed at 271nm while the band at313nm was disappeared completely and a new broad band appeared between350nm and425nm.The red-shifted band was observed presumably due to the delocaliza-tion of electrons from the nitrile nitrogen on formation of complex species with Fe3þ(Fig.S9†),which was supported by comparing the variations in the bond lengths of the DFT optimized structure of BTP-1and Fe3þ.BTP-1complex(Fig.S10†).Moreover,the red-shift can also be explained due to the intramolecular charge transfer(ICT)process and the lowering of the band gap between HOMO and LUMO on complexation with Fe3þ(Fig.S11†).This effective ICT induced by the electron push–pull system(Lin et al., 2008).As in–CN group,the presented nitrogen is having sp hybridization so less willing to bind with metal cations as compare to sp2nitrogen and sulfur.Importantly,no distinguishable spectral changes were observed in the presence of other tested metal ions.The absorption titration of BTP-1(2mL,2Â10À5M,in CH3CN) was next performed with incremental addition of Fe3þ(Fig.2). The spectral changes with the formation of an isosbestic point at 328nm indicate the formation of a single complex species between sensor BTP-1and added Fe3þ.From the absorption titration of BTP-1,the limit of detection(LOD)and quantiﬁcation (LOQ)of Fe3þwere calculated to be0.10m M and0.32m M, respectively based on the ICH Q2B recommendations by using the equations:LOD¼3.3s/S and LOQ¼10s/S,where S and s represent the slope and the standard deviation of the intercept of regression line of the calibration curve,respectively(Fig.S12†). This detection limit is acceptable within the US EPA limit(0.3mg/ L,equivalent to5.4μM)for the detection of Fe3þin drinking water. Further,the Jobs'plot(Fig.S13†)and LCMS(Fig.S14†)analysis was performed,which suggested that there was only one type of1:1 binding interaction between the sensor BTP-1and Fe3þ.3.4.Emission spectroscopic study of BTP-1The cation binding behavior of BTP-1was also investigated by ﬂuorescence spectroscopy.We observed a remarkableﬂuores-cence enhancement of BTP-1(2mL,2Â10-5M,in CH3CN)at 554nm(λex¼314nm)upon addition of Fe3þ(20m L,1Â10-2M, in H2O)due to the inhibition of photo-induced electron transfer (PET)from electron-donating nitrogen to electron-receptor naphthalene ring(Li et al.,2011),while no signiﬁcant changes were observed in the presence of other tested metal ions(Figs.3 and S15†).Theﬂuorescence titration experiment of BTP-1with Fe3þshowed a E10-foldﬂuorescence enhancement at554nm (Fig.4).The binding constant(K)of BTP-1with Fe3þwas Fig.1.Changes in the absorbance of BTP-1(2mL,2Â10À5M,in CH3CN)in the absence and presence of5equivalents of different metal ions(20m L,1Â10À2M,in H2O).J.Nandre et al./Biosensors and Bioelectronics61(2014)612–617 614Fig.2.Absorbance titration of BTP-1(2Â10À5M,in CH 3CN)upon the addition of incremental amount of Fe 3þ(0–180m L,1Â10À3M,in H 2O).Inset shows the Benesi –Hildebrandplot.Fig.3.Fluorescence emission change of sensor BTP-1(2mL,2Â10À5M,CH 3CN)upon the addition of a particular metal ions (20m L,1Â10À2M,in H 2O),λex ¼314nm.Fig.4.Changes in ﬂuorescence emission intensity of BTP-1(2mL,2Â10À5M,in CH 3CN)upon the addition of incremental amount of Fe 3þ(0–180m L,1Â10À3M,in H 2O).J.Nandre et al./Biosensors and Bioelectronics 61(2014)612–617615determined by a Benesi –Hildebrand plot analysis of both absorp-tion (Fig.2,inset)and ﬂuorescence titrations data (Fig.S16†);additional data was obtained via a Scatchard plot from ﬂuores-cence titration data (Fig.S17†).The cation binding af ﬁnity of BTP-1was found to be E 4Â104M À1for Fe 3þ.Based on the ﬂuorescence titration,the LOD and LOQ of BTP-1for Fe 3þwas found to be 0.74nM and 2.23nM,respectively,and these values are quite better than the reported sensors (Table S1†).Further,the effect of coexisting biologically relevant metal ions on the detection of Fe 3þby BTP-1was investigated.In a CH 3CN solution of BTP-1(2mL,2Â10À5M,in CH 3CN),the addition of 2equivalents of Fe 3þ(8m L,1Â10À2M,in water)in the presence of 2equivalents of other tested metal ions (8m L,1Â10À2M,in water)caused a dramatic enhancement in the ﬂuorescence inten-sity of BTP-1with only very slight or no interference effects (Fig.S18†).Therefore,we conclude that BTP-1is a reliable,highly selective and sensitive “turn-on ”ﬂuorescent sensor for Fe 3þ.3.5.Live cells imaging study of BTP-1The ﬂuorescent behavior of BTP-1was applied for the intra-cellular detection and monitoring of Fe 3þin two liver cell lines i.e.,HepG2cells (human hepatocellular liver carcinoma cell line)and HL-7701cells (human normal liver cell line).The HepG2cells in Fig.4b and d were incubated with 0.01μM and 100μM Fe 3þfor 30min in RPMI 1640medium at 37°C,and then washed with RPMI 1640to remove excess Fe 3þ.After being incubated with BTP-1(10μM)in RPMI 1640for 10min,the cells were imaged by a confocal ﬂuorescence microscope.As shown in Fig.5,there is a signi ﬁcant intracellular ﬂuorescence increase revealed in Fig.5b and d compared with the control cells in Fig.5a,which indicates the ability of BTP-1to detect intracellular Fe 3þ.Further,to con ﬁrm that the increase in the ﬂuorescence depended on the Fe 3þchanges,the iron-supplemented cells in Fig.5b and 5d were treated with 50μM of iron chelatordesferoxamine (DFO)for 40min to remove the intracellular levels of Fe 3þ.Asexpected,Fig.5.Fluorescence confocal microscopic images of living HepG2cells incubated with Fe 3þ.(a)Cells loaded with 10μM BTP-1for 10min as control.(b)and (d)Cells loaded with 0.1μM and 100μM Fe 3þfor 30min,then 10μM BTP-1for 10min.(c)and (e)Cells were treated as (b),then loaded with 50μM of iron chelatordesferoxamine (DFO)for 40min.(f)and (g)Overlays of bright ﬁeld images and ﬂuorescence channels in (a)–(e).Scale bar is 20μm.Fig. 6.Fluorescence confocal microscopic images of living HL-7701cells incubated with various concentrations of Fe 3þ.(a)Cells loaded with 50μM of iron chelatordesferoxamine (DFO)for 40min.(b)Cells loaded with 10μM BTP-1for 10min as control.(c)–(h)Cells incubated with 0.01,0.1,1,10,100and 1000μM Fe 3þ,respectively.(i)Quanti ﬁcation of mean ﬂuorescence intensity in Fig.S20a –h correspondingly.Scale bar is 20μm.J.Nandre et al./Biosensors and Bioelectronics 61(2014)612–617616the DFO chelation shrunk the cellularﬂuorescence(Fig.5c and e), indicating that the observedﬂuorescence enhancements(Fig.5b and d)were due to the changing levels in the Fe3þ-supplemented cells.After establishing that the sensor BTP-1can detect Fe3þwithin living HepG2cells,we turned our attention to quantify the cellular Fe3þlevel changes in HL-7701cells.The relativeﬂuorescence intensity of the confocal microscopy images in Fig.6was evaluated by Image-Pro Plus software(Fig.6i).HL-7701cells were treated with different concentrations of Fe3þ,and then loaded with10μM BTP-1for10min following which the cells were washed three times with RPMI-1640before imaging(Fig.6).Cells in Fig.6b were as control,which showed almost noﬂuorescence after treated with50μM of DFO(Fig.6a).Our sensor might also detect Fe3þat basal,endogenous levels within cells.Therefore,we selected the cell-body regions in the visualﬁeld(Fig.6a–h)as the region of interest to determine the averageﬂuorescence intensity.The confocalﬂuorescence images became gradually brighter as the concentration of Fe3þincreased from0.01μM to100μM Fe3þ(Fig.6c–g),and then theﬂuorescence intensity becomes saturated after100μM Fe3þ(Fig.6h).Taken together,these quantitative assays established that the sensor BTP-1can be used for the ﬂuorescence detection of Fe3þlevel changes within living cells. The results also suggest that our sensor has good membrane permeability.4.ConclusionsIn conclusion,we have developed a simple benzo-thiazolo-pyrimidine based Fe3þ-selectiveﬂuorescent“turn-on”sensor BTP-1.Sensor BTP-1showed an excellent selectivity for Fe3þover other interfering metal ions with the detection limit down to nanomolar concentration.Also,the sensor works well in the pH range of6–8.Confocal microscopy images indicate that BTP-1can be used for detecting changes in Fe3þlevels within living cells.To the best of our knowledge,this is theﬁrst example of a chromo-ﬂuorogenic sensor based on benzo-thiazolo-pyrimidine that allows the selective detection of the Fe3þion by aﬂuorescence “turn-on”mode in live cells.Based on the bioactive molecules like benzo-thiazolo-pyrimidine,sensor BTP-1with its low cost and easy preparation,its excellent selectivity and low detection limit, suggests that this approach could potentially lead to many more sensors being designed using the benzo-thiazolo-pyrimidine as a core skeleton.AcknowledgmentsThe author Dr.U.D.Patil is grateful for theﬁnancial support from the Department of Science&Technology,New Delhi,India (Reg.no.CS-088/2013).We thank the EPSRC National Crystal-lographic Service(Southampton,UK)for data.Appendix A.Supplementary informationSupplementary data associated with this article can be found in the online version at doi:10.1016/j.bios.2014.06.017.ReferencesAu-Yeung,H.Y.,Chan,J.,Chantarojsiri,T.,Chang,C.J.,2013.J.Am.Chem.Soc.135, 15165–15173.Cairo,G.,Pietrangelo,A.,2000.Biochem.J.352,241–250.Callan,J.F.,Silva,A.P.,Magri,D.C.,2005.Tetrahedron61,8551–8588.Chen,X.,Pradhan,T.,Wang,F.,Kim,J.S.,Yoon,J.,2012.Chem.Rev.112,1910–1956. 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荧光吸收光谱和发射光谱（6）

Fluorochrome Absorption and Emission SpectraHere we present the absorption and emission spectra of the fluorochromes BD Biosciences Pharmingen conjugates to monoclonal antibodies and other proteins. Four of these fluorochromes, fluorescein isothiocyanate (FITC), R-phycoerythrin (R-PE), BD Cy-Chrome™, and PerCP, can be used with single-laser flow cytometers equipped with an argon-ion laser emitting light at 488 nm for three-color flow cytometric analysis (Fig. 1).BD Biosciences Pharmingen also offers monoclonal antibodies and proteins conjugated to allophycocyanin (APC) and avidin conjugated to Texas Red™. Both of these dyes are useful in experiments where multi-color analysis is desired using flow cytometers with dual laser capabilities (Fig 1). APC can b e excited by a helium-neon (HeNe) laser emitting light at 633 nm, by a krypton laser emitting light at 647 nm, or by a dye laser which can be conveniently tuned to emit light in the 550-650 nm range. (In a dye laser, the lasing medium is a solution of fluo rescent dye excited by a pump laser, usually an ion laser.) Texas Red ™ , available conjugated to avidin, can be excited by an argon-krypton mixed-gas laser at 568 nm, or with a dye laser, where both APC and Texas Red ™ can be used simultaneously. Because many combinations of lasers, detectors, filters and fluorochromes are possible for multi-color analysis, proper precautions need to be taken (i.e.,bandpass filters, dichroic mirrors, longpass filters, etc.) by the operator to ensure each fluorochrome is being detected by only one detector (Fig. 1). In the following descriptions, we give our recommendations for the ideal instrument set-up for use with our reagents.(summarized in Table1) Allophycocyanin (APC) is an accessory photosynthetic pigment found in bluegreen algae. Its molecular weight is approximately 105 kDa. APC has 6 phycocyanobilin chromophores per molecule, which are similar in structure to phycoerythrobilin, the chromophore in R-PE. It has a 650-nm wavelength absorption maximum (Fig. 2) and a 660-nm fluorescence emission maximum (Fig. 2). Using a 660 ± 10 nm BP filter will give optimum detection for this fluorochrome. APC can be used in flow cytometers equipped with dual lasers for multi-color analysis (Fig. 2). It can be excited by laser light between 600-640 nm. For this, we recommend a He-Ne laser at 633 nm, or a tunable dye laser tund between 600-640 nm.BD Cy-Chrome™ is a tandem conjugate system, with an absorption maximum of approximately 650 nm (Fig. 2), which combines R-phycoerythrin and a cyanine dye (MW 1.5 kDa). When excited by 488-nm light, the excited fluorochrome (R-PE) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent maximum is approximately 670 nm (Fig. 2). Using a 650-nm longpass filter will give optimum detection for this fluorochrome. The efficiency of the light energy transfer between the two fluorochromes can be seen in Fig. 2F where less than 5% of the absorbed light is lost as fluorescence at 575 nm by R-PE. Compared to other fluorescence energy-transfer systems used in flow cytometry (e.g., RED613™ , EDC, PerCP), BD Cy-Chrome™ is a superior fluorochrome for third color analysis because of its high emission intensity and broad spectrum. As with our R-PE conjugates, an average of one BD Cy-Chrome™ molecule is coupled per antibody or protein. Because of its broad absorption range (Fig. 2), BD Cy-Chrome™ is not recommended for use with du al-laser flow cytometers where excitation by both lasers is possible.Fluorescein isothiocyanate (FITC) is a fluorochrome with a molecular weight of 389 daltons and an absorption maximum at 495 nm (Fig. 2). Its excitation by 488-nm light leads to a fluorescence emission maximum around 520 nm (Fig. 2). Using a 530 ± 15 nm bandpass (BP) filter will give optimum detection for this fluorochrome. The isothiocyanate derivative (FITC) is the most widely used form for conjugation to antibodies and proteins, but other derivatives are available. FITC has a high quantum yield (efficiency of energy transfer from absorption to emission fluorescence) and approximately half of the absorbed photons are emitted as fluorescent light. The number of FITC molecules per conjugate partner (antibody, Avidin, Streptavidin, etc.) is usually in the range of three to five molecules.R-phycoerythrin (R-PE) is an accessory photosynthetic pigment found in red algae. In vivo, it functions to transfer light energy to chlorophyll during photosynthesis. In vitro, it is a 240-kDa protein with 34 phycoerythrobilin fluorochromes per molecule. The large number of fluorochromes per PE molecule makeR-phycoerythrin an ideal pigment for flow cytometry applications. Its absorption maximum is 564 nm (Fi g. 2). When excited by 488-nm light, its fluorescence emission maximum is approximately 575 nm (Fig. 2). For single-laser flow cytometer use, we recommend using a 585 ± 21 nm BP filter for optimal detection (Fig. 1). When performing multi-color analysis with a dual-laser system, a tighter window of detection is required to compensate for the other conjugates being used (e.g.,Texas Red ™ ). For this, we recommend using a 575 ± 13-nm BP filter (Fig.1). Our conjugation chemistry yields an average of one R-PE molecule per antibody orprotein. The emitted light is collected in the fluorescence-2 (FL2) channel.PE-Texas Red™ is a tandem conjugate system which combines R-PE and Texas Red™ and has an absorption maximum of approximately 564 nm. When excited by 488-nm light, the excited fluorochrome (PE) is able to transfer its fluorescent energy to the Texas Red™ molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is approximately 615 nm. Special care must be taken when using PE-Texas Red™ conjugates in conjunction with R-PE as there is considerable spectral overlap in the emission profiles of both fluorochromes.Peridinin chlorophyll protein (PerCP) is a component of the photosynthetic apparatus found in the dinoflagellate,Glenodinium . PerCP is a protein complex with a molecular weight of approximately 35 kDa. When excited by light at 488 nm from an argon-ion laser, PerCP has a excitation maximum around 490 nm, with an emission spectrum which peaks at 675 nm. The emitted light is collected in the fluorescence-3 (FL3) channel. Due to its photobleaching characteristics, PerCP conjugates are not recommended for use on stream-in-air flow cytometers.PerCP-Cy5.5 is a tandem conjugate system than combines PerCP with a cyanine d ye (Cy5.5™) and has an absorption maximum of approximately 490 nm. When excited by 488-nm light, the excited fluorochrome (PerCP) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is approximately 694 nm. Using a 650 nm longpass filter will give optimum detection for this fluorochrome. The emitted light is collected in the fluorescence-3 (FL3) channel. PerCP-Cy5.5 is recommended for use with stream-in-air flow cytometers. APC-Cy7is a tandem conjugate system that combines APC and a cyanine dye (Cy7™) and has an absorption maximum of approximately 650 nm. When excited by light from a dye or HeNe laser, the excited fluorochrome (APC) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is approximately 767 nm. It is recommended that a 750-nm longpass filter be used along with a red-sensitive detector such as the Hammatsu R3896 PMT for this fluorochrome. Special filters are required when using APC-Cy7ª in conjunction with APC. It is recommended that special precautions be taken with PharRed conjugates, and cells stained with them, to protect the fluorochrome from long-term exposure to visible light.Texas Red™ is a sulfonyl chloride derivative of sulforhodamine 101 with a molecular weight of 625 daltons. BD Biosciences Pharmingen offers Texas Red ™ , conjugated to avidin, as a useful second step for multi-color analysis. Because it em its in the long wavelengths of the deep red region (Fig 2), Texas Red ™ has little spectral overlap with FITC. When performing multi-color analysis involving both Texas Red™ and R-PE, BD Biosciences Pharmingen recommends excitation of Texas Red ™ using a d ual-laser flow cytometer equipped with a tunable dye laser to avoid "leaking" into the PE detector. If a krypton laser, emitting light at 568 nm, is used, the laser light will "leak" into the R-PE channel. Texas Red ™ can be used in conjunction with APC for multi-color analysis when both dyes are excited in the 595-605 nm range with a dye laser. Texas Red ™ has an absorption maximum of 596 nm (Fig. 2). Its emission maximum, when excited by 595-600-nm laser light, is 615 nm (Fig. 2). Using a 620 ± 10-nm bandpass filter will give optimum detection for this fluorochrome (Fig. 1).Comparative staining using a monoclonal antibody (RA3-6B2; anti-B220; Cat. No. 557390/553084**) conjugated to different fluorochromes and analyzed on either BD FACSVantage™ (upper panels) or BDFACSCalibur™ (lower panels). The numbers indicate the ratio of the median fluorescence intensity of positive cells to the negative cells (signal to noise ratio). These plots demonstrate how choices in A) fluorochrome-conjugates or B) instrumentation can affect the fluorescence intensity observed for a given population.A. The differences observed between individual fluorochrome-conjugates can be affected by the mAbconjugated. Thus while in the example above the PE-conjugate is brighter than the BD Cy-Chrome™ -conjugate, when analyzed on the BD FACSCalibur™ , for many mAbs the Cy-Chrome ™ -conjugate results in a brighter stain. Contact BD Biosciences Pharmingen Technical Services for more information on specific reagents.B. Similarly different flow cytometers utilize different lasers and different fluorescence filter sets whichcan result in differences in signal to noise ratios when using the same reagent. Note that PEreagents tend to be brighter when used on a BD FACSCalibur™ while APC reagents are brighter on a BD FACSVantage™. Note changes of the signal to noise ratio depending on fluorchrome and instrument used.Enlarge imageFigure 1."Top schematic." A single laser flow cytometer with five parameters of detection. Two detectors detect the light scatter, and three photo-multiplier tubes (PMTs) detect the fluorescent signals. The bandpass filters are set up for optimal detection with BD Biosciences Pharmingen's fluorochromes: FITC, PE, BD Cy-Chrome ™ and Becton Dickinson's PerCP."Bottom schematic." A dual laser flow cytometer with six parameters of detection. Two detectors detect the light scatter, and four PMTs detect the fluorescent signals. The bandpass filters are set up forop timal detection with BD Biosciences Pharmingen's fluorochromes FITC, PE, APC, and Texas Red ™ . The second (orange) laser light is emitted from a tunable dye head using rhodamine 6 G as the fluorescent dye for excitation. Forward light scatter (FSC), side scatter (SSC), FITC, and PE signals are all produced by the primary 488-nm argon-ion laser. APC and Texas Red ™ signals are produced by the second laser (dye head with a 488-nm argon-ion laser).Figure 2. Absorption spectra of Fluorochromes. Individual fluorochrome excitation spectra are found in gray and the corresponding emission spectra in black. Typical band pass filters are given for eachflu orochrome as used on a FACSVantage™ except for BD Cy-Chrome™ and PerCP which are shown for FACSCalibur™ configurations.TABLE 1. Comparison of individual fluorochromes with single and dual laser flow cytometry.*PerCP is highly sensitive to photobleaching and must be used with laser power <150mW++Can only be used with a dye laser#Not recommended (dull)$BD Cy-Chrome and APC cannot be simultaneously used on instruments lacking cross-beam compensation. References:Loken, M.R., 1990. Immunofluorescence Techniques in Flow Cytometry and Sorting, 2nd Ed., Wiley. pp341-353.Parks, D., L. Herzenberg, and L. Herzenberg. 1989. Flow cytometry and fluorescence-activated cell sorting. Fundamental Immunology, Second Edition. William Paul, Ed.Raven Press, Ltd, New York.Zola, H. 1995. Detection of cytokine receptors by flow cytometry. In Current Protocols in Immunology (J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, W. Strober, eds.) John Wiley and Sons, New York. Unit 6.21.Immunofluorescence and cell sorting. In Current Protocols in Immunology. (J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, W. Strober, eds) John Wiley and Sons, New York. Unit 5.1 - 5.6.5. Shapiro, H.M. 1988. Practical Flow Cytometry, 2nd Ed. Wiley-Liss, New York.。

牦牛FABP3基因克隆及组织表达谱分析

第52卷第1期2024年1月西北农林科技大学学报(自然科学版)J o u r n a l o f N o r t h w e s t A&F U n i v e r s i t y(N a t .S c i .E d .)V o l .52N o .1J a n .2024网络出版时间:2023-06-30 15:46 D O I :10.13207/j .c n k i .jn w a f u .2024.01.001网络出版地址:h t t ps ://k n s .c n k i .n e t /k c m s 2/d e t a i l /61.1390.S .20230629.1615.006.h t m l 牦牛F A B P 3基因克隆及组织表达谱分析[收稿日期] 2022-09-22[基金项目] 国家重点研发计划项目(2021Y F D 1600200);合作市牦牛种质提升与提质增效项目;甘肃省科技计划项目(20J R 5-R A 580);甘肃省科技重大项目(21Z D 10N A 001,G Z G G -2021-1);现代农业(肉牛牦牛)产业技术体系建设专项(C A R S -37);中国农业科学院科技创新工程项目(25-L Z I H P S -01) [作者简介] 戴荣凤(1998-),女,山东济南人,在读硕士,主要从事动物遗传育种研究㊂E -m a i l :r o n g f e n g d a i 123@163.c o m [通信作者] 梁春年(1973-),男,甘肃武威人,研究员,硕士生导师,主要从事动物遗传育种研究㊂E -m a i l :c h u n n i a n 2006@163.c o m戴荣凤1,2,黄纯1,2,扎老3,路建卫3,唐月琴4,梁春年1,2(1中国农业科学院兰州畜牧与兽药研究所甘肃省牦牛繁育工程重点实验室,甘肃兰州730050;2农业农村部青藏高原畜禽遗传资源育种重点实验室,甘肃兰州730050;3合作市佐盖多玛乡畜牧站,甘肃甘南747000;4合作市农畜产品质量安全检测检验中心,甘肃甘南747000)[摘要] ʌ目的ɔ分析牦牛心肌脂肪酸结合蛋白3(F A B P 3)基因的结构和功能,并检测其在成年牦牛和胎牛中的表达水平,为探究该基因在牦牛育种中的生物学功能提供理论参考㊂ʌ方法ɔ以美仁牦牛的心脏组织为试验材料,P C R 扩增F A B P 3基因,对得到的编码区序列(C D S )进行生物信息学分析;利用实时荧光定量P C R (R T -q P C R ),检测F A B P 3基因在成年牦牛和胎牛心脏㊁肝脏㊁脾脏㊁肺脏㊁肾脏及肌肉组织的表达水平㊂ʌ结果ɔ牦牛F A B P 3基因编码区序列长度为402b p,编码133个氨基酸;蛋白质的高级结构主要是β-转角和延伸链;牦牛F A B P 3蛋白不存在跨膜区域和信号肽,属于具有一定亲水性的稳定蛋白;牦牛F A B P 3基因C D S 区核苷酸及其编码氨基酸序列的同源性分析显示,F A B P 3基因在牛属动物中具有一定的保守性;系统进化树分析表明,牦牛与野牦牛和瘤牛的亲缘关系最近,与鸡的亲缘关系最远㊂R T -q P C R 结果显示,F A B P 3基因在成年牦牛和胎牛的心脏㊁肝脏㊁脾脏㊁肺脏㊁肾脏及肌肉组织中均有表达,但在胎牛肝脏㊁脾脏㊁肾脏和肌肉中的表达水平显著或极显著高于成年牦牛,在成年牦牛肺脏中的表达水平极显著高于胎牛㊂ʌ结论ɔ克隆了牦牛F A B P 3基因,探究了F A B P 3基因在牦牛中的组织表达规律,为进一步研究该基因在牦牛脂肪沉积中的作用提供了基础数据㊂[关键词] 美仁牦牛;F A B P 3;基因克隆;生物信息学;组织表达谱[中图分类号] S 823.8+5[文献标志码] A[文章编号] 1671-9387(2024)01-0001-08C l o n i n g a n d t i s s u e e x p r e s s i o n p r o f i l e a n a l ys i s o f F A B P 3ge n e i n B o s g r u n n i e n s D A I R o n gf e ng 1,2,HU A N G C h u n 1,2,Z H A L a o 3,L U J i a n w e i 3,T A N G Y u e qi n 4,L I A N G C h u n n i a n 1,2(1G a n s u P r o v i n c e Y a k B r e e d i n g E n g i n e e r i n g K e y L a b o r a t o r y ,L a n z h o u I n s t i t u t e o f H u s b a n d r y an d P h a r m a c e u t i c a l S c i e n c e s ,C h i n e s e A c a d e m y o f A g r i c u l t u r a l S c i e n c e ,L a n z h o u ,G a n s u 730050,C h i n a ;2K e y L a b o r a t o r y o f A n i m a l G e n e t i c s a n d B r e e d i n g on T i b e t a n P l a t e a u ,M i n i s t r y o f A g r i c u l t u r e a n d R u r a l A f fa i r s ,L a n z h o u ,G a n s u 730050,C h i n a ;3A n i m a l H u sb a n d r y S t a t i o n o f Z o g a i d o m a T o w n s h i p i n H e z u o C i t y ,G a n n a n ,G a n s u 747000,C h i n a ;4Q u a l i t y a n d S a f e t y I n s p e c t i o n C e n t e r o f A g r i c u l t u r a l a n d L i v e s t o c k P r o d u c t s i n H e z u o C i t y ,G a n n a n ,G a n s u 747000,C h i n a )A b s t r a c t :ʌO b j e c t i v e ɔT h e s t r u c t u r e a n d f u n c t i o n o f y a k h e a r t f a t t y a c i d b i n d i n g pr o t e i n 3(F A B P 3)g e n e w e r e a n a l y z e d a n d i t s e x pr e s s i o n l e v e l s i n a d u l t a n d f e t a l y a k w e r e d e t e c t e d t o p r o v i d e r e f e r e n c e s f o r e x p l o r i n g b i o l o g i c a l f u n c t i o n s o f t h e g e n e i n y a k b r e e d i n g .ʌM e t h o d ɔT h e F A B P 3g e n e w a s a m p l i f i e d b yP C R f r o m h e a r t t i s s u e o f M e i r e n y a k ,a n d b i o i n f o r m a t i c s a n a l y s i s w a s c a r r i e d o u t a f t e r t h e C D S r e gi o n w a so b t a i n e d.T h e e x p r e s s i o n l e v e l s o f F A B P3i n h e a r t,l i v e r,s p l e e n,l u n g,k i d n e y,a n d m u s c l e t i s s u e s o f a d u l t a n d f e t a l y a k w e r e d e t e c t e d b y r e a l-t i m e q u a n t i t a t i v e P C R(R T-q P C R).ʌR e s u l tɔT h e l e n g t h o f t h e c o d i n g r e g i o n o f F A B P3w a s402b p,e n c o d i n g133a m i n o a c i d s.T h e a d v a n c e d s t r u c t u r e o f t h e p r o t e i n i n c l u d e d m a i n l yβ-t u r n a n d e x t e n d e d c h a i n.T h e r e w a s n o t r a n s m e m b r a n e r e g i o n o r s i g n a l p e p t i d e o n y a k F A B P3 p r o t e i n,i n d i c a t i n g i t w a s a s t a b l e p r o t e i n w i t h c e r t a i n h y d r o p h i l i c i t y.H o m o l o g y a n a l y s i s o f t h e n u c l e o t i d e a n d a m i n o a c i d s e q u e n c e s o f t h e C D S r e g i o n s h o w e d t h a t t h e F A B P3g e n e w a s c o n s e r v e d i n c a t t l e.A c c o r d-i n g t o t h e p h y l o g e n e t i c t r e e,y a k h a d t h e c l o s e s t g e n e t i c r e l a t i o n s h i p w i t h w i l d y a k a n d z e b u,a n d t h e f a r-t h e s t g e n e t i c r e l a t i o n s h i p w i t h c h i c k e n.R T-q P C R r e s u l t s s h o w e d t h a t t h e F A B P3g e n e w a s e x p r e s s e d i n h e a r t,l i v e r,s p l e e n,l u n g,k i d n e y,a n d m u s c l e t i s s u e s o f a d u l t a n d f e t a l y a k.T h e e x p r e s s i o n i n l i v e r,s p l e e n, k i d n e y,a n d m u s c l e o f f e t a l c a t t l e w a s s i g n i f i c a n t l y o r e x t r e m e l y s i g n i f i c a n t l y h i g h e r t h a n t h a t o f a d u l t y a k, a n d t h e e x p r e s s i o n l e v e l i n l u n g o f a d u l t y a k w a s s i g n i f i c a n t l y h i g h e r t h a n t h a t o f f e t a l y a k.ʌC o n c l u s i o nɔT h e F A B P3g e n e o f y a k w a s c l o n e d,a n d i t s t i s s u e e x p r e s s i o n p a t t e r n w a s e x p l o r e d.T h i s s t u d y p r o v i d e d b a s i c d a t a f o r f u r t h e r s t u d y o n t h e r o l e o f F A B P3i n f a t d e p o s i t i o n.K e y w o r d s:M e i r e n y a k;F A B P3;g e n e c l o n i n g;b i o i n f o r m a t i c s;t i s s u e e x p r e s s i o n p r o f i l e牦牛(B o s g r u n n i e n s)作为生活在高寒低氧地区的特色畜种,主要分布在海拔3000m以上的青藏高原,对当地恶劣的自然环境具有较强的适应性,为当地牧民提供了基本的生产生活资料[1-2]㊂由于自然条件的限制,牦牛养殖一直以放牧为主,随着牧区人类活动的增加,牦牛养殖重数量不重质量的现状,加速了天然草场的退化及生态环境的恶化㊂另外,牦牛存在的近亲繁殖现象,导致品种退化严重,个体的生产性能有所下降[3]㊂美仁牦牛是分布在甘肃省甘南州合作市佐盖多玛草原上的一个优良类群,因其具有体格硕大㊁肉质优良㊁抗逆性强的特点成为甘南草原上的优质畜种资源㊂近年来,当地政府对其采取了一系列培育措施,如加强基础设施建设㊁重视当地养殖人员的技术培训㊁及时调整畜群结构和应用科学的繁育技术进行扩繁等[4]㊂肌内脂肪(i n t r a m u s c u l a r f a t,I M F)含量会影响肉的口感,是评价肉质性状的关键因素之一,与肉的嫩度㊁多汁性和风味等性状具有密切关系[5]㊂因此, I M F在改善肉的风味以及提高动物的经济价值方面具有重要意义㊂要对I M F的含量进行调节,就需要调节脂质代谢,促进甘油三酯在骨骼肌和脂肪细胞中的沉积[6-7]㊂脂肪酸结合蛋白3(f a t t y a c i d b i n d i n g p r o t e i n3,F A B P3)是脂肪酸结合蛋白家族(F A B P s)的成员之一,是O c k n e r等[7]在研究长链脂肪酸吸收时首次在肝和心脏等组织的细胞浆中发现的,又称为心肌脂肪酸结合蛋白(h e a r t f a t t y a c i d-b i n d i n g p r o t e i n,H-F A B P),其可以与脂肪酸结合,在脂肪酸的转运和代谢中具有重要的作用㊂目前已经证实,F A B P3基因在心脏和骨骼肌中表达较高,其次是回肠㊁胰腺和盲肠等组织[8]㊂F A B P3基因对脂质和脂肪酸的转运和沉积具有调控作用,其与食草动物的脂质特性有关[8-11]㊂在牦牛的育种中,机体的脂质代谢是人们关注的重点之一,本研究以美仁牦牛为对象,对其F A B P3基因进行了克隆和生物信息学分析,并采用实时荧光定量P C R(R e a l-t i m e q u a n t i t a t i v e P C R,R T-q P C R)技术检测F A B P3基因在成年牦牛和胎牛心脏㊁肝脏㊁脾脏㊁肺脏㊁肾脏及肌肉组织中的表达水平,旨在为进一步探究该基因在美仁牦牛中的功能奠定基础㊂1材料与方法1.1试验材料供试美仁牦牛来自甘南州合作市的佐盖多玛草原㊂选取出生后7日龄的美仁牦牛,屠宰后立即采集其心脏组织,用D E P C水冲洗之后置于冻存管中,立即放入-80ħ冰箱中保存备用㊂采集3头成年牦牛(4~5岁)和胎牛(妊娠5~6个月)的心脏㊁肝脏㊁脾脏㊁肺脏㊁肾脏和背最长肌等组织,用D E P C水冲洗之后放入冻存管中,置于液氮中保存备用㊂T r i z o l R e a g e n t,I n v i t r o g e n,美国;反转录试剂盒,T R A N,北京全式金生物技术股份有限公司; 2ˑG o T a q G r e e n M a s t e r M i x,P r o m e g a,普洛麦格生物技术有限公司;p M D19-T载体和D Hα5感受态细胞,宝生物工程有限公司(T a K a R a);琼脂糖凝胶D N A凝胶回收试剂盒,天根生化科技有限公司(T I A N G E N)㊂1.2 R N A提取和反转录取适量-80ħ冰箱中保存的牦牛组织(7日龄2西北农林科技大学学报(自然科学版)第52卷美仁牦牛胎牛的心脏组织,成年牦牛和胎牛心脏㊁肝脏㊁脾脏㊁肺脏㊁肾脏和肌肉组织)样品,放入装有液氮的研钵中进行充分研磨,在研磨过程中要保证研钵中有液氮的存在㊂然后将研磨成粉末状的组织放入预冷的1.5m L 离心管中,用T r i z o l 法[12]提取其R N A ,使用紫外分光光度计对其浓度和纯度进行检测,根据其O D 260和O D 280的比值(O D 260/O D 280=1.8~2.0)判断是否符合要求㊂采用反转录试剂盒将提取的R N A 反转录成c D N A ,于-20ħ的冰箱中保存备用㊂1.3 引物设计和牦牛F A B P 3基因C D S 区的克隆根据N C B I G e n B a n k 数据库中野牦牛(B o sm u t u s )F A B P 3基因的m R N A 序列(登录号:X M _005906169.2),确定该基因的编码区序列(c o d i n gr e g i o n s e qu e n c e ,C D S ),然后使用N C B I 的P r i m e -B l a s t 在线工具(h t t p s ://w w w.n c b i .n l m.n i h .go v /t o o l s /pr i m e r -b l a s t )设计引物,用于F A B P 3基因的扩增和R T -q P C R ㊂所用的引物信息见表1,均由擎科生物技术有限公司(西安)合成㊂以牦牛心脏的c D N A 为模板,对F A B P 3基因的C D S 区域进行P C R 扩增㊂P C R 反应体系为20μL ,其中主要包括c D N A 2μL ,2ˑG o T a qG r e e n M a s t e r M i x 10μL ,上下游引物各1μL ,无酶无菌水6μL ㊂P C R 扩增程序为:94ħ预变性3m i n ;94ħ变性30s ,58ħ退火40s ,72ħ延伸1.5m i n ,35个循环;72ħ后延伸10m i n ;4ħ保存㊂用1.2%的琼脂糖凝胶对扩增产物进行电泳检测㊂表1 试验所用引物信息T a b l e 1 I n f o r m a t i o n o f p r i m e r s u s e d i n t h i s s t u d y基因G e n e引物序列(5'ң3')P r i m e r s e qu e n c e (5'ң3')退火温度/ħA n n e a l i n g t e m pe r a t u r e 产物长度/b pL e n gt h o f t h e p r o d u c t 用途F u n c t i o n F A B P 3F :G T C T T G A A G A G C C G G C T G A AR :A A T G A G G C A A T C T G G T G C C G 58579基因克隆G e n e c l o n eF :C T T C A AG C T G G G A G T C G A G T T R :C C A T T T C C C G C A C A A G T G A T G 58137R T -q P C R β-a c t i n F :T G A T G A T A T T G C T G C G C T C G R :T A C G A G T C C T T C T G G C C C A T58153R T -qP C R 用D N A 回收试剂盒纯化回收目的片段,检测其浓度㊂将纯化后的P C R 产物,参照试剂盒推荐的反应体系与p M D -19T 载体连接过夜;随后转化为D H α5感受态细胞,在提前配好的固体培养基上进行涂板,37ħ恒温培养箱中过夜(12h )[13]㊂挑选阳性菌液,送擎科生物技术有限公司(西安)测序㊂1.4 牦牛F A B P 3基因的生物信息学分析在M E G A 7.0软件中,采用邻接法(N e i gh b o r -j o i n i n g,N J )构建系统进化树,分析牦牛与其他参比物种(表2)F A B P 3基因的同源性㊂使用相关软件进行生物信息学的分析:用N C B I 的在线工具O R FF i n d e r 搜索开放阅读框,用N C B I 的在线工具B L A S T进行同源性比对分析,用D N A M A N 软件分析核酸的理化性质,用E x P A S y 中的Pr o t P a r a m 和S W I S S -M O D E L 功能进行蛋白质理化性质分析和蛋白质三级结构预测,用T MHMM 和S i g n a l P -6.0工具预测蛋白质的跨膜区,用N e t P h o s 3.1工具预测蛋白质的磷酸化位点,用S O P M A 预测蛋白质的二级结构㊂表2 参比物种名称及其F A B P 3基因的G e n B a n k 登录号T a b l e 2 N a m e s o f r e f e r e n c e s pe c i e s a n d G e n B a n k a c c e s s i o n n u m b e r s of F A B P 3g e n e 物种S pe c i e s 登录号A c c e s s i o n n u m b e r 物种S pe c i e s 登录号A c c e s s i o n n u m b e r 黄牛B o s t a u r u sNM _174313.2野牦牛B o s m u t u sX M _005906169.2水牛B u b a l u s b u b a l i sNM _001290882.1人H o m o s a p i e n s NM _001320996.2绵羊O v i s a r i e sNM _001267884.2小鼠M u s m u s c u l u s NM _010174.2野猪S u s s c r o fa NM _001099931.1大鼠R a t t u s n o r v e gi c u s NM _024162.2瘤牛B o s i n d i c u s XM _019979493.1马E qu u s c a b a l l u s NM _001163885.2山羊C a pr a h i r c u s NM _001285701.1鸡G a l l u s g a l l u sNM _001030889.21.5 牦牛F A B P 3组织表达分析以β-a c t i n 为内参基因,设置3个生物学重复和3个技术重复,使用R T -q P C R 方法检测F A B P 3基因在成年牦牛和胎牛心脏㊁肝脏㊁脾脏㊁肺脏㊁肾脏及肌肉等组织中的表达水平,所用引物根据克隆得到的序列进行设计(表1)㊂以心脏组织表达水平为参照,确定F A B P 3基因在其他组织样本中的相对表达水平㊂运用2-ΔΔC t对R T -qP C R 的结果进行分析[14],使用S P S S 软件对其进行差异显著性检验,以P <0.05视为具有统计学意义,运用G r a ph P a d 3第1期戴荣凤,等:牦牛F A B P 3基因克隆及组织表达谱分析P r i s m 9.0进行可视化处理㊂2 结果与分析2.1 牦牛F A B P 3基因C D S 区序列的克隆牦牛F A B P 3基因C D S 区扩增结果如图1所示㊂M.D L 2000M a r k e r ;1.F A B P 3基因P C R 产物M.D L 2000M a r k e r ;1.P C R p r o d u c t i o n o f F A B P 3g e n e图1 牦牛F A B P 3基因的P C R 扩增F i g .1 P C R a m pl i f i c a t i o n o f F A B P 3g e n e i n y a k 图1表明,在579b p 的位置出现了1条清晰的条带,与预期片段大小一致㊂将扩增得到的产物连接到p M D 19-T 载体上进行克隆,得到的菌液通过擎科生物技术有限公司(西安)测序㊂对序列分析结果进行比对,结果表明牦牛F A B P 3基因C D S 区克隆成功,长度为402b p㊂2.2 牦牛F A B P 3基因C D S 区序列分析对美仁牦牛F A B P 3基因C D S 区的序列进行分析,结果发现其共编码133个氨基酸㊂利用D N AMA N 软件对F A B P 3基因序列的碱基组成进行分析,结果表明其A ㊁C ㊁G 和T 的含量分别是30.35%,22.12%,27.12%和20.41%,G+C 的含量(49.24%)与A+T 的含量(50.76%)相近,说明该基因在碱基使用方面不存在偏好性㊂核苷酸同源性分析表明,美仁牦牛与野牦牛和瘤牛的核苷酸同源性最高,均为99.75%,与黄牛㊁水牛㊁绵羊㊁山羊㊁野猪㊁马㊁人㊁大鼠㊁小鼠和鸡的核苷酸的同源性依次为99.50%,98.51%,97.01%,96.77%,93.03%,90.05%,82.30%,83.33%,83.08%和77.81%;F A B P 3氨基酸序列与野牦牛㊁瘤牛㊁黄牛和水牛的同源性均为100%,与绵羊㊁山羊㊁野猪㊁马㊁人㊁小鼠㊁大鼠和鸡的同源性分别为96.24%,96.99%,92.48%,90.98%,81.94%,85.71%,85.71%和78.20%㊂将美仁牦牛与黄牛F A B P 3基因(NM _174313.2)的C D S 区核苷酸序列进行比对,结果(图2)显示,美仁牦牛在252位(C ңT )和324位(GңA )发生了碱基突变㊂图2 美仁牦牛和黄牛核苷酸序列的比对分析F i g .2 C o m p a r a t i v e a n a l y s i s o f n u c l e o t i d e s e qu e n c e s o f M e i r e n y a k a n d c a t t l e 2.3 牦牛F A B P 3基因的系统进化树将美仁牦牛㊁瘤牛㊁野牦牛和黄牛等13个品种F A B P 3基因的C D S 区,在M E G A 7.0软件中采用邻接法(N e i g h b o r -j o i n i n g,N J )构建系统进化树,结果(图3)发现,美仁牦牛与野牦牛和瘤牛的亲缘关系最近,其次是黄牛和水牛,与鸡㊁小鼠和大鼠等的亲缘关系较远㊂4西北农林科技大学学报(自然科学版)第52卷图3 美仁牦牛与其他物种F A B P 3基因的系统进化树F i g .3 P h y l o g e n e t i c t r e e o f F A B P 3g e n e i n M e i r e n y a k a n d o t h e r s pe c i e s 2.4 美仁牦牛F A B P 3蛋白分析2.4.1 蛋白的理化性质利用E x P A S y 中的在线工具P r o t P a r a m 对F A B P 3蛋白进行分析,结果显示F A B P 3蛋白的分子质量为14.78k u,分子式为C 654H 1054N 174O 204S 5,理论等电点(p I )为6.73㊂对F A B P 3蛋白的氨基酸组成进行分析,结果(表3)显示,其有20种必需氨基酸,其中T h r 含量最高(13.5%),其次为V a l (11.3%)和L y s (9.8%),C ys 和P r o 含量最少,均为0.8%㊂F A B P 3分子中,带负电荷的氨基酸残基(A s p 和G l u )与带正电荷的氨基酸残基(A r g 和L y s )数量一致,均为17个㊂除此之外,美仁牦牛F A B P 3蛋白的消光系数为13980,脂肪系数为83.38,不稳定系数为10.56,表明该蛋白比较稳定;F A B P 3蛋白的总平均亲水指数为0.247,为稳定的亲水蛋白㊂表3 美仁牦牛F A B P 3蛋白的氨基酸组成T a b l e 3 A m i n o a c i d c o m po s i t i o n o f M e r i r e n y a k F A B P 3p r o t e i n 氨基酸A m i n o a c i d s 占比/%P e r c e n t 氨基酸A m i n o a c i d s 占比/%P e r c e n t 氨基酸A m i n o a c i d s 占比/%P e r c e n t 氨基酸A m i n o a c i d s 占比/%P e r c e n t 丙氨酸A l a3.8亮氨酸L e u6.8谷氨酰胺G l n3.8丝氨酸S e r4.5精氨酸A r g3.0赖氨酸L ys 9.8谷氨酸G l u5.3苏氨酸T h r13.5天冬酰胺A s n 3.8蛋氨酸M e t 3.0甘氨酸G l y8.3色氨酸T r p1.5天冬氨酸A s p 7.5苯丙氨酸P h e4.5组氨酸H i s1.5酪氨酸T yr 1.5半胱氨酸C ys 0.8脯氨酸P r o0.8异亮氨酸I l e5.3缬氨酸V a l11.32.4.2 蛋白信号肽与跨膜区预测美仁牦牛F A B P 3蛋白信号肽的预测结果显示,其剪切位置分值C s c o r e ㊁信号肽分值S s c o r e ㊁综合参数Y s c o r e 分别为0.153,0.119和0.105,均小于阈值0.5,表明该蛋白不存在信号肽区域㊂对F A B P 3蛋白跨膜结构区域的预测发现,美仁牦牛F A B P 3蛋白中无跨膜结构,由此表明牦牛F A B P 3蛋白不是跨膜蛋白㊂2.4.3 磷酸化位点预测利用N e t P h o s3.1工具对美仁牦牛F A B P 3蛋白进行磷酸化位点预测,结果(图4)显示,美仁牦牛F A B P 3蛋白有15个潜在的磷酸化位点,其中丝氨酸㊁苏氨酸及酪氨酸的潜在数目分别是4,10和1个㊂图4 美仁牦牛F A B P 3蛋白潜在磷酸化位点预测F i g .4 P r e d i c t i o n o f p o t e n t i a l p h o s p h o r yl a t i o n s i t e s o f F A B P 3p r o t e i n i n M e i r e n y a k 5第1期戴荣凤,等:牦牛F A B P 3基因克隆及组织表达谱分析2.4.4 高级结构预测对美仁牦牛F A B P 3蛋白的二级结构进行预测,结果(图5)显示,其二级结构包括α-螺旋(h )㊁无规则卷曲(c )㊁延伸链(e )和β-转角(t ),含量分别为23.31%,30.83%,36.09%和9.77%㊂利用S W I S S -MO D E L 对美仁牦牛F A B P 3蛋白的三级结构进行可视化处理,结果显示所构建的三级结构模型与二级结构预测结果一致,牦牛F A B P 3蛋白中的主要构成依旧是无规则卷曲和延伸链㊂图5 美仁牦牛F A B P 3蛋白的二级结构预测F i g .5 P r e d i c t i o n o f s e c o n d a r y st r u c t u r e o f F A B P 3p r o t e i n i n M e i r e n y a k 2.5 美仁牦牛F A B P 3组织表达分析R T -q P C R 结果(图6)显示,F A B P 3基因在成年牦牛和胎牛的心脏㊁肝脏㊁脾脏㊁肺脏㊁肾脏及肌肉组织中均有表达,在胎牛肝脏㊁脾脏㊁肾脏和肌肉中的表达水平显著或极显著高于成年牦牛,在成年牦牛肺脏中的表达水平极显著高于胎牛(P <0.01),二者均以心脏中的表达水平最高,但相互间并无显著差异㊂*和**分别表示成年牦牛和胎牛同一组织间差异显著(P <0.05)和差异极显著(P <0.01)*a n d **i n d i c a t e s i g n i f i c a n t (P <0.05)a n d e x t r e m e l y s i gn i f i c a n t (P <0.01)d i f f e r e n c e s b e t w e e n a d u l t y a k a n d f e t a l y a k ,r e s p e c t i v e l y图6 美仁牦牛F A B P 3基因组织表达谱分析F i g .6 T i s s u e e x pr e s s i o n p r o f i l e o f F A B P 3ge n e i n M e r i r e n y a k 3 讨论F A B P 3蛋白作为一种脂肪酸载体蛋白,对脂肪合成具有重要的调控作用,在细胞内主要参与脂肪酸的吸收㊁转运和代谢[15-16]㊂石斌刚[17]以不同月龄的天祝白牦牛为对象,对其肌肉生长和肌内脂肪沉积的相关基因进行了鉴定,发现F A B P 3是与脂肪酸和脂肪沉积有关的候选基因㊂已有研究发现,F A B P s 可以与P P A R (pe r o x i s o m e p r o l if e r a t o r -a c t i -v a t e d r e c e p t o r )家族的转录因子过氧化物酶体增殖物激活受体相互作用,促进机体对脂肪酸的摄取并调节脂质代谢[18-19]㊂Y i 等[20]在猪脂肪细胞中对F A B P 3基因进行过表达处理,发现其可以促进脂肪生成㊂艾锦新等[10]研究了黔北麻羊F A B P 3基因的多态性,并探究了其与生长性状的相关性,结果显示F A B P 3基因外显子区检测出的单核苷酸多态性位点(s i n g l e n u c l e o t i d e p o l y m o r ph i s m ,S N P )与麻羊个体的体高㊁胸围和体斜长等指标显著相关㊂文力正[11]研究了改良草原红牛和草原红牛F A B P 3基因的S N P 与肉质性状的相关性,发现该基因对改良草原红牛的肌肉剪切力有显著影响㊂L i 等[21]对F A B P 3基因的5'-调控区进行了克隆并且对其转录起始位点进行了鉴定,经过与羊㊁猪和狗等8个物种进行比对发现,维甲酸X 受体α(r e t i n o i d X r e c e pt o r a l ph a ,R X R α)作为一种核受体,可以参与调节脂肪酸储存和葡萄糖代谢基因的转录㊂上述研究表明,F A B P 3基因的遗传变异可能会影响机体的I M F 含量及生长性状,但目前F A B P 3基因在美仁牦牛中的具体作用还未见研究报道,因此本研究克隆了牦牛的F A B P 3基因,探究了F A B P 3基因在牦牛中的组织表达规律㊂结果显示,美仁牦牛F A B P 3基因编码区序列长度为402b p,核苷酸序列与牛属动物的同源性较高,说明F A B P 3基因在牛属动物中具有一定的保守性㊂系统进化树分析结果发现,美仁牦牛与野牦牛的亲缘关系最近㊂由此推测,美仁牦牛可能具有野牦牛的血统㊂本研究中,美仁牦牛F A B P 3蛋白包含133个氨6西北农林科技大学学报(自然科学版)第52卷基酸,分子质量为14.78k u,带负电荷与带正电荷的氨基酸残基数量一致,均为17个,p I为6.73㊂疏水性预测发现,疏水性氨基酸(I l e和V a l)含量显著高于亲水性氨基酸(A r g和L y s),总亲水指数为0.247㊂美仁牦牛的F A B P3蛋白不存在信号肽也不具备跨膜区域,不属于分泌蛋白,这与何会金[22]对兰州大尾羊F A B P3基因的研究结果一致㊂美仁牦牛F A B P3蛋白存在15个磷酸化位点,其中苏氨酸上的潜在磷酸化位点数量最多(10个)㊂科研人员对F A B P3基因在黔北麻羊㊁鹿㊁牛和藏猪不同组织中的表达水平进行了研究,发现其在心脏中的表达量最高,其次是背最长肌和肾脏,肺脏和肝脏组织中的表达水平最低[23-26]㊂本研究发现, F A B P3基因在成年牦牛和胎牛各组织中均有表达,表达谱与其他物种的表达图谱一致,均是以心脏中的表达量最高,其次是背最长肌;F A B P3基因在成年牦牛肝脏㊁脾脏㊁肺㊁肾脏和肌肉中的表达量与胎牛存在显著差异㊂该结果表明,F A B P3基因在牦牛中的表达具有组织特异性,而且年龄对该基因在不同组织中的表达具有重要影响㊂L i等[27]研究了猪F A B P3基因表达与I M F之间的关系,结果显示腰部肌肉的I M F含量与F A B P3基因的表达水平呈正相关㊂据此推测,肌肉中F A B P3基因表达水平高的牦牛个体可能具有更高的I M F含量㊂X i o n g等[28]整合转录组学和脂质组学的分析发现,脂肪酸含量的差异是由脂肪酸㊁氨基酸㊁碳水化合物和磷脂代谢引起的,部分原因是受到了P P A R信号通路中F A B P3基因的调节㊂因此,可以猜测牦牛F A B P3基因的表达水平可能会通过影响脂质物质的代谢,进而对牦牛的I M F含量进行调控㊂4结论成功克隆了美仁牦牛F A B P3基因的编码区序列,其长度为402b p,编码133个氨基酸㊂美仁牦牛F A B P3基因在牛属动物中具有一定的保守性,并且F A B P3蛋白为稳定的亲水蛋白㊂F A B P3基因在成年美仁牦牛和胎牛的各个组织中均有表达,其在成年牦牛肝脏㊁脾脏㊁肺脏㊁肾脏和肌肉中的表达水平与胎牛存在显著或极显著差异㊂[参考文献][1]孟庆辉,陈永杏,董红敏,等.牦牛分布特点及其种群数量[J].家畜生态学报,2017,38(3):80-85.M e n g Q H,C h e n Y X,D o n g H M,e t a l.T h e d i s t r i b u t i o n c h a r-a c t e r i s t i c s a n d p o p u l a t i o n s o f y a k[J].J o u r n a l o f D o m e s t i c A n i-m a l E c o l o g y,2017,38(3):80-85.[2] A y a l e w W,C h u 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[18] C h m u r z yńs k a A.T h e m u l t i g e n e f a m i l y o f f a t t y a c i d-b i n d i n gp r o t e i n s(F A B P s):f u n c t i o n,s t r u c t u r e a n d p o l y m o r p h i s m[J].J o u r n a l o f A p p l i e d G e n e t i c s,2006,47(1):39-48. [19] T a n N S,S h a w N S,V i n c k e n b o s c h N,e t a l.S e l e c t i v e c o o p e r a-t i o n b e t w e e n f a t t y a c i d b i n d i n g p r o t e i n s a n d p e r o x i s o m e p r o-l i f e r a t o r-a c t i v a t e d r e c e p t o r s i n r e g u l a t i n g t r a n s c r i p t i o n[J].M o l e c u l a r a n d C e l l u l a r B i o l o g y,2002,22(14):5114-5127.[20] Y i B,W a n g J,W a n g S,e t a l.O v e r e x p r e s s i o n o f B a n n a m i n i-p i g i n b r e d l i n e f a t t y a c i d b i n d i n g p r o t e i n3p r o m o t e s a d i p o g e n-e s i s i n3T3-L1p r e a d i p o c y t e s[J].C e l l B i o l o g y I n t e r n a t i o n a l,2014,38(8):918-923.[21] L i A,W u L,W a n g X,e t a l.T i s s u e e x p r e s s i o n a n a l y s i s,c l o n i n ga n d c h a r a c t e r i z a t i o n o f t h e5'-r e g u l a t o r y r e g i o n o f t h eb o v i n eF A B P3g e n e[J].M o l e c u l a r B i o l o g y R e p o r t s,2016,43(9):991-998.[22]何会金.兰州大尾羊H-F A B P基因全长c D N A克隆及生物信息学分析[D].兰州:西北民族大学,2012.H e H J.F u l l-l e n g t h c D N A c l o n i n g o f H-F A B P g e n e a n d i t sb i o n f o r m a t ic s a n a l y s i s i n L a n z h o u F a t-t a i l ed s he e p[D].L a n z h o u:N o r t h w e s t M i n z u U n i v e r s i t y,2012.[23]艾锦新,龙安炬,罗卫星,等.黔北麻羊不同组织中F A B P1㊁F A B P3基因表达水平的研究[J].中国畜牧杂志,2020,56(11):63-67,73.A i J X,L o n g A J,L u o W X,e t a l.E x p r e s s i o n l e v e l s o fF A B P1a n d F A B P3g e n e s i n d i f f e r e n t t i s s u e s o f Q i a n b e i M ag o a t[J].C h i n e s e J o u r n a l o f A n i m a l S c i e n c e,2020,56(11):63-67,73.[24]郭梦雅,汤吉伟,张芙蕊,等.鹿不同组织中H-F A B P与E-F A B P基因的表达[J].东北林业大学学报,2022,50(1):100-104.G u o M Y,T a n g J W,Z h a n g F R,e t a l.E x p r e s s i o n o f H-F A B P a n d E-F A B P g e n e s i n d i f f e r e n t t i s s u e s o f d e e r[J].J o u r n a l o f N o r t h e a s t F o r e s t r y U n i v e r s i t y,2022,50(1):100-104.[25]石鹏飞,阮涌,刘文娇,等.关岭牛F A B P3和F A B P4基因的分子特征及其组织表达分析[J].南方农业学报,2022,53(8):2281-2293.S h i P F,R u a n Y,L i u W J,e t a l.M o l e c u l a r c h a r a c t e r i s t i c s o fF A B P3a n d F A B P4g e n e s a n d t i s s u e e x p r e s s i o n a n a l y s i s o fG u a n l i n g c a t t l e[J].J o u r n a l o f S o u t h e r n A g r i c u l t u r e,2022,53(8):2281-2293.[26]黄河,帅素容,余先琼,等.藏猪心脏脂肪酸结合蛋白基因(H-F A B P)组织表达差异性研究[J].四川农业大学学报, 2007,25(4):480-483.H u a n g H,S h u a i S R,Y u X Q,e t a l.H-F A B P m R N A e x p r e s-s i o n i n d i f f e r e n t t i s s u e s o f Z a n g p i g[J].J o u r n a l o f S i c h u a nA g r i c u l t u r a l U n i v e r s i t y,2007,25(4):480-483.[27] L i X,K i m S W,C h o i J S,e t a l.I n v e s t i g a t i o n o f p o r c i n e F A B P3a n d L E P R g e n e p o l y m o r p h i s m s a n d m R N A e x p r e s s i o n f o rv a r i a t i o n i n i n t r a m u s c u l a r f a t c o n t e n t[J].M o l e c u l a r B i o l o g yR e p o r t s,2010,37(8):3931-3939.[28] X i o n g L,P e i J,W a n g X,e t a l.L i p i d o m i c s a n d t r a n s c r i p t o m er e v e a l t h e e f f e c t s o f f e e d i n g s y s t e m s o n f a t t y a c i d s i n y a k sm e a t[J].F o o d s,2022,11(17):2582.8西北农林科技大学学报(自然科学版)第52卷。

QMMM简介

QM/MMFrom Wikipedia, the free encyclopediaJump to: navigation, searchThe hybrid QM/MM (quantum mechanics/molecular mechanics) approach is a molecular simulation method that combines the strength of both QM (accuracy) and MM (speed) calculations, thus allowing for the study of chemical processes in solution and in proteins. The idea of studying the chemistry of a large molecule by uncoupled quantum mechanics with molecular mechanics was first introduced by Warshel and Bromberg in 1970 [1]. This was done by combining the results of the CFF(MM) force field and computer program [2] (which is the basis for all current molecular simulation programs [3]) with a valence bond treatment of part of the molecule.The next step of having a force field for conjugated molecules with a quantum description of the pi part was introduced by Warshel in 1972 [4]. This was done by combining the CFF (MM) forcefield and quantum program with a pi-electron part [5] (which presents the QCFF/PI Program [6]). However, this force field for conjugated molecules did not include the crucial coupling between the pi and sigma electrostatic potentials, and, more importantly, did not include any coupling with the environment (and was thus not capable of exploring true chemical reactions in proteins and in solution yet). The key breakthrough in the field which truly introduced the coupled QM/MM approach, with basically all of its modern embedding aspects, appeared in the landmark 1976 paper of Warshel and Levitt [7], which not only introduced the crucial coupling with the electrostatic environment, but also paved the way for all subsequent studies of enzymatic reactions, photobiological processes and chemical processes in the condensed phase. The original 1976 work already included the link-atom treatment, the use of a polarizable force field, and the electrostatic embedding in the protein + solvent environment. Subsequently, as the power of this approach has received increasing recognition, it has been adapted by several groups including (but not limited to): Weitao Yang (Duke University), Sharon Hammes-Schiffer (The Pennsylvania State University), and Kenneth Merz (University of Florida).A recent challenge for the QM/MM approach has been to use a high level ab initio description of the QM region, while still being able to perform extensive sampling in order to accurately and properly evaluate free energies of processes in proteins. This problem has been overcome by using simple reference potentials and performing perturbation to the QM/MM surface (see discussion in [8]).An important advantage of QM/MM methods is the efficiency. The cost of doing classical molecular mechanics (MM) simulations in the most straight forward case scales O(N2), where N is the number of atoms in the system. This is mainly due to electrostatic interactions term (every particle interacts with everything else). However, use of cutoff radius, periodic pair-list updates and more recently the variations of the particle mesh Ewald(PME) method has reduced this between O(N) to O(N2). In other words, if a system with twice many atoms is simulated then it would take between twice to four times as much computing power. On the other hand the simplest ab-initio calculations typically scale O(N3) or worse (Restricted Hartree–Fock calculations have been suggested to scale ~O(N2.7)). To overcome the limitation, a small part of the system that is of major interest is treated quantum-mechanically (for instance, the active-site of an enzyme) and the remaining system is treated classically.Also, QM/MM methods can be used to treat both light nuclei susceptible to quantum effects (such as hydrogens) and electronic states. This allows generation of hydrogen wave-functions (similar to electronicwave-functions). This methodology has been useful in investigating phenomenon such as hydrogen tunneling. One example where QM/MM methods have provided new discoveries is the calculation of hydride transfer in the enzyme liver alcohol dehydrogenase. In this case, tunneling is important for the hydrogen, as it determines the reaction rate.[9][edit] See also∙List of quantum chemistry and solid state physics software∙List of software for molecular mechanics modeling[edit] References1.^ Warshel and Bromberg, "Oxidation of 4a,4b-Dihydrophenanthrenes. III.A Theoretical Study of the Large Kinetic Isotope Effect of Deuterium inthe Initiation Step of the Thermal Reaction with Oxygen", J. Chem. Phys.52 (1970) 1262.2.^ Lifson and Warshel, "A Consistent Force Field for Calculation onConformations, Vibrational Spectra and Enthalpies of Cycloalkanes and n-Alkane Molecules", J. Chem. Phys. 49 (1968), 51163.^Levitt, M. "The Birth of Computational Structural Biology", Nat. Struct.Biol. 8 (2001), 3924.^Warshel and Karplus, "Calculation of Ground and Excited State PotentialSurfaces of Conjugated Molecules. I. Formulation and Parametrization", J. Am. Chem. Soc. 94 (1972), 56125.^Warshel and Karplus, "Calculation of Ground and Excited State PotentialSurfaces of Conjugated Molecules. I. Formulation and Parametrization", J. Am. Chem. Soc. 94 (1972), 56126.^ The Consistent Force Field and Its Quantum Mechanical Extension, A.Warshel in Modern Theoretical Chemistry, Vol. 7, edited by G. A. Segal, Plenum Press, New York, 19777.^ Warshel and Levitt, "Theoretical Studies of Enzymatic Reactions:Dielectric Electrostatic and Steric Stabilization of the Carbonium Ion in the Reaction of Lysozyme", J. Mol. Biol. 103 (1976), 2278.^ Kamerlin, Haranczyk and Warshel, "Progress in ab initio QM/MMfree-energy simulations of electrostatic energies in proteins:accelerated QM/MM studies of pKa, redox reactions and solvation free energies.", J. Phys. Chem. B. 113 (2009), 12539.^Billeter, SR; SP Webb, PK Agarwal, T Iordanov, S Hammes-Schiffer (2001)."Hydride Transfer in Liver Alcohol Dehydrogenase: Quantum Dynamics, Kinetic Isotope Effects, and Role of Enzyme Motion". J Am Chem Soc123: 11262–11272. doi:10.1021/ja011384b.∙[1]Altoè P, Stenta M, Bottoni A, Garavelli M. (2007). "A tunable QM/MM approach to chemical reactivity, structure andphysico-chemical properties prediction". Theor. Chem. Acc.118: 219–240. doi:10.1007/s00214-007-0275-9.∙Gao, J; Truhlar, Dg (2002). "Quantum mechanical methods for enzyme kinetics". Annual review of physical chemistry53: 467–505.doi:10.1146/annurev.physchem.53.091301.150114. PMID11972016.A Hybrid QuantumMechanical/Molecular MechanicalApproachHybrids are popular from the ancient time:So, it was a logical step to combine Quantum Chemical and Molecular Mechanical methods.Why Do We Need a Hybrid QM/MMApproach?Quantum chemical methods are generally applicable and allow the calculation of ground and excited state properties (molecular energies and structures, energies and structures of transition states, atomic charges, reaction pathways etc.)Molecular Mechanical methods are restricted to the classes of molecule it have been designed for and their success strongly depends on the careful calibration of a large number of parameters.The main bottleneck of quantum chemical methods is that they are CPU and memory hungry.For example, for a small peptide of 126 atoms one energy evaluation requires:*Semi-empirical PM3 methodIn general, CPU and memory requirements are:where N is a number of atoms.The development of the hybrid QM/MM approaches is guided by the general idea that large chemical systems may be partitioned into an electronically important region which requires a quantum chemical treatment and a remainder which only acts in a perturbative fashion and thus admits a classical description:The Simplest Hybrid QM/MM ModelHamiltonian for the molecular system in the Born-Oppenheimer approximation:In the presence of the external charges we have two additional terms in the Hamiltonian:Using this simple model we shall be able to calculate effect of external charges on our quantum chemical system:Computed Molecular Dipole Moments (Debye Units) in the gas phase and in water (H2O molecules are treated by MM Method) using AM1 method (J. Gao, p.Chem. 18(1997), 1061)Application:MODELING OF ADSORPTION PROPERTIES OF ZEOLITESThe main drawbacks of this simple QM/MM model are:it is impossible to optimize the position of the QM part relative to the external charges because QM nuclei will collapse on the negatively charged external charges.some MM atoms possess no charge and so would be invisible to the QM atomsthe van der Waals terms on the MM atoms often provide the only difference in the interactions of one atom type versus another, i.e. chloride and bromide ions both have unit negative charge and only differ in their van der Waals terms.So, it is quite reasonable to attribute the van der Waals parameters (as it is in the MM method) to every QM atom and the Hamiltonian describing the interaction between the QM and MM atoms can have a form:The van der Waals term models also electronic repulsion and dispersion interactions, which do not exist between QM and MM atoms because MM atoms possess no explicit electrons. Such form of the Hamiltonian was suggested for the first time by A. Warshel and M. Levitt (A. Warshel, M. Levitt // TheoreticalStudies of Enzymic Reactions: Dielectric, Electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme. //J.Mol.Biol. 103(1976), 227-49)Now we can construct a "real" hybrid QM/MM Hamiltonian:A "standard" MM force field can be used to determine the MM energy. For example, AMBER-like force field has a form:Choice of QM method... is a compromise between computational efficiency and the desired chemical accuracy.The main advantage of semiempirical QM methods is that their computational efficiency is orders of magnitude greater than either the density functional or ab initio methods.The use of ab initio methods limits the types of QM/MM studies that may be conducted at a single (X-ray structure) geometry.J. Bajorath et al., Proc.Natl.Acad.Sci. USA, 88(1991), 6423. J. Bajorath et al., Proteins: Struct.Funct.Genet., 9(1991), 217. J. Bajorath et al., Proteins: Struct.Funct.Genet., 11(1991), 263.In contrast, a number of QM/MM calculations have now been performed using semiempirical QM methods to compute enzymic reaction mechanisms, potentials of mean force, and solvation energies using minimization and Monte Carlo techniques. J. Gao and X. Xia, Science, 258(1992), 631V.V. Vasilyev, J.Mol.Struct. (Theochem), 304(1994), 129.M.A. Thomson, J.Am.Chem.Soc., 117(1995), 11341.Calibration of the QM/MM potential Crucial aspect is how the interaction between QM and MM parts is determined.In choosing the appropriate form, it is required that the balance between attractive and repulsive forces must be preserved and the QM/MM interactions must be of the correct magnitude with respect to the separate QM and MM contributionsParameterization1) Modification of the one-electron terms arising from interaction of the electron cloud of the QM fragment with the point charge of an MM atom.2) By varying the radii in the van der Waals terms.3) By varying 1) + 2)Calibration1) By hand, to find the optimum values of the parameters by calculating interaction curves for charge/ion systems and comparing them with the MP2/6-311++G** ab initio results.M.J. Field, P.A. Bash, M. Karplus, p.Chem., 11(1990), 700-733.2) Fitting calculated H-bond energies to experimental data on ion-molecular complexes in the gas phase.V.V. Vasilyev, A.A. Bliznyuk, A.A. Voityuk, Int.J.Quant.Chem. 44(1992), 897-930.3) Optimizing van der Waals parameters on QM atoms to reproduce the 6-31G(d) interaction energies for H-bonded complexes in the gas phase.P.A. Bash, L. Lawrence, A.D. MacKerell, Jr., D. Levine, P. Hallstrom, PNAS USA, 93(1996), 3698-703.4) Optimizing van der Waals parameters on QM atoms to reproduce the MP2/6-31G(dp) interaction energies for H-bonded complexes in the gas phase.J. Gao // Toward a molecular Orbital Derived Empirical Potential for Liquid Simulations // J.Phys.Chem. B 101(1997), 657-635) By varying the radii in the van der Waals terms to reproduce experimental free energies ofsolvation using MD simulations.P.L. Cummins, J.E. Gready, p.Chem., 18(1997), 1496-512.Special Case: Dividing Covalent Bonds across the QM and MM Regions1) Using a hybrid orbital on the frontier MM atomA. Warshel, M. Levitt // Theoretical Studies of Enzymic Reactions: Dielectric, Electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme. // J.Mol.Biol. 103(1976), 227-249V. Thery, D. Rinaldi, J.-L. Rivail, B. Maigret, G.G. Ferenczy, Jp.Chem. 15(1995), 2691) Using "link" atoms (hydrogens, halogens)"Link" atoms are uselectron density.(1) For QM region,Its sigma and epsilit interacts withelectrostatically.(2) Charge for "linto avoid double couinteractions.(3) WdV interactionwhich form 1-2 and 1calculated.(4) Bond stretchintorsion interactioregions are calcul1-2, 1-2-3, or 1-2least one MM atomReferencesReviewsJ. Gao, Reviews in Comp.Chem., 7(1996), 119-185- Review on QM/MM[Home] [E-mail me]。

线虫鉴定通用引物

The primer pairs used were D2A (5 -aCaagtaccgtgagggaaagttg -3' ) and D2B(5 -aatccgtgtttcaagacggg- 3' ), D3A (5-gdcccgtcttgaaacacgga -3' ) and D3B(5 -tcggaaggaaccagctacta- 3' ) (Nunn, 1992), and mtA (5 -ggcggatcctacatcgatgttgtat- 3' ) andmtB(5 -ggcggatccwkttcctctcgtact- 3' ). These primers selectively amplify metazoan ribosomal DNAs (rDNA) and do not amplify com mon con tam inants such as bacteria, fun gi, or pla nt material.Courtright EM, Wall DH, Virgi nia RA, et al. Nuclear and mitocho ndrial DNA seque nee diversity in the An tarctic n ematode Scott nema lin dsayae. Journal of Nematology, 2000, 32(2): 143.DNA extract ion and polymerase cha in react ion (PCR) assays were performed accord ing toSubbotin et al. (2000). The ITS1-5.8S-ITS2 and the D2-D3 of 28S of rDNA were amplified usi ng the following primer sets: 5367 (5 '-ttgattacgtccctgcccttt-3 ') and F195 (5 '-tcctccgctaaatgatatg-3 ); and D2A (5 -acaagtaccgtgagggaaagttg-3 'and D3B (5 C cggaaggaaccagctacta-3 'as described by Schmitz et al. (1998) and De Ley et al. (1999), respectively.Castillo P, Vovlas N, Subbotin S, et al. A new root-knot nematode, Meloidogyne baetica n.sp.(Nematoda: Heteroderidae), parasitizing wild olive in Southern Spain. Phytopathology, 2003 , 93(9): 1093-1102.D3A(5'-gacccgtcttgaaacacgga -3' ) and D3B (5 3').Al-Ba nna L, Ploeg A T, Williams on V M, et al. Discrimi natio n of six Pratyle nchus species usingPCR and species-specific primers. Journal of nematology, 2004,36(2): 142.The nu clear ribosomal internal tran scribed spacer (ITS1) segme nt was amplified with the primers rDNA2 5 '-ttgattacgtccctgcccttt -3' (Vrain et al. 1992) and rDNA1.58S 5'-acgagccgagtgatccaccg- 3' (Cherry et al. 1997). The ribosomal LSU D2-D3 expansion segment was amplified with primersD2A 5'-acaagtaccgtgagggaaagttg -3' and D3B5'-tcggaaggaaccagctacta- 3' (Courtright et al.,2000) as previously described (Al-Ba nna et al., 1997).Ska ntar AM, Carta LK. Multiple displaceme nt amplificatio n (MDA) of total gen omic DNA from Meloidogyne spp. and comparison to crude DNA extracts in PCR of ITS1,28S D2-D3 rDNA and Hsp90. Nematology, 2005, 7(2): 285-294.Several n ematode specime ns of each sample were tran sferred to an Eppe ndorf tube containing16 ul ddH 2O, 2 ul 10X PCR buffer and 2 ul proteinase K (600 ug/ml) (Promega, Benelux, The Netherla nds) and crushed for 2 min with a Vibro Mixer microhomoge ni ser (Z rich, Switzerla nd). The tubes were incubated at 65o C (1 h) and then at 95?C (15 min) . Detailed protocols for PCR, cloning and automated sequencing are described by Tanha Maafi et al. (2003). The forward D2A (5 '-acaagtaccgtgagggaaagttg -3 'and reverse D3B (5 '-tcggaaggaaccagctacta-3 'primers wereused for amplification and sequencing of the fragment of the 28S rRNA gene.Subbotin SA, Vovlas N, Crozzoli R, et al. Phylogeny of Criconematina Siddiqi, 1980 (Nematoda: Tyle nchida) based on morphology and D2-D3 expa nsion segme nts of the 28S-rRNA gene seque nces with applicati on of a sec on dary structure model. Nematology, 2005, 7(6): 927-944.The different regions of rDNA were amplified as described by Castillo et al.(2003) and Tigano et al. (2005) using the following primer sets: MelF (5 -tacggactgagataatggt- 3' ) and MelR(5'-ggttcaagccactgcga- 3' for the 18S , 5367 (5 -ttgattacgtccctgcccttt -3' ) ancF195(5-tcctccgctaaatgatatg- 3' ) for thelTSI -5.8S-ITS2 , D2A (5 '-acaagtaccgtgagggaaagttg- 3' ) and D3B (5 '-tcggaaggaaccagctacta- 3' ) for theD2-D3 region of 28S .Rius JEP, Vovlas N, Troccoli A, et al. A new root-knot nematode parasitizing sea rocket fromSpanish Mediterra nean coastal dun es: Meloidog yne dunen sis n. sp. (Nematoda: Meloidogy ni dae). Journal of nematology, 2007,39(2): 190.Cloning, characterisati on and heterologous expressi on of an astac in metalloprotease, Sc-AST,from the entomoparasitic nematode Steinernema carpocapsaeY Ji ng, D Toubarro, Y Hao, N Sim? es - Molecular and biochemical …,200 - Elsevier... Total RNA from different nematode stages and from nematodes induced for 0, 6, 12, 24, 36, 48 and 72 h was ... Primers for 18S rRNA were 18SF (5 -GCTAATCGGAAACGAAAGTC- 3' ) and 18SR (5 '-CATCCACCGAATCAAGAAAG- 3' )Primers for Sc-AST were ASTF1 (5 ...5.8SF194/F195: Tm55 CTW81/AB28:18S:Me1f/Me1r: Tm 50 C18SF/18SR:28S:D2A/D3B: Tm 55 CmtDNAC2F3/1108: Tm 50 CC2F3/MRH106: Tm 50 CIncorporating molecular identification of Meloidogyne spp. into a large-scale regional n ematode surveyTO Powers, PG Mullin, TS Harris, LA Sutton - -Journal of …,2005 ... For M. chitwoodi ide ntificati on, the first of two PCR amplificati ons was con ducted with primer setC2F3 / 1108 (5 GGTCAATGTTCAGAAATTTGTGG 3 and 5 TACCTTTGACCAATCACGCT 3) located in the COII and 16S ribosomal mitoch on drial gen es, respectively (Powers andBiometrical, biochemical, and molecular diag no sis of Portuguese Meloidog yne hispa nica isolatesCM Maleita, MJ Sim? es, C Egas, RHC Curtis ••- Plant …,2012 - Am Phytopath Society... Mitochondrial DNA from isolates PtHi3 of M. his- panica and ItE of M. ethiopica were sequeneed with the primer set C2F3 (5 -GGT CAA TGT TCA GAA ATT TGT GG- 3' ) and MRH106 (5 -AAT TTC TAA AGA CTT TTC TTA GT- 3' ) located in the COII gene and the 16S rRNA[PDF] Prevale nee, in cide nee and molecular ide ntificati on of root-k not n ematodes of tomato in Pakista nM Ahmed - Africa n Jour nal of Biotech no logy, 2012 - academicjour ... DNA amplificatio n of mtDNA with C2F3 /1108 primers yielded a 1700 bp size productfor all three species of RKNs in comparison with 520 and 750 bp for M. chitwoodiand en terolobii, respectively, which were utilized as con trol.Plant-parasitic nematodes in sugarcane fields in Kitadaito Island (Okinawa), Japan, as a pote ntial sugarca ne growth in hibitorM Kawanobe, N Miyamaru, K Yoshida … -…,2014 - ... Nematode identification using DNA sequenee data Single nematodes were handpicked usinga sterilised needle, rinsed with distilled water and placed on a glass slide. ... F194 (5-CGTAACAAGGTAGCTGTAG- 3 ) and F195 (5 -TCC TCC GCT AAA TGA TAT G-3 ) were■■一—二一一一「二*115 蹶Fig. L. Jxjcatiom an iDNA of the inicmal tmnscribed 叩HTfi}primers used in (hjs study. The neginjis iti bvcwccn ihc iDNA gcne& (IKS. 5 HS nnd28S)are (tie I IS. The and 2SS genes ait rrancJitcd.占Detection of the pinewood nematode , Bursaphelenchus xylophilus, using a real-time polymerase chain reaction assayAX Cao, XZ Liu, SF Zhu, BS Lu - Phytopathology, 2005 - Am Phytopath Society... One to four n ematodes were placed into 15 l ofidouble-distilled water on ... sterile 0.5-ml thi n- walled PCR tube containing 8 l of nematode lysis buffer ... Genus-specific primers F194 (5 - CGTAACAAGGTAGCTGTAG- 3 ' ) and5368 (5 -TTTCACTCGCCGTTACTAAGG- 3 ' ) wereDe Luca F, Troccoli A, Duncan L W, et al. Pratylenchus speijeri n. sp.(Nematoda: Pratylenchidae), a new root-lesion nematode pest of plantain in West Africa. Nematology, 2012, 14(8): 987-1004.IGS sequenee variation, group-I introns and the complete nuclear ribosomal DNA of the en tomopathoge nic fun gus Metarhizium: excelle nt tools for isolate detect ion MP Pan tou, A Mavridou, MA Typas - Fun gal Ge netics and Biology, 2003 - Elsevier... 18SF, GCGAAACTGCGAATGGCT, This work. 18SR, GTAATGATCCCTCCGCTG, This work.TW81 , GTTTCCGTAGGTGAACCTGC, Curran et al. (1994). AB28 , ATATGCTTAAGTTCAGCGGGT, Curra n et al. (1994). Ma-ITS2, GTCCACTGCCGTAAAACCCC, This work.Heterodera vallicola sp. n. (Tyle nchida: Heteroderidae) from elm trees, Ulmus jap onica (Rehd ) Sarg in the Primorsky terr计ory, the Russian Far East, with rDNA AS Eroshenko, SA Subbotin …-Russian Journal of …,... DNA fragments were sequeneed in both directions with TW81 , AB28 , 5.8SM2 (5'- CTTATCGGTGGATCACTCGG-3') or 5.8SM5 (5'-GGCGCAATGTGCATTCGA-3') primers with a BigDye Termin ator Cycle Seque ncing Ready React ion Kit (PE Applied Biosystems, UK)Nematode universal primers:Gymnodinium nolleri Ellegaard et Moestrup sp. ined.(Dinophyceae) from Danish waters, a new species producing Gymnodinium catenatum- like cysts: molecular and …M Ellegaard, Y Oshima - Phycologia, 1998 - ... 1989) conserved positions 708-727 (D3A; 5' GACCCGTCTTG AAA CACGGA-3') and 1011-992(D3B; 5' TCGGAAGGAACCAGCTACTA -3'). Double-stranded and sin gle-stranded PCR amplifications were performed in a ther mocycler with an initial denaturation step of 3 min atNuclear and mitochondrial DNA sequence diversity in the Antarctic nematode Scottnema lindsayaeEM Courtright, DH Wall, RA Virginia … - Journal of …, 200-0ncbi.n lm.n ... The primer pairs used were D2A (5- ACAAGTACCGTGAGGGAAAGTTG -3) and D2B(5-AATCCGTGTTTCAAGACGGG-3), D3A(5-GACCCGTCTTGAAA- CACGGA-3) and D3B (5-TCGGAAG- GAACCAGCTACTA-3) (Nunn, 1992), and mtA (5-GGCGGATCCTACATCGATGTTGAmiri S, Subbotin S A, Moens M. Identification of the beet cyst nematode Heterodera schachtii by PCR. European Journal of Plant Pathology, 2002, 108(6): 497-506.。