Amperometric detection of gold by differential pulse voltammetry using a DNA biosensor

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Microarray-Based Multiplexed ScanometricImmunoassay for Protein Cancer Markers Using Gold Nanoparticle ProbesDongwoo Kim,Weston L.Daniel,and Chad A.Mirkin*Department of Chemistry and International Institute for Nanotechnology,Northwestern University,2145Sheridan Road,Evanston,Illinois 60208-3113We report the use of electroless gold deposition as a light scattering signal enhancer in a multiplexed,microarray-based scanometric immunoassay using gold nanoparticle probes.The use of gold development results in greater signal enhancement than the typical silver development,and multiple rounds of metal development were found to increase the resulting signal compared to one ing these conditions,the assay was capable of detecting 300aM (∼9000copies)of prostate speciﬁc antigen in buffer and 3fM in 10%serum.Additionally,the highly selective detection of three protein cancer markers at low picomolar concentrations in buffer and 10%serum was demonstrated.The use of gold deposition may have signiﬁcant utility in scanometric detection schemes and broader clinical and research applications.Sensitive,rapid,and selective immunoassays capable of mul-tiplexed protein detection are critical for clinical applications.1For instance,in many kinds of cancers,following the disease during the course of and after treatment requires the detection of multiple protein markers.2,3Despite the importance of these assays,the standard for protein detection,the enzyme-linked immunosorbent assay (ELISA),is often not sensitive enough to diagnose some diseases.4,5In addition,multiplexed detection with ELISA has drawbacks such as overlapping spectral features and the need for complex instrumentation for signal readout.6Antibody microarrays have emerged as a promising method for multiplexed detection of protein biomarkers.6-8Typically,these microarrays are functionalized with capture antibodies,which bind the protein targets.Next,a second ﬂuorophore-labeled antibody binds the targets forming a sandwich structure detectable with typical DNA microarray detection instrumentation.Onelimitation of the technique is its sensitivity.9The use of ampliﬁca-tion methods,such as immuno-PCR or rolling circle ampliﬁcation,have been used to enhance sensitivity 9but require complicated,multistep protocols.10Polyvalent oligonucleotide gold nanoparticle (Au NP)conju-gates 11have been utilized as probes for nucleic acids,12-14proteins,15-17metal ions,18-20and cancerous cells.21In addition,these conjugates are both extraordinarily sensitive and selective labels for microarray-based DNA detection.22-24This assay,called the scanometric assay,has since become an FDA-approved detection method and has spurred the development of many related assays.16,25,26The key to its high sensitivity is the ability to amplify the light scattering of the Au NP probes with electroless silver deposition.In separate but related experiments,immuno-blots using antibody Au NP conjugates as probes have shown that gold deposition gives greater signal ampliﬁcation than silver deposition.27Given these advances,we sought to combine the multiplexing utility of protein microarrays,the high sensitivity of*To whom correspondence should be addressed.Phone:847-467-7302.Fax:847-467-5123.E-mail:chadnano@.(1)Kodadek,T.Chem.Biol.2001,8,105–115.(2)Ferrari,M.Nat.Rev.Cancer 2005,5,161–171.(3)Sidransky,D.Nat.Rev.Cancer 2002,2,210–219.(4)Barletta,J.M.;Edelman,D.C.;Constantine,N.T.Am.J.Clin.Path.2004,122,20–27.(5)Maia,M.;Takahashi,H.;Adler,K.;Garlick,R.K.;Wands,J.R.J.Virol.Methods 1995,52,273–286.(6)MacBeath,G.Nat.Genet.2002,32Suppl ,526–532.(7)Angenendt,P.Drug Discovery Today 2005,10,503–511.(8)Ekins,R.P.Clin.Chem.1998,44,2015–2030.(9)Schweitzer,B.;Roberts,S.;Grimwade,B.;Shao,W.P.;Wang,M.J.;Fu,Q.;Shu,Q.P.;Laroche,I.;Zhou,Z.M.;Tchernev,V.T.;Christiansen,J.;Velleca,M.;Kingsmore,S.F.Nat.Biotechnol.2002,20,359–365.(10)Niemeyer,C.M.;Adler,M.;Wacker,R.Trends Biotechnol.2005,23,208–216.(11)Mirkin,C.A.;Letsinger,R.L.;Mucic,R.C.;Storhoff,J.J.Nature 1996,382,607–609.(12)Elghanian,R.;Storhoff,J.J.;Mucic,R.C.;Letsinger,R.L.;Mirkin,C.A.Science 1997,277,1078–1081.(13)Storhoff,J.J.;Elghanian,R.;Mucic,R.C.;Mirkin,C.A.;Letsinger,R.L.J.Am.Chem.Soc.1998,120,1959–1964.(14)Seferos,D.S.;Giljohann,D.A.;Hill,H.D.;Prigodich,A.E.;Mirkin,C.A.J.Am.Chem.Soc.2007,129,15477–15479.(15)Nam,J.M.;Park,S.J.;Mirkin,C.A.J.Am.Chem.Soc.2002,124,3820–3821.(16)Nam,J.-M.;Thaxton,C.S.;Mirkin,C.A.Science 2003,301,1884–1886.(17)Zheng,G.F.;Daniel,W.L.;Mirkin,C.A.J.Am.Chem.Soc.2008,130,9644–9645.(18)Lee,J.S.;Han,M.S.;Mirkin,C.A.Angew.Chem.,Int.Ed.2007,46,4093–4096.(19)Liu,J.;Lu,Y.J.Am.Chem.Soc.2003,125,6642–6643.(20)Li,D.;Wieckowska,A.;Willner,I.Angew.Chem.,Int.Ed.2008,47,3927–3931.(21)Medley,C.D.;Smith,J.E.;Tang,Z.;Wu,Y.;Bamrungsap,S.;Tan,W.H.Anal.Chem.2008,80,1067–1072.(22)Taton,T.A.;Lu,G.;Mirkin,C.A.J.Am.Chem.Soc.2001,123,5164–5165.(23)Taton,T.A.;Mirkin,C.A.;Letsinger,R.L.Science 2000,289,1757–1760.(24)Cao,Y.W.C.;Jin,R.C.;Mirkin,C.A.Science 2002,297,1536–1540.(25)Xu,X.Y.;Georganopoulou,D.G.;Hill,H.D.;Mirkin,C.A.Anal.Chem.2007,79,6650–6654.(26)Niemeyer,C.M.;Ceyhan,B.Angew.Chem.,Int.Ed.2001,40,3685–3688.(27)Ma,Z.F.;Sui,S.F.Angew.Chem.,Int.Ed.2002,41,2176–2179.Anal.Chem.2009,81,9183–918710.1021/ac9018389CCC:$40.75 2009American Chemical Society 9183Analytical Chemistry,Vol.81,No.21,November 1,2009Published on Web 10/09/2009Au NP conjugate-based detection systems,and the signal ampli-ﬁcation of Au NP initiated gold reduction and subsequent deposition.Herein,we present a simple,rapid,and extremely sensitive microarray-based protein detection method called the scanometric immunoassay that uses the light scattering of antibody-oligonucleotide Au NP conjugates and Au NP initiated gold deposition for signal readout.EXPERIMENTAL SECTIONGeneral.The Verigene Reader light scattering reader system, silver enhancing solutions,and10-well manual hybridization chambers were purchased from Nanosphere,Inc.Gold(III) chloride trihydrate(520918,Aldrich)and hydroxylamine hydro-chloride(159417,Aldrich)were used for preparing gold enhancing solution.Normal donkey serum(Chemicon International,Te-mecula,CA)was used as received.Assay Buffer Preparation.The assay buffer was prepared by adding500µL of a10%bovine serum albumin(BSA)solution (DY995,R&D Systems),500µL of an aqueous10%Tween20 solution(Sigma),and500mg of poly(acrylic acid)(420344,Sigma) to Dulbecco’s phosphate-buffered saline(PBS)buffer(Invitrogen) in aﬁnal volume of50mL.Antibodies and Antigens.The proteins used in the study were prostate speciﬁc antigen(PSA)(P3338,Sigma-Aldrich),the spotted PSA antibody(ab403,Abcam),the Au NP PSA antibody (AF1344,R&D Systems),R-fetoprotein antigen(APF)(A32260H, Biodesign International),the spotted AFP antibody(10-A05,clone M19301,Fitzgerald Industries International,Inc.),the Au NP AFP antibody(70-XG05,Fitzgerald Industries International,Inc.), human chorionic gonadotropin(HCG)(A81355M,Biodesign International),the spotted HCG antibody(E20579,Biodesign International),and the Au NP-monoclonal HCG antibody(E20106, Biodesign International).Preparation of Antibody and Oligonucleotide Modiﬁed Gold Nanoparticles(Au NPs).Thirteen±1nm Au NPs were synthesized by the Frens method,28resulting in∼10nM solutions. The3′-propylthiol-T24-decanoic acid oligonucleotide was syn-thesized with standard phosphoramidite chemistry reagents purchased from Glen Research and puriﬁed with ion exchange HPLC.The oligonucleotide Au NP conjugates were synthesized by incubating3µM of the oligonucleotide with the as-synthesized Au NPs.The conjugates were salted using litera-ture procedures29to aﬁnal concentration of1.0M NaCl and puriﬁed via repeated centrifugation and resuspension in0.01% Tween20in water.The antibodies were conjugated to the oligonucleotide modiﬁed Au NPs with1-ethyl-3-(3-dimethy-laminopropyl)carbodiimide(EDC)and sulfo N-hydroxysuccin-imide(NHS).In this procedure,10µL of0.01%Tween20 solutions containing0.5pmol of the particles were prepared. FiveµL of a30mM sulfo-NHS solution in a0.1M2-(N-morpholino)ethanesulfonic acid(MES)buffer at pH5,followed by5µL of15mM EDC solution in0.1M MES were added to these particles.This mixture was agitated for15min,and then, the particles were puriﬁed from excess reagent via centrifuga-tion and resuspension three times in5mM MES buffer supplemented with0.01%Tween20.After theﬁnal centrifuga-tion and supernatant removal,the particles were isolated in10µL of oily suspension.To this solution,5µg of antibodies in 10mM PB were added from a1mg/mL st,5µL of 0.1M phosphate buffer(PB),pH7.4,were added to the mixture,and the solution was agitated overnight at room temperature.The conjugates were puriﬁed by repeated cen-trifugation and resuspension in Dulbecco’s PBS with0.025% Tween20and0.1%BSA.Finally,BSA was added to aﬁnal concentration of1%,and the conjugates were passivated overnight.Microarray Preparation.An arrayer equipped with125µm diameter pins(GMS417,Affymetrix)was used for the preparation of the antibody microarrays.The microarrays were fabricated by spotting250µg/mL solutions of the antibodies in0.1M phosphate buffer(PB),pH8.0,with150mM NaCl and0.001%Tween20on to the surface of NHS ester-activated Codelink slides(SurModics Inc.).Six replicate spots for single analyte detection or three replicate spots of each antibody for multiplexed detection were arrayed at deﬁned locations.The slides were then incubated overnight at4°C under an N2atmosphere.They were then passivated by incubating them with a0.2%(v/v)solution of ethanolamine(411000,Aldrich)in50mM borate buffer,pH) 9.5overnight at4°C.Finally,they were then washed with Nanopure water(>18MΩ,Barnstead International)and spin-dried for one minute.The microarray of the oligonucleotide-modiﬁed Au NP conju-gates for SEM imaging was prepared by spotting∼400conjugates to the surface of glass slides(Codelink,SurModics).30Three replicate spots were arrayed at deﬁned locations.The glass slides were dried,and the diameters of Au NP probes were increased with silver or gold staining solution,gently washed with Nanopure water,and spin-dried.The slides were sputtered with20nm of Au/Pd before imaging.All scanning electron microscopy(SEM) images were obtained using a LEO Gemini SEM.Scanometric Immunoassay Protocol.The antibody mi-croarray was assembled with a10-well manual hybridization chamber.Antibody spots on the microarray were arrayed at deﬁned locations across the glass slides so that multiple tests could be performed on the single slide by isolating reaction sites with silicone gaskets to create individual wells.Each well of the chamber wasﬁlled with50µL of antigen solution and allowed to incubate for1h at room temperature with shaking at1200rpm. After washing the chambers three times with assay buffer,50µL of150pM Au NP probes in assay buffer were incubated with the slides for1h at room temperature.The concentration of each of the Au NP probes was150pM in multiplexed detection experi-ments.The chamber was again washed three times and then disassembled.The slide was rinsed with Dulbecco’s PBS with0.1% Tween20and Nanopure water and spin-dried for one minute.The slide was then developed with silver or gold enhancing solution (1:1(v:v)mixture of5mM HAuCl4and10mM NH2OH)for5 min and imaged with a Verigene Reader system.RESULTS AND DISCUSSIONAs a proof-of-concept detection experiment,we created a microarray sandwich assay for prostate speciﬁc antigen(PSA),(28)Frens,G.Nature-Phys.Sci.1973,241,20–22.(29)Hurst,S.J.;Lytton-Jean,A.K.R.;Mirkin,C.A.Anal.Chem.2006,78,8313–8318.(30)Andreeva,L.V.;Koshkin,A.V.;Letiedev-Stepanov,P.V.;Petrov,A.N.;Alﬁmov,M.V.Colloids Surf.,A2007,300,300–306.9184Analytical Chemistry,Vol.81,No.21,November1,2009Scheme 1.PSA was chosen as an initial analyte because of its importance as a prostate cancer marker,31and since many assays have been developed for this analyte,16,32-36there is a good basis for comparison.In a typical experiment,an antibody microarray was fabricated by spotting monoclonal capture antibodies to the surface of N -hydroxysuccinimide-activated glass slides (CodeLink,SurModics).Six spots,all with antibodies for PSA,were used in each assay well.The use of six spots allow one to obtain statistically signiﬁcant data in each assay.The slides were then passivated with ethanolamine.Probes were prepared by ﬁrst modifying 13nm diameter Au NPs with 3′-propylthiol and 5′-decanoic acid modiﬁed oligonucleotides and then covalently immobilizing antibodies for PSA via carbodiimide coupling.37The assay began by incubating the test solution with PSA at a designated concentration for 1h at room temperature on the chip with capture antibodies (assay buffer:Dulbecco’s PBS with 0.1%Tween 20,0.1%BSA,and 1%poly(acrylic acid)).Since each chip had ten different wells (in addition to six capture spots in each well),multiple separate assays can be carried out at once (top tobottom,Figure 1).After washing the slide with assay buffer,150pM of the Au NP probes in assay buffer were incubated with the microarray-bound targets for 1h at room temperature.The slides were washed again.To increase the light scattering signal of the immobilized Au NP probes,gold or silver was catalytically deposited on them using electroless deposition techniques (left to right,Figure 1).Finally,the light scattering was quantiﬁed with a Verigene Reader system,which is a device that captures evanescent wave-induced light scattering from the ampliﬁed Au NPs.In a conventional scanometric detection experiment,electroless silver deposition is used to grow Au NP probes on oligonucleotide microarrays,23Figure 1a.When PSA was used as the analyte under the conditions described above,the limit of detection (LOD)is 3pM when silver was the amplifying agent.Interestingly,the silver-plated Au NP conjugates could be used as nucleation agents to perform a second silver deposition on the same microarray,which improves the LOD to 30fM,Figure 1b.Others have shown that a second round of silver development increases the limit of detection of immunoblots 27and immunosorbent assays.38The increase in signal arises from particle growth (vide infra),because on the nano-and microscale light scattering intensity increases dramatically with particle diameter.39A third round of silver deposition did not signiﬁcantly improve the assay LOD due to increased background signal (data not shown).Methods of electroless deposition using HAuCl 4and NH 2OH have been used to increase the diameter of Au NPs in solution 40(31)Lilja,H.;Ulmert,D.;Vickers,A.J.Nat.Rev.Cancer 2008,8,268–278.(32)Oh,B.K.;Nam,J.M.;Lee,S.W.;Mirkin,C.A.Small 2006,2,103–108.(33)Schweitzer,B.;Wiltshire,S.;Lambert,J.;O’Malley,S.;Kukanskis,K.;Zhu,Z.R.;Kingsmore,S.F.;Lizardi,P.M.;Ward,D.C.Proc.Natl.Acad.Sci.U.S.A.2000,97,10113–10119.(34)Yu,X.;Munge,B.;Patel,V.;Jensen,G.;Bhirde,A.;Gong,J.D.;Kim,S.N.;Gillespie,J.;Gutkind,J.S.;Papadimitrakopoulos,F.;Rusling,J.F.J.Am.Chem.Soc.2006,128,11199–11205.(35)He,L.;Musick,M.D.;Nicewarner,S.R.;Salinas,F.G.;Benkovic,S.J.;Natan,M.J.;Keating,C.D.J.Am.Chem.Soc.2000,122,9071–9077.(36)Goluch,E.D.;Nam,J.M.;Georganopoulou,D.G.;Chiesl,T.N.;Shaikh,K.A.;Ryu,K.S.;Barron,A.E.;Mirkin,C.A.;Liu,b Chip 2006,6,1293–1299.(37)Hermanson,G.T.Bioconjugate Techniques ;Academic Press:San Diego,CA,1996.(38)Shim,S.Y.;Woo,J.R.;Nam,E.J.;Hong,H.J.;Mook-Jung,I.;Kim,Y.H.;Nam,J.M.Nanomedicine 2008,3,485–493.(39)Jain,P.K.;Lee,K.S.;El-Sayed,I.H.;El-Sayed,M.A.J.Phys.Chem.B2006,110,7238–7248.(40)Brown,K.R.;Natan,ngmuir 1998,14,726–728.Scheme 1.Scanometric Immunoassay9185Analytical Chemistry,Vol.81,No.21,November 1,2009and in immunoblots.27Interestingly,a microarray developed with these reagents resulted in an LOD of 30fM,comparable to that of two sequential silver depositions,Figure 1c.An additional treatment with the gold development solution improved the LOD to 300aM,Figure 1d.A third deposition of gold increased the light scattering signal but did not improve the LOD due to increased background signal,Figure 1e.Experiments where combinations of gold and silver deposition were used resulted in limits of detection less than that of two gold depositions (data not shown).As one moves to more complex matrixes,assay LODs are often challenged due to increased background.When this assay was carried out in 10%serum,the LOD was 3fM with two gold depositions,Figure S1in the Supporting Information.This LOD is approximately 3orders of magnitude lower than that of commercially available ELISA assays for PSA (approximately picomolar concentration).41One of the unique features of a multistage development is that it allows for quantiﬁcation over a large concentration range in addition to increased sensitivity.With one gold deposition,the dynamic range of this assay in buffer is between 30fM and 3pM,and with two,it is between 300aM and 300fM,Figure 1.Therefore,with two serial gold depositions,this scanometric assay is capable of PSA detection over a 4order of magnitude concentration range.To better understand the reason why multiple gold depositions provide better signal than one silver deposition,the growth of Au NP probes were investigated by scanning electron microscopy(SEM)after various metal deposition procedures.In a typical experiment,a microarrayer was used to deposit ∼400Au NPs per spot on glass slides,30and then,the size of the Au NP probes were measured after silver or gold development.With silver,the average diameters of the probes were 100±25,270±130,and 550±140nm after one,two,and three developments,respectively.With gold,the developed probe diameters are 420±100,1400±470,and 2700±710nm after one,two,and three depositions,respectively.These data indicate that repeated metal depositions increase the average probe rger nano-and micro-structures scatter light better than smaller ones,39,42,43which correlates with increased light scattering intensity as seen in Figure 1.The greater signal ampliﬁcation observed when gold deposition is used versus silver deposition likely arises from their different growth mechanisms.Typically,silver deposition causes the auto-catalytic reduction of silver on the Au NPs,23increasing the size of the structure,which results in signal enhancement,Figure 2a.The gold development solution,however,likely leads to the continuous nucleation of new Au NPs by the probe Au NPs in addition to autocatalytic growth.These newly nucleated particles aggregate on the probe Au NPs,resulting in signal enhancement and gold microstructures that are larger than those developed(41)Ward,A.M.;Catto,J.W.F.;Hamdy,F.C.Ann.Clin.Biochem.2001,38,633–651.(42)Yguerabide,J.;Yguerabide,E.E.Anal.Biochem.1998,262,157–176.(43)Yguerabide,J.;Yguerabide,E.E.Anal.Biochem.1998,262,137–156.Figure 1.Scanometric identiﬁcation (left)and the corresponding quantization (right)of the net signal intensities of various concentrations of PSA in buffer after (a)one silver deposition,(b)two silver depositions,(c)one gold deposition,(d)two gold depositions,and (e)three gold depositions.The light scattering signal was saturated at 65536(216)units.The gray scale images from the Verigene Reader system were converted into colored ones using GenePix Pro 6software (Molecular Devices).The exposure time was 500ms.Figure 2.Representative SEM images of Au NP probes developed with (a)three silver depositions and (b)three gold depositions.9186Analytical Chemistry,Vol.81,No.21,November 1,2009by silver,Figure 2b.The nucleation of new particles by existing Au NPs has been observed in the seed-mediated synthesis of Au NPs.44After the origin of the increased signal using gold development was determined,the scanometric immunoassay was challenged with detecting three protein cancer markers using multiple gold depositions.Multiplexed protein analysis is becoming increasingly important for disease diagnosis,and high selectivity is critical for the success of multiplexing assays.2In a typical experiment,antibodies to PSA,human chorionic gonadotropin (hCG),a testicular cancer marker,and R -fetoprotein (AFP),a hepatic cancer marker,were spotted onto a microarray chip.Next,the target antigens were incubated in the wells.After washing,antibody modiﬁed oligonucleotide Au NPs speciﬁc for PSA,hCG,or AFP were used to sandwich the antigens.The selectivity of the system was tested by detecting eight different combinations of antigens.In the ﬁrst well,all three antigens were present.In the next seven wells,different combinations of targets were mixed.The concen-trations of each of the target antigens were kept constant at 1.4pM.After two serial gold depositions the presence of the target in each combination was clearly indicated by the high intensity signal,Figure 3.In the absence of the protein cancer marker,little signal was observed.This indicates that the assay is capable of highly selective antigen detection.The differences in spot intensity for the different antigens are likely a result of differences in the binding afﬁnity of the antibodies.45Finally,the assay demonstrated high selectivity in 10%serum,Figure S2in the Supporting Information.CONCLUSIONSIn conclusion,we have used multiple gold depositions as a signal enhancing mechanism in a simple,rapid,and ultrahigh sensitivity scanometric assay based on antibody microarrays and Au NP probes.Multiple gold depositions are an alternative light scattering ampliﬁcation tool for scanometric assays that provide greater signal than the typical single silver deposition.This greater signal arises because the developed probe diameters are much larger and,thus,scatter light better than probes developed by one silver deposition.Gold-developed structures are likely larger than silver developed structures due to the unique growth mechanism of gold deposition.Although this work focused on the detection of protein cancer markers,the use of multiple gold developments should improve the signal from any scanometric assay,including those for DNA,23metal ions,46and the biobarcode assay.16Ultimately,gold-based signal enhancement could have signiﬁcant utility in detection schemes as well as in broader clinical and research applications.ACKNOWLEDGMENTC.A.M.acknowledges the NCI-CCNE and the NSF for support of this work,and he is also grateful for a National Security Science and Engineering Faculty Fellows award.D.K.acknowledges the Korea Research Foundation Grant (KRF-2007-357-C00053)funded by the Korean Government (MOEHRD)for postdoctoral fellow-ship support.D.K.and W.L.D.contributed equally to this work.SUPPORTING INFORMATION AVAILABLEAdditional information as noted in text.This material is available free of charge via the Internet at .Received for review August 14,2009.Accepted September 23,2009.AC9018389(44)Jana,N.R.;Gearheart,L.;Murphy,C.J.Chem.Mater.2001,13,2313–2322.(45)Stoeva,S.I.;Lee,J.S.;Smith,J.E.;Rosen,S.T.;Mirkin,C.A.J.Am.Chem.Soc.2006,128,8378–8379.(46)Lee,J.S.;Mirkin,C.A.Anal.Chem.2008,80,6805–6808.Figure 3.Scanometric identiﬁcation of three protein cancer markers for eight different samples in buffer after two gold depositions.The concentration of each antigen was 1.4pM.(1)All targets present;(2)hCG and PSA;(3)hCG and AFP;(4)PSA and AFP;(5)AFP;(6)PSA;(7)hCG;(8)no targets present.The gray scale images from the Verigene Reader system were converted into colored ones using GenePix Pro 6software (Molecular Devices),and the exposure time was 200ms.9187Analytical Chemistry,Vol.81,No.21,November 1,2009。

基于纳米金表面等离子体的高灵敏过氧化氢光学检测方法

基于纳米金表面等离子体的高灵敏过氧化氢光学检测方法李娜娜;黄嘉荣;詹求强【摘要】A colorimetric method was developed for the determination of trace amount of H2 O2 in acidic 2-( N-mor-pholino) ethanesulphonic acid buffer medium, based on surface plasma of gold nanoparticles.The detecting sys-tem, whose limit of detecting resolution with naked eyes was measured previously, was used to accurately quantify the concentration( c) of H2 O2 by spectral analysis after stabilization enhancement.The results show that blue-col-oured solutions with aggregated gold nanoparticles could be obtained when c <100 μmol/L, while the solutions were red-coloured with non-aggregated gold nanoparticles when c>120μmol/L.The resolution limit of human eyes to this detection system was able to distinguish H2 O2 concen tration difference of 20 μmol/L.However, the color distinction of solutions was unstable and has a quick change in 45 mins.The blue solution with c of 60 to 100μmol/L turned red gradually.The OD570 of solutions also demonstrates that the reaction was constantly changing in 3 h after detection.Moderate L-glutathione was added to terminate the reaction after 10 min to improve the detec-tion stability, which can stabilize the color distinction and effectively keep a constant OD570 .The spectra of the nan-oparticle dispersions show the absorption peak at 550 nm and there is a positive correlation between intensity and the H2 O2 concentration when c<100 μmol/L.The absorption peak shifted to 540 nm when c>120 μmol/L.By the spectral analysis, a linear relativity ofregressive curves of OD630/545 and the concentration of H2 O2 was found. Then solutions with c from 100 to 120 μmol/L were detected and accurately quantified H2 O2 with concentration difference of 2μmol/L to break the limit of detecting resolution.T he detection system could provide a preferential platform for trace detection.%在酸性2-吗啉乙磺酸介质中，建立基于纳米金表面等离子体的微量过氧化氢（ H2 O2）的比色检测体系，探讨裸眼检测途径下的分辨极限，提高体系稳定性并利用光谱分析精确定量H2 O2浓度（ c ）．结果表明，当c＜100μmol／L时，纳米金为团聚态，呈现蓝色；c＞120μmol／L时，纳米金为分散态，呈现红色．该方法的裸眼检测极限可分辨浓度差为20μmol／L．反应溶液的显色结果并不稳定，在反应后45 min 内变化较快，c为60～100μmol／L的反应溶液也将逐渐由蓝色变为红色．溶液在570 nm处的吸光度（ OD570）检测证明，反应体系在检测后3 h内仍持续变化．反应10 min后，加入适量半胱甘肽终止反应，可使反应溶液的显色结果稳定，提高体系稳定性．实验测定不同浓度H2 O2反应产物的吸收光谱发现，c＜100μmol／L的反应孔产物在波长约550 nm处有吸收峰，且吸收强度与c成正相关．而c＞120μmol／L的反应产物吸收峰逐渐往短波长方向偏移，最终峰值约为540 nm．分析反应体系吸收光谱结果证明，各反应产物在630 nm和545 nm 处吸光度的比值（ OD630／545）与c呈良好的线性关系．实验对c为100～120μmol／L的H2 O2溶液进行检测，定量浓度差为2μmol／L的H2 O2溶液，降低裸眼检测途径下该方法的分辨极限，为微量样品检测提供更好的平台．【期刊名称】《华南师范大学学报（自然科学版）》【年(卷),期】2015(000)004【总页数】5页(P41-45)【关键词】纳米金;过氧化氢;裸眼检测【作者】李娜娜;黄嘉荣;詹求强【作者单位】华南师范大学华南先进光电子研究院，广州510006;华南师范大学华南先进光电子研究院，广州510006;华南师范大学华南先进光电子研究院，广州510006【正文语种】中文【中图分类】O65纳米金表面等离子体因其独特的光学、化学性质和良好的生物相容性，近年来被广泛用于光学探针、电化学传感和生物传感等领域[1-2]. 纳米金具有明显的表面等离子体共振吸收峰，其波长与纳米金的粒径、周围的介质、粒子的间距等密切相关，导致纳米金溶胶颜色差异较大[3]. 纳米金比色法则通过纳米金溶液颜色的变化检测微量样品. 在纳米金比色法中，微量的待测样品可改变纳米金颗粒的聚集情况，从而导致溶液产生颜色变化，用肉眼即可分辨.结合抗体或其他靶向试剂(如蛋白质、多肽和核酸)，纳米金比色法被广泛用于金属离子[4-5]、DNA[6-7]、生物酶[8-9] 等目标分子的微量检测和分析. 与传统荧光分子相比，纳米金具有很高的消光系数，其检测灵敏度可达到荧光分子的103～104倍[10-11]. 纳米金比色法以其简单快速、灵敏度高、肉眼可分辨且不需要借助复杂仪器等卓越优势，在生化检测、环境检测等领域具有广阔的应用前景[12-13].过氧化氢(H2O2)在生化领域应用广泛，尤其常用于传统的酶联免疫吸附检测技术中. 利用纳米金比色法设计检测微量H2O2的方法，可得到较高的检测灵敏度[14]. 结合酶联免疫吸附技术，该方法可直观快速、灵敏地检测多种微量目标物质. 将传统酶联免疫吸附技术和纳米金测定H2O2的方法相结合，已经成为一种新型的检测艾滋病病毒技术[15]，检测灵敏度可达到10-18 g/mL，比目前检测技术高10倍以上.该技术用过氧化氢酶标记HIV-1衣壳抗原p24，浓度极低的p24即可引起H2O2浓度的减小和纳米金颗粒的不规则聚集，进而使溶液颜色呈现蓝色；阴性结果则是纳米金粒子仍保持为单分散形态，溶液颜色显示为红色，用肉眼就可以分辨出以上的颜色变化. 运用这个技术,在釆用常规核酸检测方法都无法检出的情况下，研究人员可以在HIV患者血液中检测出超低浓度的p24. 但该技术具有一定的局限性：(1)反应体系稳定性较差，其结果随反应时间的变化较快，为检验带来不便；(2)只能定性分析所测样品浓度在检测极限范围内外，却无法精确定量. 本文在此基础上，应用纳米金表面等离子体建立微量H2O2的检测体系，提高反应体系的稳定性并探索精确定量方法，并降低检测的分辨极限，为微量样品检测提供更好的平台.1.1 仪器与试剂紫外-可见-近红外分光光度计(PerkinElmer，Lambda 950)；酶标仪(Bio-Rad，iMark)；电子透射显微镜；磁力搅拌器；pH计；2-吗啉乙磺酸(西格玛奥德里奇上海贸易有限公司)；氢氧化钠(国药试剂，分析纯)；过氧化氢(阿拉丁试剂有限公司，分析纯)；氯金酸(阿拉丁试剂有限公司，Au≥48%).1.2 实验方法取1.95 g 2-吗啉乙磺酸粉末溶于80 mL去离子水中，在磁力搅拌下用1 mol/L过氧化氢溶液调节pH 6.5，转移至100 mL 容量瓶定容，为100 mmol/L 2-吗啉乙磺酸储备液，室温储存备用. 另外，将1 g 氯金酸粉末溶于25.4 mL去离子水中，为100 mmol/L氯金酸储备液，避光存于4 ℃中，保存时间勿超过15 d. 称取0.012 3 g还原型半胱甘肽粉末溶于100 mL去离子水中，配制成400 μmol/L 半胱甘肽溶液，4 ℃保存.使用1 mmol/L 的2-吗啉乙磺酸溶液配置浓度为20～200 μmol/L的H2O2溶液以及0.2 mmol/L 氯金酸溶液，现配现用.于96孔板中，每孔各加100 μL氯金酸溶液和100 μL H2O2溶液，静置反应10 min. 向各反应孔加入10 μL半胱甘肽溶液，并于紫外-可见-近红外分光光度计或者酶标仪下读取吸光度. 反应系统稳定情况检测部分未加半胱甘肽溶液.2.1 裸眼检测体系的辨色极限及金颗粒电镜图2-吗啉乙磺酸可作为一种弱还原剂，与氯金酸溶液反应将生成团聚态的纳米金颗粒，反应溶液呈现裸眼可辨的蓝色. 此时，如果加入过量的H2O2作为辅助还原剂，将促进反应生成分散态的纳米金颗粒，使反应溶液呈现红色. 因此，纳米金表面等离子体可作为检测微量H2O2的灵敏探针.实验分别检测0、20、40、60、80、100、120、140、160、180及200μmol/L 的H2O2溶液，反应10 min后辨色(图1). 当H2O2的浓度c<100μmol/L时，反应溶液肉眼观察显示蓝色；当c>120 μmol/L时，反应溶液呈现红色. 该检测体系裸眼检测H2O2极限可分辨浓度差为20 μmol/L.其中，取加入了60 μmol/L和160 μmol/L H2O2的反应孔产物分别作为蓝色和红色反应孔的代表，制备铜网样品，用电子透射电镜观察结果(图2). 蓝色反应孔的反应产物为团聚态的的纳米金颗粒，红色反应孔的反应产物为分散态的纳米金颗粒，与实验原理相符.2.2 反应体系稳定性分析及其终止2.2.1 反应体系的稳定性分析实验发现，反应体系在3 h内稳定性差，且在反应进行45 min内颜色变化较快，对下一步检测和定量分析带来困难. 随着反应时间增加，c>120 μmol/L的反应孔所生成的分散态纳米金颗粒将逐渐增加，红色逐渐加深；c<100 μmol/L的反应孔也将生成分散态的纳米金颗粒，逐渐呈现红色，具体辨色结果见图3A. 反应进行15 min后，c为100 μmol/L和80 μmol/L的反应孔开始略微变红；反应进行20 min后，此2孔与更低过氧化氢浓度的反应孔颜色有明显区别，呈现红色，并逐渐加深(图3A). 此外，c<60 μmol/L的反应孔所呈现的蓝颜色也随着反应时间不断加深. 其中，c=60 μmol/L的反应孔在反应进行45 min后也已变为红色.由于纳米金溶液在波长为570 nm处有特征吸收，实验也通过检测溶液在570 nm 处的吸光度 (OD570) 来监测纳米金生成的稳定情况. 图3B为不同反应时间各反应孔的吸光度OD570变化曲线. c>180 μmol/L的反应孔在反应10 min后OD570保持不变，反应稳定；而c<160 μmol/L时，反应孔的OD570则在反应45 min 内变化较快，之后达到稳定. 此外，随着反应孔中所加入H2O2浓度的降低，反应体系的OD570需要的稳定时间明显增加，与辨色结果中较低H2O2浓度反应孔逐渐变红的现象相吻合.2.2.2 半胱甘肽对反应体系的终止效果为了方便下一步的检测和量化分析，实验使用还原型半胱甘肽作为反应体系的终止剂，以提高反应体系的稳定性. 由于还原型半胱甘肽对纳米金颗粒表面具有较好的亲和性，反应体系中的半胱甘肽将附着于纳米金表面，从而阻止了金颗粒的进一步团聚，终止反应. 反应进行10 min后，向各反应孔中加入10 μL的半胱甘肽溶液，所得不同反应时间各反应孔情况见图4A. 加入半胱甘肽后，各反应孔的辨色结果不再随时间改变，c<100 μmol/L的反应孔保持蓝色，c>120 μmol/L的反应孔保持红色. 不同反应时间各反应孔的吸光度OD570变化曲线见图4B. 加入半胱甘肽后，各反应孔在反应15 min后的OD570基本不变，反应稳定，与辨色结果中各反应孔颜色稳定的现象相吻合. 实验表明，还原型半胱甘肽溶液可有效终止各孔的反应，使纳米金颗粒的状态稳定. 2.3 检测结果吸收光谱及定量分析为了精确定量H2O2浓度，实验使用紫外-可见-近红外分光光度计测定不同H2O2浓度反应孔生成物的吸收光谱(图5A). c<100 μmol/L的反应孔产物在波长约550 nm处有吸收峰，且吸收强度与c成正相关. 而c>120 μmol/L的反应孔产物吸收峰逐渐往短波长方向偏移，最终峰值约为540 nm. 实验对各反应孔产物在630 nm和545 nm处吸光度的比值(OD630/545)进行分析，得到不同浓度H2O2的定量分析曲线(图5B). 各反应孔产物的OD630/545与c(μmol/L)呈良好的线性关系，其回归方程为OD630/545 =-0.003 02c+1.011 77.2.4 裸眼分辨极限下的检测分析根据定量分析曲线，可有效地对裸眼分辨极限下的检测样品进行定量分析. 实验设计了加入100、102、104、106、108、110、112、114、116、118和120μmol/L 过氧化氢的反应孔，辨色结果见图6. c=100 μmol/L的反应孔呈现蓝色，c=120 μmol/L的反应孔呈现红色，而中间浓度的反应孔呈现从蓝色过渡到红色的辨色结果，裸眼难以区分. 实验中各反应孔的吸光度OD630/545如图7所示，OD630/545与过氧化氢浓度c(μmol/L)呈良好的线性关系. 应用OD630/545可精确定量H2O2浓度，降低裸眼检测途径下该方法的分辨极限.本实验在酸性2-吗啉乙磺酸介质中，建立基于纳米金表面等离子体的微量过氧化氢的检测体系，其裸眼检测H2O2极限可分辨浓度差为20 μmol/L. 实验使用半胱甘肽有效终止反应，提高了体系的稳定性，并利用光谱分析精确定量H2O2浓度，降低裸眼检测途径下该方法的分辨极限. 该检测方法操作简单、灵敏度高，便于结合传统酶联免疫吸附技术，在微量生物分子检测领域有广阔应用前景.【相关文献】[1] Saha K, Agasti S S, Kim C, et al. 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Multiplex Biosensor Using Gold Nanorods

Multiplex Biosensor Using Gold Nanorods Chenxu Yu and Joseph IrudayarajDepartment of Agricultural and Biological Engineering and Bindley Biosciences Center,Purdue University, 225South University Street,West Lafayette,Indiana47907Gold nanorods(GNRs)with different aspect ratios werefabricated through seed-mediated growth and surfaceactivation by alkanethiols for the attachment of antibodiesto yield gold nanorod molecular probes(GNrMPs).Mul-tiplex sensing was demonstrated by the distinct responseof the plasmon spectra of the GNrMPs to binding eventsof three targets(goat anti-human IgG1Fab,rabbit anti-mouse IgG1Fab,rabbit anti-sheep IgG(H+L)).Plas-monic sensors are highly specific and sensitive and canbe used to monitor refractive index changes caused bymolecular interactions in their immediate vicinity withpotential to achieve single-particle biosensing.This tech-nique can play a key role in developing novel optical biosensors for both in vivo and in vitro detection and single-receptor kinetics.Gold nanoparticles(GNRs)possess optical properties that make them uniquely suitable for biosensing applications.Their optical properties strongly depend on both the particle size and shape and are related to the interaction between the metal conduction electrons and the electric field component of the incident electromagnetic radiation,which leads to strong,char-acteristic absorption bands in the visible to infrared part of the spectrum.1In aqueous solutions,gold nanostructures exhibit strong plasmon bands depending on their geometric shape and size.For spherical particles,a strong absorption band around520nm due to the excitation of plasmons by incident light can be readily observed.1For nanorods,two distinct plasmon bands,one associ-ated with the transverse(∼520nm)mode and the other with the longitudinal mode(usually>600nm),could be observed.1 Plasmon modes have also been reported for more complex structures such as prisms and quadrupoles.2Applications3-11based on wavelength shifts due to changes in dielectric properties in the vicinity of the particles(known as nanoSPR,3or localized surface plasmon resonance4)achieved by connecting biological receptor molecules(i.e.,antibodies)to activated gold nanoparticles with a compatible chemical tether exists.The subsequent interac-tion of these gold nanoparticle-based molecular probes to their corresponding targets(antigens)completes the sensing modality.Most of the existing work utilizing the nanoSPR mechanism was based on binding-induced aggregation of spherical particles. The concept here is that,when the separation distance between particles is comparable to or smaller than their radii,the oscillation of the plasmons from adjacent particles can become coupled, lowering their vibration frequency that appear as absorption bands red-shifted to longer wavelengths.The red-shift is dependent upon the number of particles and their spatial arrangement within the aggregate.12A50-100-nm red-shift with a significant broadening of the absorption bands due to overlapping of shifted modes of vibration was observed.3Mirkin and co-workers used gold nano-particles to develop a one-pot colorimetric sensor for DNA detection utilizing the aggregation of DNA-capped gold nano-spheres due to DNA hybridization and reported a sharp melting transition capable of detecting insertions,deletion,and mismatches at a single-base resolution.13-15Lee and Perez-Luna11constructed epoxy-functionalized gold nanospheres through a series of steps to subsequently react with hydroxyl moieties of the R-D-glucopy-ranosyl groups of carboxylated dextran chain.The interaction of this material with three proteins was then investigated through changes in the plasmon spectra of gold nanoparticles.This work*Towhomallcorrespondenceshouldbeaddressed.E-mail:josephi@.(1)Perez-Juste,J.;Pastoriza-Santos,I.;Liz-Marz’an,L.M.;Mulvaney,P.Coord.Chem.Rev.2005,249,1870-1901.(2)Millstone,J.E.;Park,S.;Shuford,K.;Qin,L.;Schatz,G.C.;Mirkin,C.A.J.Am.Chem.Soc.2005,127,5312-5313.(3)Nath,N.;Chilkoti,A.J.Fluoresc.2004,14,377-389.(4)Haes,A.;van Duyne,R.P.J.Am.Chem.Soc.2002,124,10596-10604.(5)Nath,N.;Chilkoti,A.Anal.Chem.2002,74,504-509.(6)Yonzon,C.R.;Jeoung,E.;Zou,S.;Schatz,G.C.;Mrksich,M.;Van Duyne,R.P.J.Am.Chem.Soc.2004,126,12669-12676.(7)Ghosh,S.K.;Nath,S.;Kundu,S.;Esumi,K.;Pal,T.J.Phys.Chem.B2004,108,13963-13971.(8)Dahlin,A.;Zach,M.;Rindzevicius,T.;Kall,M.;Sutherland,D.S.;Hook,F.J.Am.Chem.Soc.2005,127,5043-5048.(9)Raschke,G.;Kowarik,S.;Franzl,T.;Sonnichscen,C.;Klar,T.A.;Feldmann,J.;Nichtl,A.;Kurzinger,K.Nano Lett.2003,3,935-938.(10)Raschke,G.;Brogl,S.;Susha,A.S.;Rogach,A.L.;Klar,T.A.;Feldmann,J.;Fieres,B.;Petkov,N.;Bein,T.;Nichtl,A.;Kurzinger,K.Nano Lett.2004, 4,1853-1857.(11)Lee,S.;Perez-Luna,H.Anal.Chem.2005,77,7204-7211.(12)Quinten,M.;Kreibig,U.Surf.Sci.1986,172,557-577.(13)Elghanian,R.;Storhoff,J.J.;Mucic,R.C.;Letsinger,R.L.;Mirkin,C.A.Science1997,277,1078-1081.(14)Jin,R.;Wu,G.;Li,Z.;Mirkin,C.A.;Schatz,G.C.J.Am.Chem.Soc.2003,125,1643-1654.(15)Taton,T.A.;Mirkin,C.A.;Letsinger,R.L.Science2000,289,1757-1760. Figure1.Schematic illustration of a gold nanorod molecular probe.Anal.Chem.2007,79,572-579572Analytical Chemistry,Vol.79,No.2,January15,200710.1021/ac061730d CCC:$37.00©2007American Chemical SocietyPublished on Web12/16/2006revealed that the binding of these proteins is concentration-dependent over a wide range (∼100nM to 50µM),and a simple and convenient colorimetric method to monitor biospecific interac-tions was suggested.The refractive index-dependent plasmon spectral peak shift of nonaggregated spherical gold nanoparticles was also used to develop a solution-phase immunoassay to monitor the binding kinetics of antibody -antigen interactions in real time.16,17Mono-clonal antibodies specific to human ferritin,human chorionic gonadotropin,and human heart fatty acid binding protein func-tionalized to gold particles gave rise to a red-shift in the SPR extinction resulting in an increase in the extinction intensity at 600nm upon binding to the target.The assay was performed in an automated clinical analyzer offering tremendous potential for high sample throughput.There are two ways to evaluate changes in the plasmon spectra caused by target binding:wavelength change of the plasmon peak or extinction/absorption intensity change at a fixed wavelength(i.e.,600nm).When spherical particles coated with Fab segments of monoclonal antibodies specific to a single epitope on the ligand were constructed,aggregation was not observed.17Since the wavelength shift of the plasmon peak was only 2∼3nm (also observed in our own experiments),which is too small a value for detection purposes (low signal/noise ratio),the latter (intensity change at 600nm)was often used as an indicator of target binding events.However,this approach has an inherent problem:the change in extinction intensity could also occur due to a change in particle concentration.When multiple washing steps are incorporated to remove unbound analytes in a “lab-in-a-tube”setup (in some of the one-pot colorimetric study proposed earlier),uncertain changes in the nanoparticle concentration can occur and contribute to the “noise”component of extinction intensity changes,thus making it difficult to monitor biospecific interactions events.Observation of the wavelength shift of the plasmon bands on the other hand is solely determined by changes in the dielectric properties (i.e.,refractive indexes)in the immediate vicinity of the nanoparticles;concentration change has a minimal or no effect on this parameter.Hence the wavelength shift of the plasmon band(16)Englebienne,P.Analyst 1998,123,1599-1603.(17)Englebienne,P.;Van Hoonacker,A.;Valsamis,J.Clin.Chem.2000,46,2000-2003.Figure 2.Absorption spectra of gold nanorods with different aspectratios.Figure 3.TEM micrographs of gold nanorods with mean aspect ratios 2.8and 4.5.Analytical Chemistry,Vol.79,No.2,January 15,2007573has a greater potential for biosensing.In order to induce a large enough shift for detection purposes,when spherical particles are used,the sensors are designed based on the controlled aggrega-tion concept.11,13-15Since aggregation results in a large wavelength shift with a significant widening of the plasmon peak,the resulting spectral resolution is too poor to distinguish multiple targets or for specific detection purposes.Van Duyne and co-workers 6demonstrated that anisotropic silver nanoparticles (tetrahedron)experienced a large shift in the plasmon maximum upon target binding without aggregation,and a microarray was constructed using these silver nanoparticles for molecular fingerprinting.We believe that gold nanorods can be used to overcome this “aggregation deficit”as effectively;furthermore,because gold nanorods with different aspect ratios could be easily fabricated,unique “multiplexing”advantage could be realized.When anisotropic particles such as nanorods are used to create gold nanorod molecular probes (GNrMPs),single-particle sensors could be devised.Significant changes in the plasmon spectra in response to a change in the refractive index in the vicinity of GNrMPs could be observed and utilized to sense specific target-binding events,as illustrated in Figure 1.The longitudinal plasmon bands are extremely sensitive to changes in the dielectric properties of the surroundings,for example,for nanorods with an aspect ratio of 3suspended in liquid matrix,a change in refractive index of 0.1unit of the liquid matrix causes a red-shift of ∼40nm from the longitudinal plasmon band.1The sensitivity increases as the aspect ratio of the nanorods increases,as demonstrated by El Sayed and co-workers,both experimentallyand theoretically.18-20Small changes in the aspect ratio can lead to drastic changes in the transmitted colors/plasmon spectra,suggesting significant multiplexing potential when carefully de-signed GNrMPs are deployed for simultaneous detection of multiple targets.In this work,we will demonstrate,for the first time,multiplex biosensing using GNrMPs,and the detection of target binding monitored via direct spectral changes induced by changes in refractive index in the vicinity of individual particles,not by aggregation/assembly of the nanoparticles.GNRs fabricated through seed-mediated growth normally have CTAB caps preferentially attached to their {1,1,0}and {1,0,0}side faces.The attachment of CTAB to the {1,1,1}side faces is weaker and as such this face is more exposed and accessible to the chemical linkers.21-23There have been several reports in which alkanethiols are introduced onto the {1,1,1}side faces of gold nanorods,through hydrogen bonds between alkanethiol mol-ecules 23or through binding of biomolecules to the alkanethiol layer 21,22to result in a head-to-tail chainlike formation of gold nanorod aggregates.In this study,a thiol functionalization procedure was utilized to selectively bind recognition agents to(18)Lee,K.S.;El-Sayed,M.A.J.Phys.Chem.B 2006,110,19220-19225.(19)Jain,P.K.;Eustis,S.;El-Sayed,M.A.J.Phys.Chem.B 2006,110,18243-18253.(20)Link,S.;El-Sayed,M.A.J.Phys.Chem.B 2005,109,10531-10532.(21)Chang,J.;Wu,H.;Chen,H.;Ling,Y.;Tan,mun.2005,1092-1094.(22)Caswell,K.K.;Wilson,J.N.;Bunz,U.H.F.;Murphy,C.J.J.Am.Chem.Soc.2003,125,13914-13915.(23)Thomas,K.G.;Barazzouk,S.;Ipe,B.I.;Shibu,J.S.T.;Kamat,P.V.J.Phys.Chem.B 2004,108,13066-13068.Figure 4.Plasmonic spectra of gold nanorods with AR )2.3and 3.5,before and after SAM formation.Table 1.Concentration of IgGs in the Supernatant (nmol/10mL)after 1h o Immobilization onto the Gold Nanorods (n )3)GNrMPaspect ratio 2.3aspect ratio 3.5aspect ratio 4.5aspect ratio 6.5IgG Fabs 0.82(0.080.90(0.070.87(0.080.85(0.08binding ratio1.18(0.081.1(0.071.13(0.081.15(0.08574Analytical Chemistry,Vol.79,No.2,January 15,2007gold nanorods that can bind to their respective ligands for multiplex biosensing.Although recently gold nanorods have been investigated as possible bio/optical sensors that utilize the two-photon or three-photon excited luminescent emission,24,25using them as nanoSPR sensors for multiplexing purposes has not been demonstrated.EXPERIMENTAL SECTIONFabrication of Gold Nanorods.A seed-mediated growth procedure slightly modified from that suggested by Nikoobakht and El-Sayed 26was used to fabricate gold nanorods with aspect ratio between 2.5and 7.Hexadecyltrimethylammoniumbromide (C 16TAB,99%)and benzyldimethylammoniumchloride hydrate (BDAC,99%),sodium borohydride (99%),L -ascorbic acid,gold-(III)chloride hydrate (>99%),and silver nitrate (>99%)were all purchased from Sigma-Aldrich (St.Louis,MO)and used without further purification.Nanopure deionized and distilled water (18.2M Ω)was used for all experiments.Nanorod fabrication was initiated by adding small gold particles as seeds (∼4nm)into aqueous solution containing the CTAB capping agent,gold ions (present in HAuCl 4),reducing agents (in ascorbic acid),and silver ions (in AgNO 3).By adjusting the concentrations of the chemicals in the solution,nanorods of different aspect ratios were fabricated.To synthesize nanorods with aspect ratios larger than 4.5,a two-surfactant system was used.Here,the seeds were grown in aqueous solution containing BDAC and CTAB at different ratios,gold ions,silver ions,and reducing agents.By adjusting the relative content of these ingredients,gold nanorods with aspect ratio up to 7were fabricated.The concentration of nanorods was estimated by measuring the average distance between adjacent particles present in the TEM images.At least 150∼200particles from each of the images were analyzed to yield a mean interparticle distance.It should benoticed that this estimation is rough and cannot be considered as an accurate indication of particle concentration.The nanorod growth reaction was terminated after 3h by removing the reaction solution by centrifugation at 5000rpm for 15min.Some small spherical particles were also removed since they were retained in the supernatant.The nanorods were(24)Wang,H.;Huff,T.B.;Zweifel,D.A.;He,W.;Low,P.S.;Wei,A.;Cheng,J.X.Proc.Natl.Acad.Sci.U.S.A.2005,102,15752-15756.(25)Li,C.;Male,K.B.;Hrapovic,S.;Luong,mun.2005,3924-3926.(26)Nikoobakht,B.;El-Sayed,M.A.Chem.Mater .2003,15,1957-1962.Figure 5.Functionalization efficiency of activated and nonactivated goldnanorods.Figure 6.Detection of single epitope targets by GNrMPs.(a)GNrMP 1;(b)GNrMP2.Figure 7.Head-to-tail aggregation caused by streptavidin binding to biotinylated GNRs.Analytical Chemistry,Vol.79,No.2,January 15,2007575resuspensed in 0.005M CTAB solutions and found to remain stable for up to 100days.All subsequent characterization,activation,and functionalization were conducted with these na-norod samples.Functionalization of Gold Nanorods To Make GNrMPs.Once synthesized,the nanostructures can be functionalized by chemical and biological elements and deployed as sensors.Biofunctionalization constitutes a two-step process:in step 1,termed the activation step,a chemical anchor layer was formed on the nanorod surface to provide active functional groups to which biological molecules (i.e.,antibodies)can be covalently attached;and in step 2,the functionalization step,biomolecules were covalently linked to the anchor layer to produce GNrMPs for target specific sensing.Partial activation of the freshly made gold nanorods was done by incubating the nanorods with alkanethiol compounds to form an alkanethiol monolayer on the {1,1,1}side faces of the rod by self-assembly.Such a process will retain the CTAB capping at the {1,1,0}/{1,0,0}side faces.At the {1,1,1}side faces,the alkanethiol self-assembled monolayers (SAMs)was formed to present active groups (i.e.,COOH)that can be used to tether antibodies through NH -CO bonds.The chemical modification/activation of GNRs was achieved as follows:11-mercaptoundecanoic acid (MUA)was purchased from Sigma-Aldrich;0.5mL of 20mM ethanol solution of MUA was added into 5mL of the gold nanorod solution and stirred mildly for 24h under room temperature.Nanorods were then collected by centrifugation at 5000rpm for 15min and resus-pended in a 0.005M CTAB solution to yield a final concentration of ∼100nM.Once the MUA SAM was formed,Fab segments of human and mouse IgGs were then attached to the activated nanorods as follows:to 5mL of the activated nanorods (∼100nM),1mL of freshly prepared 0.4M 1-ethyl,3-(3-dimethylaminopropyl)carbo-diimide (Sigma-Aldrich)and 0.1M 4-(4-maleimidophenyl)butyric acid N -succinimidyl ester (Sigma-Aldrich)solution was added and sonicated for 25min at 4°C.The resulting nanorods were then collected by centrifugation at 5000rpm for 5min and resuspended in a 5mL of PBS buffer (pH 7.4)containing 0.005M CTAB.IgGFabs suspended in PBS were then added to the resulting nanorod suspension (the concentration of IgG Fab was ∼200nM)and then incubated for 1h under constant sonication at room temperature.The functionalized nanorods were subsequently collected by centrifugation at 5000rpm for 5min.After three rounds of vigorous washing,the collected nanorods were sonicated in 0.005M CTAB solution for 10min,the supernatant after each washing step was collected and added together,and the protein content of the combined supernatant was measured using a Bio-rad protein assay (Bio-rad Laboratories,Hercules,CA)with bovine serum albumin as a protein standard.The amount of IgGs bound to the nanorods was determined by subtracting the IgGs left in the supernatant from the original amount.A control experiment was conducted with nonactivated nanorods to evaluate the physisorp-tion of IgGs to the CTAB capped side faces via electrostatic interaction.Characterization of the Gold Nanorods.The yield and aspect ratios of the gold nanorods was determined using transmis-sion electron microscopy (TEM),acquired with a Philips CM-100TEM (Philips,Eindhoven,Netherlands)operating at 100kV,200-µm condenser aperture,70-µm objective aperture,and a spot 3.TEM grids were prepared by placing 1µL of the nanorod solution in a 400-mesh Formvar-coated copper grid and evaporat-ing the solution at room temperature.Images were then captured using a Tietz F415slow scan digital camera at 4K resolution.At least 150-200nanorods could be counted and measured per grid to calculate the mean aspect ratio of the nanorods after the synthesis step.Absorption spectra of GNrMP samples through each stage of experiments were measured using a Jasco V570UV -visible -NIR spectrophotometer (Jasco,Inc.,Easton,MD),in the wavelength range between 400and 1500nm.The measured spectra were normalized by rescaling the maximum absorbance of the longi-tudinal plasmon peak to 1.RESULTS AND DISCUSSIONNanorod Fabrication.Gold nanorods of aspect ratios in the range between 2.8and 7were made following the single0and double-surfactant protocols discussed earlier.Figure 2shows the absorption spectra of nanorods with aspect ratios of 2.8,3,4.5,5.5,and 7,respectively.It can be clearly seen that small changes in aspect ratio introduce a significant red-shift of the longitudinal plasmon band of the GNR colloids,implying a significant potential for multiplexing.Within the range of this study,a linear correlation could be established between the aspect ratio of gold nanorods and the absorbance wavelength of the longitudinal plasmon bands;hence,the aspect ratio of gold nanorods could be easily deduced from their plasmon spectra (data provided in Supporting Informa-tion).The concentrations of nanorods were estimated through TEM imaging.A droplet (1µL)of the nanorod suspension was spread on a TEM grid for observation.The evaporation of water during sample drying pushes the nanorods toward the rim of grid and resulted in a more densely packed distribution of the nanorods in the TEM view field,and the observed interparticle distances tend to be smaller than the real values,which leads to a larger concentration value being calculated.Therefore,the concentration so estimated is indeed an upper limit of the real concentration of the nanorods;they were found to be quite consistent (∼25-30Figure 8.Detection of double-epitope targets by GNrMPs of aspect ratio 7.0.576Analytical Chemistry,Vol.79,No.2,January 15,2007nM)regardless of the aspect ratios of the nanorods as long as the quantity of seed used was constant.Figure 3shows TEM images of the nanorods of aspect ratios 3and 4.5.Counting particles in the images taken for each type indicated that the final particles contain 95and 96.5%rods,respectively,confirming the validity of the protocol.Another observation is that the width of the nanorods remained ap-proximately the same;hence,an increase in aspect ratio was predominately determined by elongation of nanorods.It should be noted that as the ratio reaches 7,the longitudinal peak red-shifts to ∼1011nm,well beyond the visible and into the NIR region of the spectrum where biological samples such as cells only have weak absorption and their interference with the plasmon signal is negligible (there is a weak and broad band ∼800-1000nm due to small amount of shorter rods,fragmented rods,and particles of other shapes,which is not important in the context of this work).Hence,these GNRs have a major advantage compared to the existing fluorescence and illumination labels where interfer-ences due to sample autofluorescence could pose a significant problem in target detection.Functionalization of Gold Nanorods To Make GNrMPs.Functionalization of gold nanorods takes advantage of the well-known affinity between gold and thiol compounds.However,because the {1,1,0}/{1,0,0}side faces of the rods are covered by CTAB,only the {1,1,1}side faces are easily utilized by alkanethiols to form SAMs,for attachment of recognition agents (antibodies in this work).It should,however,be noted that the formation of alkanethiol SAM introduces a red-shift of the plasmon peaks due to the changes in refractive index at the surface of the gold nanorods.Figure 4shows the spectra of two types of nanorod (aspect ratios 2.3and 3.5,respectively)suspensions before and after alkanethiol (MUA)attachment.A red-shift of 5-7nm in the longitudinal peaks is clearly identifiable,indicating the alkanethiol SAM formation.Repeated measurement of spectra of the same nanorod sample over an elongated period of time (3days)yields a maximum peak drift of 0.5nm,toward both red and blue ends,showing that the 5-7-nm red-shift can only be caused by SAM formation.Once the MUA SAM is formed,biomolecules can be covalently attached via the NH 2bond of the antibodies to the COOH terminus of the MUA SAM.A further red-shift of the plasmon peak can be observed due to the antibody functionalization.The same two type of rods,after the human IgG Fab attachment,showed a significant shift (of up to 20nm)compared to the unmodified rods (data provided in Supporting Informtion).The sensitivity of the plasmon spectra of the nanorods to the attachment of layers of molecules forms the basis of molecular biosensors using single particles.If IgG Fabs only attach to MUA SAM at the {1,1,1}side faces,the number of IgG Fabs attached to each nanorod could be estimated.According to the TEM images,the edges of the nanorods with the {1,1,1}side faces have diameters ∼7-10nm and IgG Fabs are of cylindrical shape with a size of ∼6nm.27Therefore,under ideal conditions,two IgG Fabs can attach to each side of one gold nanorod and yield a 2:1IgG Fab/rod ratio.From the length (∼20-50nm)and nanorod material stiffness,it could be reasoned that the IgG Fabs bound to the ends of a rod could only bind to different individual target molecules to provide a highly sensitive and specific platform.Although IgGs can only covalently attach to the MUA-activated sites,physisorption of IgGs to the CTAB capped side faces is also possible.The isoelectric points for IgG Fabs are ∼6;28hence,at a pH of ∼7.4during the functionalization reaction,the IgG Fabs are negatively charged,and they will bind to the positively charged CTAB cap via electrostatic interaction.However,physisorbed IgGs(27)Kienberger,F.;Mueller,H.;Pastushenko,V.;Hingterdorfer,P.EMBO Rep.2004,5,579-583.(28)Bremer,M.B.;Duval,J.;Norde,W.;Lyklema,J.Colloids Surf.,A 2004,250,29-42.Figure 9.Multiplexing detection of various targets using GNrMPs.(a)One target;(b)two targets;(c)three targets.Analytical Chemistry,Vol.79,No.2,January 15,2007577are not as strongly attached to the GNrMP surfaces as covalently bound ones;under vigorous washing after functionalization,a significant portion of these would be removed.In order to obtain GNrMPs that have consistent IgG coating,the MUA activation route is preferred,especially when a low IgG/nanorod ratio is used.Table1provided a binding ratio of the IgGs to four different gold nanorods determined by measuring the remaining IgG contents in the supernatant after the functionalized GNrMPs were collected by centrifugation.The binding ratio here is defined as the average number of IgG molecules attached to each individual gold nanorod.For the concentration ratio used in our experiment (IgG Fab:gold nanorod)2:1),if only covalent binding occurs, under a coupling efficiency of100%,this binding ratio would be close to2.However,in reality,the binding ratio appears to be not very high(∼1.18)and rather independent of the aspect ratio of the GNRs;roughly1IgG Fab molecule/gold nanorod is obtained.A separate experiment was conducted to investigate the effect of increased IgG concentrations on the functionalization of the nanorods(aspect ratio2.1,100nM).As the IgG/nanorod ratio increases from2:1to20:1,the binding ratio increases from1.2to 5.5and levels off,as shown in Figure5.Also it is observed that, at low IgG/nanorod ratio,the binding ratio of IgGs to activated gold nanorods is quite low(∼0.2).Binding to activated nanorods is consistently higher than to nonactivated rods;at high IgG/ nanorod ratio,on average two more IgG Fab molecules are bound to each nanorod,due to the covalent binding to the MUA-activated sites.Nevertheless,the GNrMPs used for target detection in this work had a binding ratio of∼1.2,so a1:1binding to target molecules was expected.Whether or not the IgG Fab is covalently bound to the MUA-activated site is not of critical importance.Detection of Biological Targets Using GNrMPs.GNrMPs were made using human IgG Fab and gold nanorods of aspect ratio 2.3(GNrMP1)and gold nanorods of aspect ratio 3.5 (GNrMP2).It has been reported that a head-to-tail aggregation of gold nanorods could be realized when two nanorods are connected by binding to the same ligand(for example,a biotin-streptavidin binding).If the ligand has only one active binding site to which the GNrMP can interact,head-to-tail aggregation will not occur and the plasmon spectral changes observed will be due to binding events from individual GNrMPs.Figure6depicts the detection of goat anti-human IgG Fabs using GNrMP1and2via successive plasmon shifts.The plasmon peaks near520nm are not very sensitive to the refractive index change induced by target binding,and the red-shifts of these peaks are below3nm,thus offering no opportunity for the detection of specific target binding.The longitudinal peaks of the nanorods are extremely sensitive to the refractive index changes induced by target binding,suggesting that they are excellent reporters of target specific binding events and have the potential to achieve single-molecule sensitivity if measured using microspectroscopy. The sensitivity of the longitudinal peaks to the refractive index changes in their vicinity increases significantly with the aspect ratio of the rods,a red-shift of11and27.5nm is observed due to target binding to GNrMP1and2with aspect ratios of2.3and 3.5,respectively.In order to ensure that the plasmon spectral changes observed are not due to head-to-tail aggregation,a comparison study was done using the biotin-streptavidin model as the recognition-target pair.Earlier work13has reported an expected head-to-tail aggregation of streptavidin with partially biotinylated gold nano-rods,18because of the four active biotin-binding sites of the streptavidin molecules.As shown in Figure7,the longitudinal peak of gold nanorods(aspect ratio2.0)experienced a small red-shift after partial biotinylation and a subsequent red-shift of∼100nm together with significant band-broadening after20µg/mL strepta-vidin was added to the solution,which could primarily be due to significant aggregation of the nanorods.Further,the transverse peak experienced a∼40-nm red-shift as well.Thus,we conclude that significant aggregation introduces a large red-shift in both the transverse and longitudinal bands together with significant paring the characteristics of Figures4and 7,it is obvious that the plasmon shifts observed in Figure4are not due to aggregation.A head-to-tail aggregation of gold nanorods requires multiple epitopes in the targets(to serve as ligand bridges)oriented favorably for simultaneous head-to-tail coupling.Whole IgG molecules have two antigen-binding sites;however,if the targets are also immunoglobulins,the Y-shape of the two molecules will introduce a spatial hindrance that would render the head-to-tail connection of the molecules an unfavorable process.Thus,even if whole IgG molecules(2binding sites)are used as targets instead of IgG Fabs(1binding site),aggregation should not be significant and the single-particle-based target detection scheme proposed should be possible.Figure8shows the results obtained with a GNrMPs(aspect ratio of7)containing a monoclonal sheep IgG whole molecule as a probe agent binding to a rabbit anti-sheep IgG whole molecule target.Clearly,aggregation is not a dominant phenomenon in this illustration.The main advantage of GNrMPs is the multiplexing potential offered by the aspect ratio dependence of the wavelength of the longitudinal bands.To this date,this advantage has not been fully utilized.To demonstrate multiplex detection of various targets,a system using three GNrMP-target pairs was constructed(Table 2).A3-mL aliquot of a mixture of an equal amount of each of the three GNrMPs was placed in three separate test tubes,and an equal amount(1mL at20µg/mL)of sample1(containing target 1),sample2(a mix of target1and target2),or sample3(a mix of target1,target2,and target3)was separately added and theTable2.GNrMP/Target Pairs Investigated in Detection ExperimentsGNrMP no.1no.2no.3 GNR aspect ratio 2.1 4.5 6.5recognition agent of GNrMPs monoclonal humanIgG1Fabmonoclonal mouseIgG1Fabmonoclonal sheepIgG(H+L)target monoclonal goatanti-human IgG Fab monoclonal rabbitanti-mouse IgG Fabmonoclonal rabbitanti-sheep IgG(H+L)578Analytical Chemistry,Vol.79,No.2,January15,2007。

Amperometric-biosensors-based-on-gold-nanoparticles-decorated-multiwalled-carbon-nanotubes-poly(dial

Sensors and Actuators B 147 (2010) 593–598Contents lists available at ScienceDirectSensors and Actuators B:Chemicalj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /s nbAmperometric biosensors based on gold nanoparticles-decorated multiwalled carbon nanotubes-poly(diallyldimethylammonium chloride)biocomposite for the determination of cholineXia Qin,Huicai Wang,Xinsheng Wang,Zhiying Miao,Lili Chen,Wei Zhao,Miaomiao Shan,Qiang Chen ∗The Key Laboratory of Bioactive Materials,Ministry of Education,College of Life Science,Nankai University,No.94Weijin Road,Tianjin 300071,Chinaa r t i c l e i n f o Article history:Received 7January 2010Received in revised form 4March 2010Accepted 4March 2010Available online 11 March 2010Keywords:Choline biosensor Choline oxidaseMulti-wall carbon nanotubes Gold nanoparticlesPoly(diallyldimethylammonium chloride)a b s t r a c tA novel amperometric choline biosensor based on the nanocomposite ﬁlm composed of choline oxidase (ChOx),multi-wall carbon nanotubes (MWCNTs),gold nanoparticles (GNp)and poly(diallyldimethylammonium chloride)(PDDA)was developed for the speciﬁc detection of choline.GNp-decorated MWCNTs (MWCNTs–GNp)was synthesized by a classical chemical method,and GNp was attached on MWCNTs through the speciﬁc interaction between –SH and Au.The enzyme ChOx would be attached to the MWCNTs–GNp matrix and also be adsorbed electrostatically with PDDA which was employed not only as a dispersant but also a binder material.The microscopic structure and composi-tion of the synthesized MWCNTs–GNp were characterized by transmission electron microscopy (TEM)and energy-dispersive X-ray spectroscopy (EDX),and properties of the resulting choline biosensors were monitored by electrochemical measurements.Taking the sensitivity and selectivity into consideration,0.35V versus Ag/AgCl was selected to detect choline.The resulting biosensor exhibited a wide linear range of 0.001–0.5mM choline,with a remarkable sensitivity of 12.97␮A/mM,a detection limit of 0.3␮M estimated at a signal-to-noise ratio of 3and fast response time (within 7s).Moreover,it showed good reproducibility,anti-interferant ability and long-term stability.This work presented a feasible approach for further research in biosensing and other surface functionalizing.© 2010 Elsevier B.V. All rights reserved.1.IntroductionThe quantitative determination of choline in biological samples such as human bile,serum,amniotic ﬂuid,brain extracts and phar-maceutical products is very important in clinical analysis [1–4].The chromatographic methods can be used for these analyses,but it is time consuming and expensive.Amperometric enzyme-based biosensors have received considerable attention due to its conve-nience,high sensitivity and selectivity [5,6].Thus,the development of choline biosensors utilizing choline oxidase (ChOx)is an active research area,ChOx catalyses the oxidation of choline to betaine and hydrogen peroxide as follows [Eq.(1)]:Choline +O 2+H 2O ChOx−→betaine +2H 2O 2(1)Quantiﬁcation of choline is achieved via electrochemical oxidation of the liberated H 2O 2[Eq.(2)]:H 2O 2→O 2+2H ++2e −(2)∗Corresponding author.Tel.:+862223506173;fax:+862223506122.E-mail address:qiangchen@ (Q.Chen).Nevertheless,the oxidation of H 2O 2usually requires a relatively high positive potential (usually over +0.6V vs.SCE),leading to interferences from various electroactive species,such as ascorbic acid (AA),uric acid (UA),and 4-acetamidophenol (AP).It is there-fore highly desired to design and prepare a functional material for the modiﬁcation of the electrode surface;one of the pur-poses is to efﬁciently lower the H 2O 2oxidation potential,and the other is for enzyme immobilization and maintenance of the enzy-matic activity [7,8].The emergence of nanotechnology offers great opportunities to improve the sensitivity,long-term stability,and anti-interference ability of the biosensing systems [9].For example,Goyal et ing fullerene-C 60-modiﬁed electrodes well achieved the above goals [10–13].Carbon nanotubes (CNTs)as new class nanomaterials are extremely attractive in electrochemical applications,especially in electrochemical biosensors because of their unique structure,high chemical stability and high surface-to-volume ratio [14].It was reported in many literatures that single-wall carbon nan-otubes (SWNTs)-modiﬁed electrodes exhibited good stability and markedly enhanced current response [15–17].Gold nanoparticles (GNp)are used increasingly in many electrochemical applications since they have the ability to enhance the electrode conductivity and facilitate the electron transfer,thus improving the analytical0925-4005/$–see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.snb.2010.03.010594X.Qin et al./Sensors and Actuators B147 (2010) 593–598selectivity and sensitivity[18,19].Recently,integration of CNTs and GNp with a synergistic effect has shown particular promise in chemical sensors[20–23].For example,Xu et al.synthesized CNTs–GNp hybride by microwave radiation,and applied it for the determination of trace mercury(II)[22].Li et al.reported that GNp could be in situ noncovalently deposited on PSS-functionalized multiwalled carbon nanotubes(MWCNTs),and the as-prepared nanocomposite exhibited good biocompatibility with glucose oxi-dase[23].PDDA,a positively charged ionic polymer withﬁlm-forming and adhesion ability,is widely recognized as an ideal candidate for enzyme immobilization and is often used to modify substrate surface and colloids,exploiting the electrostatic attraction for their deposition[24,25].Moreover,PDDA can act as a dispersant to dis-perse CNTs or stabilizing agents for colloids because of electrosteric stabilization[26,27].The key idea of this paper is to develop an ampero-metric biosensor constructed with GNp-decorated MWCNTs (MWCNTs–GNp)for interference-free determination of choline. First,the MWCNTs–GNp hybride as the immobilization matrix, choline oxidase(ChOx)as the enzyme,the immobilization of ChOx was carried out by adsorption of ChOx on MWCNTs–GNp hybride, which can not only adsorb redox enzymes without loss of biologi-cal activity but also act as“molecular wire”facilitating the electron transfer.Second,the cationic polymer PDDA which was employed to disperse the enzyme immobilized MWCNTs–GNp composite as a thinﬁlm can also play the role of adsorbing ChOx.By a combi-nation of MWCNTs,GNp,ChOx and PDDA,a novel amperometric choline biosensor was produced by simple solvent casting.The properties of the resulting choline biosensors were measured by amperometric experiments.2.Experimental2.1.ReagentsCholine oxidase(ChOx,EC 1.1.3.17,from Alcaligenes species,13units/mg),gold nanoparticles(GNp,ca.5nm), poly(diallyldimethylammonium chloride)(PDDA,MW: 40,000–50,000),cysteamine,N-hydroxysuccinimide(NHS)and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)were pur-chased from Sigma–Aldrich(USA).Multi-wall carbon nanotubes (MWCNTs)(10–30nm diameter)were supplied by Nanoport Co. Ltd.(Shenzhen,China).Uric acid,ascorbic acid and acetaminophen were obtained from Tianjin damao Chemical Reagent Co.(China). Choline chloride was obtained from Beijing Chemical Reagent Company(China).All other reagents were of analytical grade and used without further puriﬁcation.All aqueous solutions were prepared with doubly distilled water.2.2.Preparation of GNp-decorated multiwalled carbon nanotubesFig.1shows an illustration of the pretreatment of MWCNTs and the preparation of the MWCNTs–GNp hybride.MWCNTs were chemically shortened by ultrasonic agitation in a3:1(v/v)mix-ture of concentrated sulfuric acid and nitric acid for about4h. The resulting MWCNTs were separated and washed with distilled water by centrifugation(10,000rpm)until the pH of the resulting MWCNTs solution became neutral.MWCNTs thus obtained were water-soluble,since they were disconnected and functionalized with carboxylic acid groups on their tips and any defect on the side walls during the oxidation process[28,29].The carboxyl on the MWCNTs could create an active ester using water-soluble coupling agent namely EDC/NHS,which was used to assemble cysteamine by the carboxyl–amine coupling,following to anchor GNp.The reac-tion was carried out by ordinal immersing the activated MWCNTs in a1:1(v/v)EDC/NHS mixture(50mg mL−1EDC and50mg mL−1 NHS),cysteamine,each for1h,and washed with distilled water by centrifugation after each incubation without drying-procedure, MWCNTs modiﬁed by thiol function group were obtained,and then transferred into GNp for1h to assemble GNp onto the MWCNTs sur-face modiﬁed by thiol group[30].Ultimately,the MWCNTs–GNp hybride was synthesized and washed thoroughly with water by centrifugation and dried.2.3.Electrode preparation and modiﬁcationThe enzyme solution was prepared by dissolving5mg of ChOx in0.5ml of PBS at pH7.6,when the enzyme was negatively charged, because the isoelectric point(p I)of ChOx lies around pH 4.5. Enzyme immobilization with MWCNTs–GNp(1mg)was achieved by mixing thoroughly with30␮L of the enzyme.Due to the abil-ity of MWCNTs–GNp to adsorb biomolecules,the enzyme became electrostatically and hydrophobically adsorbed onto the surface of MWCNTs–GNp during mixing.The enzyme–MWCNTs–GNp mix-ture was then dispersed in0.1mL of1wt%PDDA solution with the aid of ultrasonic agitation for5min to form a homogeneous bio-composite colloidal solution.For comparison,electrodes without MWCNTs or GNp were prepared similarly.The Pt electrode used in experiments was a3mm diameter platinum disk insulated in a7mm diameter Teﬂon rod.The Pt elec-trode was thoroughly polished using an alumina powder(0.05␮m diameter),etched for3min in a1:3:4(in volume)mixture of HNO3:HCl:H2O and then sonicated for4min in distilled water.The enzyme biocomposite electrodes were prepared by cast-ing6␮L of MWCNTs–GNp–ChOx–PDDA,GNp–ChOx–PDDA or MWCNTs–ChOx–PDDA biocomposite colloidal solutions on the surface of Pt electrode followed by air drying for about1h,rinsed by water several times before use.All the resulting modiﬁed elec-trodes were thoroughly washed and stored in PBS at4◦C when not in use.2.4.Apparatus and measurementsThe electrochemical measurements were performed at room temperature in a conventional one compartment cell using a 283Potentiostat–Galvanostat electrochemical workstation(EG&G PARC with a software M270)(USA)linked to a personal com-puter for data acquisition and potential control.A conventional three-electrode system comprising the biocompositeﬁlm modi-ﬁed Pt electrode(3mm diameter)as a working electrode,a Pt wire(1mm diameter)as an auxiliary electrode and an Ag/AgCl (saturated KCl)as a reference electrode was employed for all elec-trochemical experiments in a0.1M phosphate buffer solution(PBS, pH7.6).In steady-state amperometric experiment,a magnetic stir-rer provided the convective transport.Transmission electron microscopy(TEM)images were obtained by using Tecnai G2F20instrument(Philips Holland)equipped with an energy-dispersive X-ray spectroscopy(EDX)analyzer. MWCNTs–GNp was dispersed in an alcohol medium and then trans-ferred to a carbon-coated Cu grid for TEM and EDX observation. 3.Results and discussion3.1.Charaterizations of MWCNTs–GNp hybrideThe morphology and structure of the MWNTs–GNp were inves-tigated by TEM.Fig.2A and B shows the TEM images of the MWCNTs–GNp hybride under different magniﬁcation,respec-tively.As can be seen,GNp was attached on the tips or side walls ofX.Qin et al./Sensors and Actuators B 147 (2010) 593–598595Fig.1.Schemes of the pretreatment of MWCNTs and the preparation of the MWCNTs–GNp hybride.MWCNTs,and no free nanoparticles could be observed in the back-ground of TEM images,suggesting the validation of the approach to prepare MWCNTs–GNp nanocomposite,which could be attributed to the presence of –SH providing active sites for the adsorption of GNp,because GNp can strongly bound to the functional groups,such as –CN,–NH 2,or –SH [31,32].The results of EDX in Fig.2C validated the composition of the MWCNTs–GNp.The presence of gold peaks originated from GNp anchored on MWCNTs,while the existence of oxygen and sulfur peaks should be attributable to car-boxyl and thiol groups modiﬁed on MWCNTs,respectively.And Cu peaks arised from Cu grid.To further characterize the MWCNTs–GNp nanocomposites,cyclic voltammograms were obtained with the modiﬁed Pt elec-trode in 0.5M H 2SO 4.As shown in Fig.2D,dash line shows the response of a Pt electrode modiﬁed with clean MWCNTs.No voltammetric peaks were observed in both of the forward and the backward direction.The solid line represents the response of MWCNTs–GNp nanocomposites conﬁned on Pt electrode.A clean reduction peak was observed at around +0.85V corre-sponding to the reduction of the gold surface oxide [33].This observation also conﬁrmed the attachment of GNp onto the MWC-NTs.Fig.2.(A,B)TEM images of the MWCNTs–GNp hybrid at different magniﬁcation.(C)EDX spectrum of MWCNTs–GNp.(D)Cyclic voltammograms of the MWCNTs–GNp hybrid in 0.5M H 2SO 4with scan rate of 50mV/s;the dash line represents the voltammogram of MWCNTs without attachment of the GNp.596X.Qin et al./Sensors and Actuators B147 (2010) 593–598Fig. 3.Amperometric response of(A)GNp–ChOx–PDDA/Pt,(B) MWCNTs–ChOx–PDDA/Pt and(C)MWCNTs–GNp–ChOx–PDDA/Pt electrodes upon successive addition of choline in pH7.6,0.1M PBS at+0.6V vs.Ag/AgCl.3.2.Electrochemical characterization of the modiﬁed electrodes3.2.1.Amperometric responseFig.3shows the amperometric response of biocomposite ﬁlms modiﬁed Pt electrode at+0.6V versus Ag/AgCl upon suc-cessive addition of choline.In order to check the catalytic performance of the MWCNTs–GNp hybrid to H2O2,which was generated during the course of ChOx-catalyzed oxidation of choline in presence of dissolved oxygen,we compared the GNp–ChOx–PDDA(curve a),MWCNTs–ChOx–PDDA(curve b)and MWCNTs–GNp–ChOx–PDDA(curve c)modiﬁed Pt electrodes with the same amount of successive addition of choline,which are 0.01mM,0.02mM,0.04mM,0.06mM,0.08mM.Experiments showed that MWCNTs–GNp–ChOx–PDDA/Pt electrode exhibited the highest amperometric response toward the enzymatic product H2O2,even larger than their response summation.In particu-lar,it ampliﬁed the current response by∼3times compared with GNp–ChOx–PDDA/Pt electrode and∼2times compared with MWCNTs–ChOx–PDDA/Pt electrode.This phenomenon could be ascribed to the synergistic effect of MWCNTs and GNp for electron transfer and electrocatalytic activity,and the larger surface areas of MWCNTs–GNp available for binding more enzyme than that of sole MWCNTs or GNp also contributed to the higher performance.This result is consistent with the pervious literature reports[34–36].A hydrodynamic voltammogram was then carried out for determining the optimum potential necessary for the chronoam-perometric determination of choline.The experiment was performed by the injection of0.25mM solution of choline at the MWCNTs–GNp–ChOx–PDDA/Pt electrode in pH7.6PBS(0.1M).The working electrode was operated at the applied potential range of 0–0.6V versus Ag/AgCl with a potential step of0.05V(data not shown).The oxidation started at about0.21V,showed a sharp increase then and leveled off around0.35V with a maximum at 0.55V,similar with the results in our previous study[36].Taking the sensitivity and selectivity into consideration,the applied poten-tial of0.35V was chosen for the oxidation detection of choline in this work.3.2.2.CalibrationAs shown in Fig.4A,the resulting choline biosensor achieved 95%of the steady-state current within7s.The current reached a saturation value at high choline concentration,showing the characteristics of the Michaelis–Menten kinetics.Linear regres-sion equation for the choline in the range of0.001–0.5mMwas Fig.4.(A)Amperometric response of the MWCNTs–GNp–ChOx–PDDA/Pt electrode upon successive addition of choline in pH7.6,0.1M PBS at+0.35V vs.Ag/AgCl and (B)the calibration curve of the above electrode.Inset:Lineweaver–Burk plot of1/i ss vs.1/C.Error bars=±standard deviation and n=5.Y(␮A)=0.393+12.974X(choline,mM),and the correlation coefﬁ-cient was0.992.The sensitivity or the slope of the calibration plot was12.97␮A/mM choline with the detection limit of0.3␮M when signal-to-noise ratio was3.The sensitivity of the resulting biosen-sor was higher than those of the previously reported values[37–39] and our earlier reported values[40–43],and the detection linear range of choline was much wider as compared with our previously work.The high sensitivity and the expanded linear range might be due to the extra active surface area provided by the MWCNTs–GNp hybrid and large amount of ChOx entrapped in the nanocompos-ite.Additionally,the PDDA membranes with the MWCNTs–GNp hybride could provide a friendly microenvironment to retain the biological activity of the enzyme and that the nanohybride could act as a high conducting nanowire connecting biocompositeﬁlm domains to the electrode surface also contributing to the high per-formance of the biosensor.The apparent Michaelis–Menten constant(K appm),which gives an indication of the enzyme–substrate kinetics for the choline biosen-sor can be calculated from the Lineweaver–Burk equation:1i ss=K appmi max1C+1i maxwhere i ss is the steady-state current after the addition of sub-strate,i max is the maximum current measured under saturatedX.Qin et al./Sensors and Actuators B147 (2010) 593–598597substrate condition and C is the bulk concentration of the substrate. The K appm determined in the study(Fig.4B,inset)was calculated to be0.42mM,which was lower than the literature value of free ChOx(0.87mM)[44],close to the reported value for ChOx of the multilayer ChOx/PEIﬁlm-based sensor(0.436mM)[43],and much lower than the reported value for ChOx in the HFBI self-assembled ﬁlm matrix(1.30mM)[45].These results revealed that there was no substantial loss of ChOx activity,indicating the immobiliza-tion procedure in the present study was biocompatible and the biosensor possesses higher biological afﬁnity to choline.It has been reported that the CNTs have excellent electrocatalytic ability,and the CNTs attached to the enzyme could improve the electron trans-fer between the active redox center of the enzyme and CNTs,which could accelerate the regeneration of enzyme and increase the rel-ative activity of the enzymes[46].3.2.3.Effect of interference on choline monitoringThe general problem in the electrochemical detection of choline is the interference from physiological species such as AA,UA, and AP,etc.We have studied the selectivity of the present choline biosensor against these possible interfering species through measuring the amperometric response to0.25mM choline and successive addition of physiological levels of various interfering species(0.1mM AA,0.1mM AP and0.5mM UA)at the potential of0.35V versus Ag/AgCl(data not shown).The current of the three interfering substances was only about0.15␮A,0.2␮A,and0.10␮A, respectively,indicating that no noticeable changes in current were detected for the interferences in choline pared to the high sensitivity of the resulting choline biosensor,the interfer-ence current can be negligible.The high selectivity of the biosensor could be mainly attributed to the relative low working potential for detection minimizing the responses of common electroactive interferences.3.2.4.Reproducibility and stability of the enzyme electrodeThe reproducibility of the proposed electrode was examined at0.25mM choline solution at+0.35V versus Ag/AgCl.The rela-tive standard deviation was3.7%(n=5).The electrode-to-electrode reproducibility was also examined between5different electrodes in the above solutions,and the relative standard deviation was calculated to be6.2%,indicating a reasonable reproducibility.In order to demonstrate long-term stability,the amperomet-ric response of the electrode was measured to0.25mM choline every day in PBS(0.1M,pH7.6).All electrodes,when not in use, were stored at4◦C under dry conditions.It remained82.5%of its initial current response for choline after1month of storage,sug-gesting the stability and biocompatibility of the PDDA membrane with MWCNTs–GNp matrix render a favorable microenvironment for preserving the activity of ChOx.4.ConclusionIn summary,we have developed a facile route to fabricate a novel amperometric choline biosensor based on nanohybride MWCNTs–GNp with PDDA as the binder.Investigation shows that the as-prepared MWCNTs–GNp nanohybrids not only exhibit efﬁ-ciency for enzyme immobilization but also are of great importance for amplifying the sensor response and enhancing the sensor sensitivity.In addition,the biosensor facilitated low-potential amperometric detection,which was able to efﬁciently monitor choline and reduce the problem of interferences derived from AA, UA,and AP,etc.Meanwhile,the resulting choline biosensor exhib-ited a good reproducibility and long-term stability.The proposed method in this work can provide promising platforms for other biosensor construction to detect glucose,ethanol,and so on.AcknowledgementsTheﬁnancial supports from National Natural Science Founda-tion of China(Grant No.30772746,Grant No.30870665and Grant No.30900325)and Natural Science Foundation of Tianjin(Grant No.08JCZDJC20600)are well acknowledged.References[1]L.Campanella,M.Mascini,G.Palleschi,M.Tomassetti,Determination ofcholine-containing phospholipids in human bile and serum by a new enzyme 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[43]H.B.Shi,Z.Song,J.D.Huang,Y.Yang,Z.X.Zhao,J.-I.Anzai,T.Osa,Q.Chen,Effects of the type of polycation in the coatingﬁlms prepared by a layer-by-layer deposition technique on the properties of amperometric choline sensors, Sens.Actuators B:Chem.109(2005)341–347.[44]M.Ohta-Fukuyama,Y.Miyake,S.Emi,T.Yamano,Identiﬁcation and propertiesof the prosthetic group of choline oxidase from Alcaligenes sp.,J.Biochem.88 (1980)197–203.[45]Z.X.Zhao,H.C.Wang,X.Qin,X.S.Wang,M.Q.Qiao,J.-I.Anzai,Q.Chen,Self-assembledﬁlm of hydrophobins on gold surfaces and its application to electrochemical biosensing,Colloid Surf.B:Biointerfaces71(2009)102–106.[46]D.R.Shobha Jeykumari,S.S.Narayanan,Functionalized carbonnanotube–bienzyme biocomposite for amperometric sensing,Carbon47 (2009)957–966.BiographiesXia Qin is currently a PhD student of Nankai University in China.Her current inter-ests are in the area of electrochemistry and biosensors.Huicai Wang is currently a postdoctor of Nankai University in China.He obtained the doctor degree in Zhejiang university in2007.His current interests are in the area of electrochemistry and biosensors.Xinsheng Wang is currently a PhD student of Nankai University in China.His current interests are in the area of electrochemistry and biosensors.Zhiying Miao is currently a PhD student of Nankai University in China.His current interests are in the area of electrochemistry and biosensors.Lili Chen is currently a graduate student of Nankai University in China.Her current interests are in the area of electrochemistry and biosensors.Wei Zhao is currently a graduate student of Nankai University in China.Her current interests are in the area of electrochemistry and biosensors.Miaomiao Shan is currently a graduate student of Nankai University in China.Her current interests are in the area of electrochemistry and biosensors.Qiang Chen is a professor of Nankai University in China.He received his BSc in physical chemistry from the College of Chemistry,Nankai University in1984and received his MS(in1991)and PhD(in1994)at the Faculty of Pharmaceutical Sciences from Tohoku University,Japan.His research interests are biosensor and biological electrochemistry.。

博士生导师-南通大学公共卫生学院

姓名：于春梅导师简介：于春梅，副教授，苏州大学博士，营养与食品卫生系副主任。

美国南卡罗来纳大学访问学者。

近五年来主持并完成国家自然科学基金、江苏省高校自然科学基金及南通市应用研究项目各1项。

在研江苏省自然科学基金及南通市应用基础研究各1项。

现为江苏省“333工程”高层次人才培养对象，江苏省“青蓝工程”优秀青年骨干教师，江苏省“六大人才高峰”及南通大学“创新人才”培养对象。

近五年发表SCI论文23篇(第一或通讯作者14篇)。

申请国家发明专利2项。

获南通市科学技术进步一等奖1项，南通市自然科学优秀学术论文一等奖及三等奖各1项。

曾获南通大学优秀教育工作者、优秀共产党员、巾帼先进个人等称号。

研究方向：生物与纳米电分析化学。

该研究该研究将纳米技术、细胞生物学、电化学等多学科进行交叉，以电化学及其他分析方法为手段，致力于建立各种分析平台、信息的加工与整合系统，为生命体系自身的各种化学复杂过程提供高灵敏度、高选择性、在线动态跟踪、单细胞实时分析、单分子检测技术等有效的分析方法。

对生命过程中的电子转移现象、肿瘤细胞的电化学行为、疾病相关的生物小分子等进行特异性识别和诊断。

同时，在纳米尺度上了解生物大分子的结构及功能，利用纳米生物传感器获得生化反应信息等，以满足生命分析化学领域原位、活体、实时和在线分析的要求。

荣誉获奖：(1) 于春梅朱振坤王莉，可抛型纸基电极对白血病细胞的研究及抗癌药物敏感性测定，南通市人民政府，南通市自然科学优秀学术论文，一等奖，2015.(2) 于春梅季万余苟莉莉，基于｛血红蛋白/银纳米粒子｝n多层组装膜的pH开关效应，南通市人民政府，南通市自然科学优秀学术论文，三等奖，2013.(3) 顾海鹰于春梅刘扬张勤惠何红范红，Fe3O4磁性纳米粒子的表面功能化修饰、生物兼容性及其应用研究，南通市人民政府，南通市科技进步奖，一等奖，2011.主持在研项目：1. 江苏省自然科学基金面上项目，BK20151267，基于微囊包裹血红蛋白阵列的人工红细胞研究，2015/07~2018/062. 南通市应用研究项目：微纳米结构用于血红蛋白氧载体的应用研究，MS12015046，2015/06~2018/063. 江苏省“333工程”高层次人才培养对象，2016年4. 江苏省“青蓝工程”优秀青年骨干教师项目，2015/01~2016/125. 江苏省“六大人才高峰”项目，2014-SWYY-045，2015/01~2016/126. 南通大学创新人才资助项目，2015/04~2018/03已完成科研项目：1. 国家自然科学基金青年基金，81001263，基于核壳纳米材料的酚类雌激素电化学传感器研究，2011/01~2013/12，主持2. 江苏省高校自然科学基金，10KJB150015，核壳纳米材料的制备及其在环境雌激素电化学传感器中的应用，2011/01~2013/12，主持3. 南通市应用研究项目，BK2011020，纳米Fe3O4的表面修饰及其在构建基于蛋白质多层膜可控生物传感器中的应用，2011/06~2013/09，主持4. 国家自然科学基金面上项目，21175075，基于"血细胞/纳米粒子" 生物器件的构建及其应用基础研究，2012/01~2015/12，已结题，参加(3/8)5. 国家自然科学基金面上项目，20875051，基于"{血红蛋白/Au@Fe3O4}n"多层组装的生物分子电子器件研究，2009/01~2011/12，已结题，参加(2/9)近五年发表论文：(1) Chunmei Yu, Qiuhong Wang, Dongping Qian, Weibo Li, Ying Huang, Fangting Chen, Ning Bao, Haiying Gu*. An ITO electrode modified with electrodeposited graphene oxide and gold nanoclusters for detecting the release of H2O2from bupivacaine-injured neuroblastoma cells. Microchimica Acta, 2016, 183: 3167-3175.(2) Weibo Li, Dongping Qian, Qiuhong Wang, Yubin Li, Ning Bao, Haiying Gu*, Chunmei Yu*. Fully-drawn origami paper analytical device for electrochemical detection of glucose, Sensors and Actuators B, 2016, 231: 230–238.(3) Weibo Li, Dongping Qian, Yubin Li, Ning Bao, Haiying Gu*, Chunmei Yu*. Fully-drawn pencil-on-paper sensors for electroanalysis of dopamine, Journal of Electroanalytical Chemistry, 2016, 769: 72–79.(4) Qiuhong Wang, Weibo Li, Ning Bao, Chunmei Yu*, Haiying Gu*. Low-potential amperometric determination of NADH using a disposable indium-tin-oxide electrode modified with carbon nanotubes. Microchimica Acta, 2016, 183: 423–430.(5) Qiuhong Wang, Weibo Li, Dongping Qian, Yubin Li, Ning Bao, Haiying Gu*, Chunmei Yu*. Paper−based analytical device for detection of extracellular hydrogen peroxide and its application to evaluate drug−induced apoptosis, Electrochimica acta, 2016, 204: 128–135.(6) Lijun Sun, Yannan Lu, Zhongqin Pan, Tingting Wu, Xiaojun Liu, Ning Bao, Chunmei Yu, Hong He, Haiying Gu. Layer-by-layer assembly of hemoglobin-coated microspheres for enhancing the oxygen carrying capacity. RSC Advances, 2016, 6, 59984–59987.(7) Yuanhong Wang, Chunmei Yu, Haiying Gu, Yifeng Tu*. The hemoglobin-modified electrode with chitosan/Fe3O4 nanocomposite for the detection of trichloroacetic acid. Journal of Solid State Electrochemistry, 2016, 20:1337–1344.(8) Zhongqin Pan, Tingting Wu, Yang Liu, Chunmei Yu, Ning Bao, Haiying Gu*, Oxygen carrying capability evaluation based on direct electrochemistry of highly loaded hemoglobin spheres, Surfaces and Interfaces, 2016, 6: 50-55.(9) Yannan Lu#, Tingting Hu#, Tingting Wu, Xiaojun Liu, Ning Bao, Chunmei Yu, Hong He, Haiying Gu*, Construction of electrochemical avenue for evaluating oxygen-carrying performance of a microsphere-based oxygen carrier with bovine serum albumin protection layer, Journal of Electroanalytical Chemistry, 2016, 781: 327-331.(10) Chunmei Yu, Qiuhong Wang, Weibo Li, Yubin Li, Shuxian Liu, Ning Bao, Haiying Gu. Paper-based cell impedance sensor and its application for cytotoxic evaluation. Nanotechnology, 2015, 26: 325501.(11) Chunmei Yu, Li Wang, Chun Zhu, Ning Bao, Hai-ying Gu*. Detection of cellular H2O2 in living cells based on horseradish peroxidase at Au nanoparticles decorated graphene oxide interface. Sensors and Actuators B: Chemical, 2015, 211: 17–24.(12) Chunmei Yu, Zhenkun Zhu, Qiuhong Wang, Wei Gu, Ning Bao, Haiying Gu*. Disposable indium-tin-oxide sensor modified by gold nanorod-chitosan nanocomposite for the detection of H2O2 in cancer cells. Chemical Communication, 2014, 50: 7329–7331.(13)Chunmei Yu, Zhenkun Zhu, Li Wang, Qiuhong Wang, Ning Bao, Haiying Gu*. A new disposable electrode for electrochemical study of leukemia K562 cells and anticancer drug sensitivity test. Biosensors and Bioelectronics, 2014, 53: 142–147.(14) Chunmei Yu, Li Wang, Zhenkun Zhu, Ning Bao, Haiying Gu*. Trans-membrane electron transfer in red blood cells immobilized in a chitosan film on a glassy carbon electrode. Microchimica Acta, 2014, 181:55–61.(15) Hao Jing, Qingfeng Zhang, Nicolas Large, Chunmei Yu, Douglas A. Blom, Peter Nordlander, Hui Wang*. Tunable plasmonic nanoparticles with catalytically active high-index facets. Nano Letters 2014, 14 (6): 3674–3682.(16)Chunmei Yu, Yidan Wang, Li Wang, Zhenkun Zhu, Ning Bao, Haiying Gu*.Nanostructured biosensors built with layer–by–layer electrostatic assembly of hemoglobin and Fe3O4@Pt nanoparticles. Colloids and Surfaces B, 2013, 103:231–237.(17) Yuanhong Wang#, Chunmei Yu#, Zhongqin Pan, Yufei Wang, Jianwei Guo, Haiying Gu*. A gold electrode modified with hemoglobin and the chitosan@Fe3O4nanocomposite particles for direct electrochemistry of hydrogen peroxide. Microchimica Acta, 2013, 180, 659–667.(18) Zhongqin Pan, Chuanguo Shi, Hong Fan, Ning Bao, Chunmei Yu, Yang Liu, Rong Lu, Qinghui Zhang, Haiying Gu*. Multiwall carbon nanotube CdS/hemoglobin multilayer films for electrochemical and electrochemiluminescent biosensing, Sensors and Actuators B: Chemical, 2012, 174, 421-426.(19) Chunmei Yu, Wanyu Ji, Lili Gou, Ning Bao, Haiying Gu*.The pH-sensitive switchable behavior based on the layer-by-layer films of hemoglobin and Ag nanoparticles, Electrochemistry Communications, 2011, 12, 1202-1205.(20) Chunmei Yu, Lili Gou, Xiaohui Zhou, Ning Bao, Haiying Gu*.Chitosan-Fe3O4 nanocomposite based electrochemical sensors for the determination of bisphenol A, Electrochimica Acta, 2011, 56, 9056-9063.(21) Lili Gou, Chuanguo Shi, Chunmei Yu, Zhongqin Pan, Ning Bao, Haiying Gu*. Applying pure water plugs for electroosmotic flow monitoring in microchip electrophoresis, Sensors and Actuators B: Biointerfaces, 2011, 160(1): 1485-1488.(22) Zhongqin Pan, Hong Fan, Chuanguo Shi, Ning Bao, Chunmei Yu, Haiying Gu*. Direct electrochemistry of hemoglobin on CdS: Mn nanoparticles, Microchimica Acta, 2011, 173, 277-283.(23) Yang Liu, Ting Han, Chao Chen, Ning Bao, Chunmei Yu,Haiying Gu*.A novel platform of hemoglobin on core-shell structurally Fe3O4@Au nanoparticles and its direct electrochemistry, Electrochimica Acta, 2011, 56, 3248-3257.。

胶体金技术的应用及展望PPT课件

This paper mainly introduces the design of an intelligent temperature control system which realizes the function of temperature measurement and control by using single bus digital temperature sensor DS18B20 and single chip microcomputer. The core components of the system are AT89C51 m sensor.
肌梗死）. The determination assays for human myoglobin（肌红蛋白）,
cardiac troponin complex（肌钙蛋白复合物） and the MB isoform of the
enzyme creatine kinase are presented. Cyclic voltammetry measurements
机理
根据胶体金的一些物理性状，如高电子密度、颗粒大小、形状及颜色反应，加上结合物的免疫和生物学特性，因而使胶体金广泛地应用于免疫学、组织学、病理学和细胞生物学等领域。
This paper mainly introduces the design of an intelligent temperature control system which realizes the function of temperature measurement and control by using single bus digital temperature sensor DS18B20 and single chip microcomputer. The core components of the system are AT89C51 m sensor.

纳米金比色检测葡萄糖

/ac Naked Eye Detection of Glucose in Urine Using Glucose Oxidase Immobilized Gold NanoparticlesChangerath Radhakumary and Kunnatheeri Sreenivasan*Laboratory for Polymer Analysis,Biomedical Technology Wing,Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram695012,Indiab Supporting InformationC hemical sensors are miniaturized devices that can deliverreal-time and online information on the presence of speciﬁc compounds or ions in complex matrixes.1Metallic nanoparticle based sensing has emerged as an important colorimetric tool for the detection of biomolecules linked to the onset of diseases to aid in early diagnosis.Among them,colloidal gold is extensively used for molecular sensing due to the wide opportunities it oﬀers in the design of easy to perform methods.2The synthesis of gold colloid with an organic monolayer in a one-step process is also exploited, and this monolayer provides the extraordinary stability to the nanoparticles along with the additional surface properties.3À8 Optical sensing based on the plasmon resonance absorption exhibited by nanoparticles has been used with a view to develop analytical tools in clinical diagnosis.9À11It is well-known that the gold nanoparticles display surface plasmon resonance(SPR) absorption bands at a speciﬁc wavelength(∼519nm).The frequency and width of the SPR absorption depend on the size and shape of the metal nanoparticle as well as on the dielectric constant of the metal itself and of the medium surrounding it.12 On aggregation of the gold nanoparticles,the absorption maxima shifts to longer wavelengths resulting in the color change of the colloid from wine red to blue due to mutually induced dipoles that depend on interparticle distance and aggregate size.13In the present study,we use functionalized gold nanoparticles for urine glucose sensing.We focus on urine because it is the most informativeﬂuid that can be obtained noninvasively and is used routinely to diagnose and monitor a variety of medical conditions.14The glucose level in blood is used as a clinical indicator of diabetes.The presence of glucose in urine is a more dangerous condition,as it is an indication of worsening of diabetes. According to the World Health Organization,over150million people in the world were aﬀected with diabetes in the year2004 and it is expected to climb further to366million in2030.The aﬀected population has to be tested for blood glucose levels daily for an eﬀective treatment.In order to avoid the inconveniences caused by drawing blood intravenously or by hand pricking,a preliminary screening of the patients with high level diabetes (having renal glycosuria)15,16can be done instantly by checking their urine glucose levels.The visible color change of the functionalized gold nanopar-ticles on interacting with urine glucose enable the patients to have a self-checking method at home and seek immediate medical attention.Recently,Malhotra et al.and Li et al.have studied in length the characteristics of glucose oxidase(GOD) immobilized GNPs.17,18These authors have shown enhanced stability of GOD by the process of immobilization.Ma and Ding have reported that,while free GOD in solution only retains about 22%of its relative activity at90°C,the immobilized GOD on gold nanorods retains about39.3%activity.19Li et al.also suggested that such GOD/GNPs bioconjugates can be consid-ered as a catalytic nanodevice to construct a nanoreactor basedReceived:December18,2010Accepted:February25,2011inaccessible to the bulk of the population.on a glucose oxidation reaction for a biotechnological purpose.18 Interestingly,the feasibility of the use of these entities in the sensing of glucose has not been attempted.Zhang et al.have reported the use of modiﬁed glassy carbon electrode for the application of glucose sensing.The GNPs was deposited on the electrode surface through the glucose oxidase catalyzed oxidation of glucose which in turn was measured by diﬀerential pulse stripping voltammetry.20Very recently,Jiang et al.have demon-strated the application of a AuNP based colorimetric assay for the simple but eﬀective detection of glucose in the rat brain by taking advantage of the cascade reactions of GOD catalyzed oxidation of glucose and the Fenton reaction of H2O2,as well as the oxidative cleavage of ssDNA with OH radicals.21There has been an ever increasing demand for the develop-ment of simple,cost-eﬀective methodologies in an easy to read out format for the detection of clinically relevant molecules to aid in diagnosis.Such approaches are extremely important,particu-larly in third world countries where high tech diagnostics aids are inaccessible to bulk of the population.14Simple procedures which could be performed at home without the need of sophisticated expensive instrumentation possibly bring radical changes in rural health care management.Visual detection of disease speciﬁc marker molecules in biologicalﬂuids such as urine,saliva,or blood is an attractive approach to address these issues.A color change observable by the naked eye in response to the concentration of an analyte can be an indication of a disease condition warranting further medical attention.Herein,we report conjugation of GOD,the enzyme speciﬁc toβ-D glucose onto thiol capped GNPs,using carbodiimide chemistry and its use in the colorimetric detection of glucose in urine.’EXPERIMENTAL SECTIONMaterials.Gold chloride,glucose oxidase(GOD),trisodium citrate,Tween20,16-mercaptohexadecanoic acid(16-MHDA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC),and N-hydroxysuccinimide(NHS)were obtained from Sigma-Al-drich,Bangalore,India.The glucose standard used is from the glucose kit supplied by Enzyme Technologies Pvt.Ltd., Mumbai,India.Preparation of Gold Nanoparticles.GNPs were synthesized as reported by Turkevich et al.22Briefly,to a boiling solution of 20mL of1.0mM HAuCl4,2mL of a1%solution of trisodium citrate dihydrate was added under constant stirring.The contents were removed from the hot plate when the solution turned red. The cooled contents were kept under refrigeration until its usage.Preparation of Alkane Thiol Modified Gold Colloids.The surface modification of gold colloids using alkane thiols was done as per Aslan and Luna.2Briefly,equal volumes of gold colloid and Tween20(2mg/mL)in phosphate buffer at pH7.0were gently mixed and allowed to stand for a minimum of20min to allow for the physisorption of Tween20to the gold nanoparticles.Then,a solution of0.5mM16-MHDA in methanol was added to the above solution and allowed to stand for4h.Excess Tween20and thiols were removed from the surface modified gold colloids by centrifugation for20min at14000rpm with an ultracentrifuge (Sigma3À30K,Germany).The absorbance spectra of the colloidal gold and the modified colloids with Tween20,16-MHDA was taken with a UVÀvisible spectrophotometer (Varian,Cary-Win Bio,Melbourne,Australia)using1cm path length polystyrene cuvettes.Conjugation of GOD on Alkanethiol Modified Gold Col-loids.Immobilization of GOD with16-MHDA capped gold nanoparticles was done on the basis of the EDC/NHS chemistryvia the formation of an amide linkage between the carboxyl groups of the16-MHDA and the primary amine groups of the GOD.The thiolated gold colloids(3mL)were activated with 5mL of a mixture of EDC and NHS,both10mM in phosphate buffer at pH7.0by incubating the mixture at room temperature for30min.Then,2mL of GOD solution(1mg/mL in phosphate buffer)was added to the above mixture and incubated at room temperature for2h and then kept overnight at4°C. After centrifugation at14000rpm,the GOD immobilized gold nanoparticles were resuspended in3mL of phosphate buffer(pH 7.0)and kept refrigerated until use.Fourier Transform Infrared Spectroscopy(FT-IR)of the Nanoparticles.The FT-IR spectra of the citrate stabilized GNPs and GOD capped GNPs were recorded in the range of 600À4000cmÀ1on a Nicolet5700FT-IR spectrometer (Nicolet Inc.,Madison,USA)using a Diamond ATR accessory.Particle Size and Zeta Potential Determination.The tech-nique of dynamic light scattering(DLS;Malvern Instruments Ltd.,Malvern,UK)was used for the determination of the size of the nanoparticles.All measurements were performed at a fixed angle of90°at25°C.The zeta potential of a particle is the overall charge that the particle acquires in a particular medium and is also measured using the same equipment at25°C.Transmission Electron Microscopy.Transmission electron microscopy(TEM)images were obtained on a Hitachi,H7650 microscope(Tokyo,Japan).The gold colloid and the GOD capped gold colloid were deposited onto a200mesh copper grid coated with a Formvar film and dried overnight.Glucose Sensing Using GOD Functionalized Gold Col-loids.The shift in the SPR absorption maxima of the GOD functionalized GNPs on adding predetermined quantities of glucose standard is recorded using a UVÀvisible absorption spectrophotometer,and a calibration curve was plotted with the wavelength against the optical density.The urine samples,which did not contain glucose,were collected from a clinical laboratory. These fluids were spiked with glucose and used as the test samples.The corresponding shift in the absorption maxima was used to calculate the amount of glucose present.Selectivity of the Method.The selectivity of the method was investigated by checking the shift in SPR absorption maxima of the GOD functionalized GNPs on interacting with normal urine samples and the same spiked with albumin/creatinine in the ratio of40mg/mM and cysteine200μM/L.To confirm the precision and recovery of the probe,each set of experiments was carried out in triplicate,and similar results within the maximum error of 2À3%were obtained.’RESULTS AND DISCUSSIONThe surface plasmon resonance makes the absorption cross section of the nanoparticles several orders of magnitude stronger than the most strongly absorbing molecules and the light scattering cross section several orders of magnitude stronger than the organic dyes.23Hence,most of the applications of gold nanoparticles as sensors are based on detecting the shift in surface plasmon peak either due to change in the local dielectric constant of the nanoparticles by adsorbed biomolecules or due to analyte induced aggregation of the nanoparticles.24À29Both these eﬀects rely on the selectivity provided by the functionalized capping agents.Functionalization of GNPs.The typical plasmon absorptionpeak of the gold colloid was observed at 519nm (Supporting Information,Figure S1),indicating the formation of spherical GNPs.30,31This is further substantiated in Figure S2(Supporting Information)which shows the TEM images of the GNPs at a magnification of 500nm,and the nanoparticles displayed an average size of 11to 12nm.After adding Tween 20,the SPR absorption maximum of the gold colloid was shifted to 523(1nm due to the physical adsorption of the surfactant on the GNPs and was consistent with the reported shifts of the band upon formation of dielectric layers around colloidal metals.2The absorption maxima further shifted to 524(1nm upon chemisorption of 16-MHDA,indicating the formation of a thicker monolayer around the nan-oparticles,as reported by Aslan etal.2The presence of a physisorbed layer of Tween 20on the surface of colloidal gold could prevent irreversible aggregation of gold nanoparticles during and after chemisorption of alkane thiols.2On conjugating the ÀCOOH group of the 16-MHDA with the amino group of GOD,the SPR was further red-shifted to 534(1nm.The spectral shift is not accompanied by any broadening,confirming nonaggregation of the particles at this stage.Fourier Transform Infrared Spectroscopy (FT-IR).We re-corded FT-IR spectra of the nanoparticles to get further insight on the surface modification (Supporting Information,Figure S3).Citrate stabilized GNPs showed peaks at 1509and 1399cm À1,characteristic of citrate ions.GOD conjugated GNPs showed intense peaks at 1654and 1549cm À1,typical of amide I (ÀC d O)and amide II (N ÀH bending)bands of the enzyme and a strong ÀC ÀO Àstretching band at 1074cm À1.The peak at 3278cm À1was assigned the N ÀH/O ÀH stretching frequency of the GOD.Effect of Glucose on the SPR Absorption Maximum of the GOD Conjugated GNPs.On adding varied amount of glucose (from 10to 100μg/mL),the SPR absorption band was found to shift from 535to 569nm,reflecting the formation of aggregates having enhanced sizes.The reaction is instant,and no incubation time is required between glucose and GOD conjugated GNPs.The corresponding spectra are shown in Figure 1.The red shift in the plasmon peak was found to vary almost linearly with the concentration of glucose,suggesting the possi-bility of the use of this methodology for the quantitative estimation(inset in Figure 1).The magnitude of wavelength shift was con-comitantly increased with concentration of glucose,and beyond 90μg/mL,it is leveled o ﬀ,indicating the saturation of the reactive sites.The lowest amount of glucose that can produce a red shift in the SPR absorption maxima was found to be 10À90μg/mL with a detection limit of 5μg/mL.Particle Size and Zeta Potential Determinations.The particle sizes and zeta potentials of the GNPs before and after the addition of glucose (50and 100μg/mL)are given in Table 1.The average particle size of GNPs was 22.5(0.7nm,and the same was increased to 158nm on conjugating with GOD.On adding 50μg/mL glucose,the particles size was increased to 231.7nm and the size was enlarged to 1202nm on the addition of 100μg/mL glucose,showing the tendency toward the formation of aggregates.The formation of aggregated assembly is reflected again in the color change of the solution from red to blue (Figure2).As shown in Table 1,the zeta potential of the GNPs was reduced from À48(0.41mV to À21.4mV,on conjugating with GOD.When 50μg/mL glucose was added to GOD functionalized GNPs,the charge was reduced to À14.1mV and subsequently to À5.85mV on the addition of 100μg/mL glucose.The considerable reduction in zeta potential also indicates the tendency for the formation of aggregates.GOD oxidizes glucose to gluconic acid and H 2O 2.H þions resulted from the formation of gluconic acid could reduce anionic character of the enzyme,bringing down the negative zeta potential.At acidic pH,nanoparticles have shown less zeta potential due to the increased chemical potential of H þions.32The magnitude of the measured zeta potential is an indication of the net charge over the nanoparticles and can be used toFigure 1.UV Àvisible absorption spectra of GOD conjugated GNP showing red shift on reacting with di ﬀerent quantities of glucose standard.(Inset is the graphical relationship of wavelength shift against increasing quantities of glucose.)Table 1.Size and Charge of the Nanoparticlessampleparticle size (nm)zeta potential (mV)gold nanoparticles 22.5(0.7À48(0.41Au-GOD158(2À21.4(0.1Au-GOD-glucose 50μg/mL 231.7(9À14.1(0.3Au-GOD-glucose 100μg/mL1202(26À5.9(0.5Figure 2.Color of (A)GNPs and (B)GOD-GNP on reacting with g 100μg/mL glucose.predict the long-term stability of the particles.If all the particles in suspension have either a large negative or a positive zetapotential,there will not be any tendency for them to come closer due to strong repulsion.33However,if the particles have low zeta potential values,the probability of them coming together to form an aggregate is higher.The GOD functionalized GNPs synthe-sized in our laboratory did not show any sign of aggregation even after 2months under refrigerated conditions.Transmission Electron Microscopic (TEM)Analysis.We measured the size of the particles using TEM.Figure 3A shows the size of the GOD conjugated GNPs as 44À47nm.Figure 3B ÀD displays the sizes of the GOD functionalized GNPs on interaction with 5,100,and 150μg/mL glucose,respectively.The size of the nanoparticles increased on adding increasing quantities of glucose.When 50μg/mL glucose was added,the size was increased to 73À75nm;with 100μg/mL glucose,it became 102to 145nm,and a total aggregate was formed on adding 150μg/mL glucose.TEM,however,showed lower values for the size of the nanoparticles compared to DLS measurements (Table 1).DLS measurement records higher values since the light is scattered by the core particle and the layers formed on the surface of the particles.TEM,on the other hand,shows the size of metallic core only.He et al.have also made similar observations earlier.34Glucose induced aggregation of GOD conjugated GNPs leading to color change is well supported by the absorption,particle size measurements,and TEM investigations.Glucose Sensing in Urine Samples.We spiked a urine sample with glucose (50μg/mL),and the absorption peaks are shown in Figure 4.The peak maximum showed a shift of 21nmexactly as in the case of the aqueous standard sample (see Figure 1),demonstrating the feasibility of extending the method for the estimation of glucose in real samples.Rather than quantitative estimation,it seems that the method provides a quick qualitative screening of samples.It is apparent from Figure 2that a concentration of 100μg/mL glucose showed a visible color change of the solution to conclude that the glucose content of the test solution is g 100μg/rmation of this kind could be used for further assessment of the health status of the individual.The optical detection method discussed here may be useful for rural populations where clinical laboratory facilities are limited.Figure 3.TEM images of (A)GOD conjugated GNP,(B)50μg/mL glucose added,(C)100μg/mL glucose added,and (D)150μg/mL glucose added.Figure 4.Wavelength shift with a urine sample spiked with 50μg/mL glucose.导致纳米金聚集的原因Selectivity of the Method.To check the cross reactivity of the GOD functionalized GNPs,we conducted the following studies.Diabetic patients are at increased risk of renal diseases. The principal feature of diabetic nephropathy is proteinuria which is defined as the albumin/creatinine ratio being g30 mg/mmol.35We spiked the urine sample with100μL of aqueous solution containing albumin/creatinine in the ratio40mg/mM to mimic the urine samples of patients with diabetic nephro-pathy.It is also reported that the concentration of cysteine in urine is200μM/L.36A urine sample(100μL)spiked with cysteine is also added to Au-GOD to check its selectivity toward glucose.The SPR absorption maximum of the Au-GOD shifted only on adding50μL of100mg/dL glucose standard.No red shift for albumin/creatinine or cysteine was found,confirming that the GOD functionalized GNPs were highly specific to glucose and had no interference with any other molecules present in the urine.The SPR absorption pattern and the corresponding color images in the inset are shown in Figure S4 (Supporting Information).’CONCLUSIONSWe devised a simple method for the detection of glucose in ﬂuids like urine using GOD immobilized gold nanoparticles.The color of the solution was found to change from red to blue in the presence of∼100μg/mL glucose.Plasmon absorption peak was red-shifted proportionally up to100μg/mL glucose,and good correlation was obtained between the wavelength shift and concentration pointing out the feasibility of the use of the method for the quantitative measurement of glucose in urine. The color change observable by the naked eye can advanta-geously be used for a preliminary screening at home itself and may be referred for more focused investigations.The method seems to be a potential one,particularly for the rural population in third world countries.’ASSOCIATED CONTENTb Supporting Information.Additional information as noted in text.This material is available free of charge via the Internet at .’AUTHOR INFORMATIONCorresponding Author*E-mail:sreeni@sctimst.ac.in.Phone:091À471À2520248.Fax: 091À471À2341814.’ACKNOWLEDGMENTWe are grateful to the Director of SCTIMST and Head of the Biomedical Technology Wing,SCTIMST,for providing all the facilities for conducting this study.We are also thankful to Ms.S. 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荧光共振能量转移于葡萄糖特异检测与细胞成像

S Supporting Information *
ABSTRACT: In this paper, we have developed a biofriendly and high sensitive apo-GOx (inactive form of glucose oxidase)-modiﬁed gold nanoprobe for quantitative analysis of glucose and imaging of glucose consumption in living cells. This detection system is based on ﬂuorescence resonance energy transfer between apo-GOx modiﬁed AuNPs (Au nanoparticles) and dextran-FITC (dextran labeled with ﬂuorescein isothiocyanate). Once glucose is present, quenched ﬂuorescence of FITC recovers due to the higher aﬃnity of apo-GOx for glucose over dextran. The nanoprobe shows excellent selectivity toward glucose over other monosaccharides and most biological species present in living cells. A detection limit as low as 5 nM demonstrates the high sensitivity of the nanoprobe. Introduction of apo-GOx, instead of GOx, can avoid the consumption of O2 and production of H2O2 during the interaction with glucose, which may exert eﬀects on normal physiological events in living cells and even lead to cellular damage. Due to the low toxicity of this detection system and reliable cellular uptake ability of AuNPs, imaging of intracellular glucose consumption was successfully realized in cancer cells.

基于纳米金与纳米银簇间表面等离子增强能量转移效应特异性检测microRNA

基于纳米金与纳米银簇间表面等离子增强能量转移效应特异性检测microRNA王红亚;尹斌成;叶邦策【摘要】There is high demand for a sensitive method for miRNA detection in clinical diagnosis. In this work, we developed a method for miRNA detection based on the surface plasmon-enhanced energy transfer ( SPEET ) between gold nanoparticles ( AuNPs ) and silver nanoclusters ( AgNCs ) , coupled with DNA polymerase and nicking enzyme-assisted isothermal amplification for target recycling. Two DNA probes ( Probe a and Probe b) were assembled onto the surface of AuNPs to form Probe b-Probe a-AuNP conjugates. Probe a consisted three domains:the complementary sequence of miRNA, the specific site of the nicking enzyme, and the self-assembly sequence for AgNCs. The 3′ end of Probe a was modified with thiol as a binding site for AuNPs. The SPEET of AgNCs and AuNPs was inhibited when miRNA was added to produce the dumbbell shaped template by polymerase. The template could promote synthesis of AgNCs, resulting in replacement and subsequently recycling of the target molecule for signal amplification. In comparison with the traditional method of miRNA detection with commercial RT-PCR kits, this method avoided the processof reverse transcription and was easy to perform. In addition, this method with a detection limit of 2. 5×10-11 mol/L was cost-effective, label-free, and highly selective for detecting miRNA, and could be applied to the analysis of miRNA in biological samples.%microRNAs(miRNAs)的灵敏检测对临床诊断具有十分重要的意义.本研究采用偶联DNA聚合酶和核酸内切酶介导的恒温扩增反应实现靶标循环再生的策略,利用纳米金(AuNPs)与纳米银簇(AgNCs)间表面等离子增强能量转移效应,开发了一种miRNA定量检测方法.在AuNPs表面组装两种探针(Probe a和Probe b)制备响应元件Probe b-Probe a-AuNP,其中Probe a通过3′端巯基共价偶联到AuNPs表面,此外具有靶标miRNA互补序列、核酸内切酶酶切序列和Probe b互补序列,Probe b为荧光AgNCs合成模板.靶标miRNA存在时,启动酶级联恒温扩增反应,导致Probe b脱离AuNPs表面,抑制了Probe b为模板合成的AgNCs与AuNPs间表面等离子增强能量转移效应,使得反应体系荧光信号增强.本方法的检出限为2.5×10-11 mol/L,与miRNAs商业化检测试剂盒相比,避免了逆转录反应,而且操作简单,检测成本低,可应用于生物样本中miRNAs分析.【期刊名称】《分析化学》【年(卷),期】2017(045)012【总页数】8页(P2018-2025)【关键词】microRNA检测;纳米金;纳米银簇;表面等离子增强能量转移效应【作者】王红亚;尹斌成;叶邦策【作者单位】石河子大学化学和化学工程学院,石河子832000;华东理工大学生物反应器工程国家重点实验室,上海200237;华东理工大学生物反应器工程国家重点实验室,上海200237;石河子大学化学和化学工程学院,石河子832000;华东理工大学生物反应器工程国家重点实验室,上海200237【正文语种】中文MicroRNAs(miRNAs)是一类内源性的具有调控功能的非编码RNA，其长约19～25个核苷酸，通过碱基互补配对的方式识别靶mRNA，并根据互补程度的不同指导沉默复合体降解靶mRNA或者阻遏靶mRNA的翻译[1,2]。

智能化金纳米颗粒自组装及其生物医学的应用进展

correlates of response to nivolumab in Japanese patients withesophageal cancer[J]. Cancer Sci, 2020,111(5):1676-1684. ［111］Huang J, Xu B, Mo H,et al. Safety, activity, and biomarkers of SHR-1210, an Anti-PD-1 antibody, for patients with advancedesophageal carcinoma[J]. Clin Cancer Res, 2018,24(6):1296-1304.［112］Wang X, Zhang B, Chen X,et al. Lactate dehydrogenase and baseline markers associated with clinical outcomes of advancedesophageal squamous cell carcinoma patients treated withcamrelizumab (SHR-1210), a novel anti-PD-1 antibody[J].Thorac Cancer, 2019,10(6):1395-1401.［113］Xu J, Zhang Y, Jia R,et al. Anti-PD-1 antibody SHR-1210 combined with apatinib for advanced hepatocellular carcinoma,gastric, or esophagogastric junction cancer: an open-label,dose escalation and expansion study[J]. Clin Cancer Res,2019,25(2):515-523.智能化金纳米颗粒自组装及其生物医学的应用进展朱明芮　毛秋莲　赵燕　史海斌苏州大学放射医学与辐射防护国家重点实验室，苏州大学医学部放射医学与防护学院，江苏省高校放射医学协同创新中心，苏州 215123通信作者：史海斌，【摘要】　金纳米材料形貌多样，因具有独特的光学性质及良好的生物相容性特点，近年被广泛应用于生物医学基础研究。

基于金-石墨烯修饰的高灵敏甲胎蛋白电化学免疫传感器

基于金-石墨烯修饰的高灵敏甲胎蛋白电化学免疫传感器朱强;余红霞【摘要】Based on gold nanparticles (AuNPs) and gold-grephene (Au-Gra) nanomaterials, an highly sensitive am-prometric immunosensor forα-1-fetoprotein was successfully prepared. Firstly, AuNPs were electrodeposited on the surface of bare gold electrode. Then NiNPs were dropped on the electrode and served as electrochemical redox probe, next Au-Gra was fabricated to combine anti-AFP. The electrochemical characteristics of the immunosensor were demonstrated by cyclic voltammetry (CV). The immunosensor exhibited a good linear range from 0.1 to 100 ng/mL with a detection limit of 0.03 ng/mL (S/N=3). The proposed immunosensor was simple, rapid and sensitive, and may provide a promise method for clinical detection. of AFP.%以具有大比表面积和良好生物相容性的纳米金颗粒(AuNPs)和金-石墨烯复合物(Au-Gra)为基底,成功制备了高灵敏甲胎蛋白免疫传感器。

纳米金与生物分子的相互作用及生物传感检测

第6卷第3期2009年6月Vol．6No．3June 20090引言生物传感器（Biosensor ）是以生物分子为识别元件，通过生物特异性识别过程来分析和检测各种生命物质和化学物质的器件。

由于具有灵敏度高、特异性好、快速、准确和方便的特点，生物传感器已被广泛应用于食品工业、环境监测、临床医学和军事等领域。

生物传感器的识别元件和传导元件决定了传感器的选择性和灵敏度，因此，不断开发和应用新功能的传感材料，选择并优化现有的传感材料是改善传感器性能的重要措施之一。

近些年来，纳米材料的快速发展和应用为改善生物传感器的性能和拓宽应用领域创造了有利条件，也不断催生新型纳米生物传感器的发展，其中，纳米金颗粒由于具有良好的光电性能以及生物兼容性，在生物分析领域中得到了广泛应用。

利用纳米金体系对DNA 检测是近十几年来发展起来的一种简便、快速的传感方法，目前仍然以光学比色分析为主要手段。

同时，基于纳米金高效猝灭荧光的性质可以用荧光法检测分析；纳米金颗粒的拉曼光谱研究是基于纳米金光学性质的检测方法；而基于纳米金的电学性质，在电导分析、电化学分析等方面也有广泛的应用；基于纳米金颗粒的质量变化而建立的质量型传感方式也显收稿日期：2009-01-08*基金项目：国家自然科学基金(20704043，20805055),上海市科委项目(0852nm00400，07ZR14136),上海市启明星计划(08QA14078,08QH14029)纳米金与生物分子的相互作用及生物传感检测*刘刚1，潘敦1，刘丽2，宋世平1，王丽华1，樊春海1(1.中国科学院上海应用物理研究所，上海201800)（2.上海大学材料学院高分子系，上海201800）摘要：系统地分析了纳米金与生物分子的相互作用，并从光学比色分析、荧光分析、电化学检测、质量变化检测等几个方面入手，详细介绍了纳米金在DNA 检测领域中的应用。

关键词：纳米金；DNA ；生物传感；生物检测Interaction of Gold Nano Particles and Biomoleculesand their Application in BiosensorJIU Gang 1,PAN Dun 1,LIU Li 2,SONG Shi-ping 1,WANG Li-hua 1,FAN Chun-hai 1(1.Shanghai Institute of Applied Physics,Chinese Academy of Sciences,Shanghai 201800,China)(2.School of Materials Science and Engineering,Shanghai University,Shanghai 201800,China)Abstract:Systematically studied the interaction between gold nanoparticles and bimolecules and introduced the applica －tion of gold nanoparticles in DNA sensing area through optical colorimetric analysis,fluorescence analysis,electrochemi －cal detection,the quality change detection.Keywords:Gold nanoparticles;DNA;biosensing;biological detection中图分类号：TB34文献标识码：A 文章编号：1812-1918（2009）03-0006-05纳米材料与应用Nanomaterial &Application 6纳米科技Nanoscience ＆Nanot echnologyNo．3June 2009第6卷第3期2009年6月图1DNA 分子对纳米金颗粒间距的调控及生物传感策略示出较大的潜力。

制备特定尺寸的纳米金颗粒方法及性能表征_郑海霞

777国家自然科学基金(21002006)和北京市教委项目(AJ2010-08)资助收稿日期: 2010-07-14; 修回日期: 2011-01-12; 网络出版日期: 2011-07-08 网络出版地址: /kcms/detail/11.2442.N.20110708.1757.005.html北京大学学报(自然科学版), 第47卷, 第5期, 2011年9月Acta Scientiarum Naturalium Universitatis Pekinensis, Vol. 47, No. 5 (Sept. 2011)制备特定尺寸的纳米金颗粒方法及性能表征郑海霞黄博能胡君曼龚䶮†北京服装学院材料科学与工程学院, 北京100029; † 通信作者, E-mail: clygy@摘要通过化学还原法制备出不同粒径的纳米金颗粒。

利用紫外可见分光光度计和透射电子显微镜对纳米金颗粒的形貌及尺寸进行表征。

讨论了还原剂种类、还原剂用量、试剂加入顺序、反应温度等因素对纳米金颗粒稳定性、粒径、形貌和分散性的影响。

结果表明: Na 3C 6H 5O 7为还原剂制得纳米金颗粒粒径在15~20 nm 之间, NaBH 4为还原剂制得的纳米金颗粒粒径在3~10nm 之间, 柠檬酸钠与氯金酸的摩尔比为1.5:1时最佳, Na 3C 6H 5O 7为还原剂时, 采用HAuCl 4溶液加入到加热的Na 3C 6H 5O 7与聚乙烯吡咯烷酮(PVP)混合溶液比Na 3C 6H 5O 7溶液加入到加热的HAuCl 4与PVP 混合溶液制得的纳米金溶胶的颗粒分散性好, 粒径小且更均一。

关键词纳米金制备; 柠檬酸钠还原剂; 硼氢化钠还原剂; 聚乙烯吡咯烷酮(PVP); 透射电子显微镜中图分类号 O611Synthesis and Characterization of Gold Nanoparticles withVarious DiametersZHENG Haixia, HUANG Boneng, HU Junman, GONG Yan †School of Material Sciences and Engineering, Beijing Institute of Fashion Technology, Beijing 100029;† Corresponding author, E-mail: clygy@Abstract Gold nanoparticles with various diameters were synthesized by chemical reduction. UV-Vis spectroscopy and transmission electron microscopy (TEM) were used to characterize the morphology and the size of the prepared Au nanoparticles. The effects of factors, such as the type of the reducing agent, the amount of the reducing reagent, reagent adding order and reaction temperature on the stability, radius, morphology and dispersion of Au nanoparticles were studied. The results show that the size of Au nanoparticles prepared with Na 3C 6H 5O 7 as a reductant was within the range of 15−20 nm, and the size of Au nanoparticles prepared with NaBH 4 as a reducing agent was within the range of 3−10 nm. The optimum molar ratio of Na 3C 6H 5O 7 and HAuCl 4 was 1.5:1. The gold nanoparticles prepared after adding HAuCl 4 to the hot mixture of Na 3C 6H 5O 7 and poly vingl pyrrolidone (PVP) solution were better dispersed, smaller in size and more uniform, compared with that prepared after adding Na 3C 6H 5O 7 to the hot mixture of HAuCl 4 and PVP solution.Key words preparation of gold nanoparticles; Na 3C 6H 5O 7 reducing agent; NaBH 4 reducing agent; poly vingl pyrrolidone (PVP); transmission electron microscopy (TEM)纳米金具有独特的生化与物化性质, 使其在免疫分析、生物传感器、DNA 的识别与检测、基因治疗等许多方面有独特的作用[1−6]。

金银分析方法纯金分析

10 纯金分析（ Analysis of pure gold） (1)10.1 概述 (1)10.2 火试金法测定金（Determination of Gold by Fire Assaying） (1)10.2 原子吸收光谱法测定银（Determination of Silver by Atomic Absorption Spectrometry） (2)10.3 原子吸收光谱法测定铁（Determination of Iron by Atomic Absorption Spectrometry） (3)10.4 原子吸收光谱法测定铜、铅、铋和锑（Determination of Copper， Lead， Bismuth andAntimony by Atomic Absorption Spectrometry） (4)10.5 原子发射光谱法测定微量杂质元素（Determination of Impurity Elements bySpectrographic Method） (5)10.5.1 金属棒状法（双电弧法）（Metal rod shape method） (5)10.5.2 粉末石墨电极小孔法（粉末电弧法）（Powder graphite electrode small holemethod） (6)10.6电感耦合等离子发射光谱法测定杂质元素（Determination of Impurity Elements byInductively Coupled Plasma-Atomic Emission Spectrometry Method） (6)10.6.1 乙醚萃取ICP-AES法测定铂、钯、铑等12种杂质元素（Determination of ImpurityElements by aether extraction ICP-AES Method） (6)10.6.2 乙酸乙酯萃取ICP-AES法测定银、铜、铁等10种杂质元素（Determination ofImpurity Elements by ethyl acetate extraction ICP-AES Method） (8)10.6.3 标准基体匹配 ICP-AES法直接测定银、铜、铁等10种杂质元素（Determination ofImpurity Elements by standard matrix matching ICP-AES Method） (9)10.7 直读光谱法测定银、铜、铁等8种杂质元素（Determination of Impurity Elements byDirect-reading Spectrometry Method） (9)10.8 电感耦合等离子质谱法测定银、铜、铁等19种杂质元素（Determination of ImpurityElements by Inductively Coupled Plasma-Mass Spectrometry Method ） (10)参考文献 (11)10 纯金分析（Analysis of pure gold）10.1 概述纯金是黄金工业的主要产品之一。

生物样品中碘的分析方法概述

生物样品中碘的分析方法概述崔俐俊;范国荣;廖跃华【摘要】碘是具有重要生物效应的微量元素之一,与人体的生长发育、新陈代谢密切相关,自然界中碘分布广泛并以多种形式存在,对于人体、食物、药物及环境中碘含量的分析是人们极为关注的问题.本文综述了近年来有关碘特别是生物样品中碘的分析方法进展.%Iodine was one of the most important trace elements in human nutrition. It was essential for the biosynthesis of thyroid hormones, which was closely related with the mental development, growth and basic metabolism. Iodine spead and exist widely in many forms. The analysis methods of iodine in human body, food, medicine and environmental received many attentions. The analysis methods of iodine in biological matrix were reviewed to give some references for the further research.【期刊名称】《药学实践杂志》【年(卷),期】2011(029)006【总页数】5页(P408-411,415)【关键词】碘;生物样品;分析方法;综述【作者】崔俐俊;范国荣;廖跃华【作者单位】上海医疗器械高等专科学校,上海200093;第二军医大学药学院药物分析学教研室,上海200433;上海市药物代谢产物研究重点实验室,上海200433;上海医疗器械高等专科学校,上海200093【正文语种】中文【中图分类】TQ460.7+2碘是具有重要生物效应的微量元素之一，与人体的生长发育、新陈代谢密切相关。

金的等离子共振等综述

a b s t r a c t
This review describes the fundamental aspects of laser–gold nanoparticle (Au NP) interaction that leads to nanoscale energy deposition to the surroundings through light ampliﬁcation and heat generation. Besides the importance of the primary process in physics and chemistry, application of the light–NP interaction has attracted signiﬁcant interest from various areas ranging from analytical chemistry to material chemistry and biomedicine. Here we consider both mechanistic and application aspects. Our attention is focused on pulsed-laser-induced fast processes that revealed the heating–cooling dynamics of electrons, lattice (particle), and particle’s environment. On the application side, we focus on material fabrication and processing that beat diffraction-limited resolution. Together, we will shed a light on the essence of research activities carried out in the past 10 years. In addition to an abundance of latest information obtained from currently available literature, this review includes ﬁgures obtained by our own calculations to provide readers with a better understanding of the basics of the optical properties and energy and heat-transfer processes of Au NPs, which are not familiar to photochemists. © 2012 Elsevier B.V. All rights reserved.

Method for Detecting the Presence of a Target Anal

专利名称：Method for Detecting the Presence of aTarget Analyte in a Test Spot发明人：William Cork,Tim Patno,Mark Weber,DaveMorrow,Wesley Buckingham申请号：US11530110申请日：20060908公开号：US20070041624A1公开日：20070222专利内容由知识产权出版社提供专利附图：摘要：An apparatus and method for imaging metallic nanoparticles is provided.Preferably, the invention provides for an apparatus and method for detection of goldcolloid particles and for accurate reporting to the operator. The apparatus includes a substrate holder for holding the substrate, a processor and memory device, an imaging module, an illumination module, a power module, an input module, and an output module. The apparatus may have a stationary substrate holder and imaging module which are proximate to one another. The apparatus provided for a compact sized system without the need for complex motorized devices to move the camera across the substrate. Further, the apparatus and method provide for automatic detection of the spots/wells on the substrate, automatic quantification of the spots on the substrate, and automatic interpretation of the spots based on decision statistics.申请人：William Cork,Tim Patno,Mark Weber,Dave Morrow,Wesley Buckingham地址：Lake Bluff IL US,Chicago IL US,Cary IL US,Chicago IL US,Chicago IL US国籍：US,US,US,US,US更多信息请下载全文后查看。

QuantiFERONGoldb...

QuantiFERON Gold blood test for TuberculosisThe QuantiFERON®‐TB Gold test (QFT‐G) is a whole‐blood test for use as an aidin diagnosing Mycobacterium tuberculosis infection, including latenttuberculosis infection (LBTI) and tuberculosis (TB) disease.At HealthPartners, the QFT‐G blood test is the preferred method of testing forpersons 5 years of age and older. The Tuberculin Skin Test (TST) is thepreferred method of testing for persons younger than 5. If the patient prefersthe skin test, the TST can be administered. The QFT‐G blood test replaces theTST and should not be used in addition to the TST.QFT‐G may be especially useful in patients suspected of having possible false‐positive Tuberculin Skin Testing (TST) due to previous BCG vaccination orenvironmental (non‐tuberculosis) mycobacterial infection. The test specificallydetects responses to two proteins that are made by M. tuberculosis, thebacterium that causes TB. These reactions are absent in all BCG preparationsand environmental mycobacterium (with few exceptions). As a result, QFT‐G ismore specific than Tuberculin Skin Testing for the diagnosis of M. tuberculosisinfection.A positive response to the QFT‐G does not mean the person has ACTIVE TB. Itsimply means they have been exposed to the M. tuberculosis bacterium. Theymay have latent infection, active infection, or treated infection. Clinicalassessment and other diagnostic tests are needed to confirm a diagnosis ofactive TB, such as a chest x‐ray and sputum smear and cultures.The advantages of using the QFT‐G blood test are:•Requires only a single patient visit.•Results can be available sooner than the TST.•Results are more accurate and specific to TB.•Results are easily found in EPIC.•Does not boost responses measured by subsequent tests.•Prior BCG vaccination does not cause a false‐positive result.•Insurance companies are paying for this testing.The disadvantages of using the QFT‐G blood test are:•Blood samples must be processed within 12‐16 hours of collection while white blood cells are still viable.•Limited data on the use of QFT‐G blood tests for:o Children younger than 5 years of age.o Persons recently exposed to M. tuberculosis.o Immunocompromised personso Serial testingFor more detailed information regarding the testing and treatment of TB, please refer to the TB Policy & Procedure found in Compliance 360.References:1.Hauck FR, Neese BH, Panchal AS. Identification and management of latenttuberculosis infection. Am Fam Physician 2009; 79:879-86.2.Mazurek GH, Jereb J, Lobue P, Iademarco MF, Metchock B, Vernon A.Guidelines for using the QuantiFERON-TB Gold test for detectingMycobacterium tuberculosis infection, United States. MMWR RecommRep. 2005; 54:49–55.3.Pai, M. Interferon-gamma release assays for latent tuberculosis infection. In:UpToDate, Basow, DS (Ed), UpToDate, Waltham, MA, 2011.Questions: Please reply to this e‐mail, and your questions(s) will be directed to the author of this Pearl, Dr. Jonathan Sellman.Pearl Archive: All Pearl recommendations are consistent with professional society guidelines,and reviewed by HealthPartners Physician Leadership.。

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