胶体金免疫色谱试纸条及微芯片

ORIGINAL PAPER

Microchip based and immunochromatographic strip assays for the visual detection of interleukin-6and of tumor necrosis factorαusing gold nanoparticles as labels

Yan Man&Xuefei Lv&Javed Iqbal&Guang Peng&

Da Song&Congxiao Zhang&Yulin Deng

Received:8June2014/Accepted:8September2014

#Springer-Verlag Wien2014

Abstract The enzyme-linked immunesorbent assay(ELISA) is most frequently used for the determination of interleukin-6 (IL-6)and tumor necrosis factor-α(TNF-α).However,this technique is time-consuming and requires a substantial sample volume.We have developed(a)a new kind of colloidal gold-based immunochromatographic strip,and(b)a colloidal gold-based immunochromatographic microchip for simultaneous, rapid,and visual detection of IL-6and TNF-α.The lowest limit of detection is62.5ng mL?1for IL-6,and30ng mL?1for TNF-α.Both the strip method and the microchip method are highly specific,user-friendly,and can be read out without the need for instrumentation.

Keywords Immunochromatographic strip.IL-6.TNF-α. Immunochromatographic microchip.Simultaneous detection Introduction

Interleukin-6(IL-6)is a well know pleiotropic cytokine main-ly produced by the immune system cells,vascular endothelial cells and adipocytes.It has a wide range of biological activ-ities in T cells,B cells,hepatocytes,hematopoietic and neural cells[1,2].Its critical role in the immune response,the acute phase response,hematopoiesis,oncogenesis,and inflamma-tion is of utmost importance[3–7].Tumor necrosis factor-α(TNF-α)is another important inflammatory cytokine pro-duced by macrophages,lymphoid cells,neuronal tissue and many other tissues.Numerous studies have shown that the expression of IL-6and TNF-αwas associated with a number of age-related chronic diseases,i.e.,cardiovascular disease, diabetes,cancer,physical disabilities,and cognitive decline [8].So,it is necessary to establish a rapid,simple,sensitive, and specific detection method both for IL-6and TNF-α.The sensitivity is defined as the percentage of positive test re-sponses and specificity as percentage of negative tests.

Presently,enzyme-linked immunosorbent assay(ELISA)is the conventional method used for the detection IL-6and TNF-α[9–13].However,this method is laborious,time-consuming and requires complicated cleanup steps,costly equipment and more sample quantity.All these limitations make ELISA unsuitable for the rapid diagnosis of plasma cytokines.

The immunochromatographic strip technique has been de-veloped as a popular platform for rapid diagnosis since it was first introduced in the https://www.360docs.net/doc/7f19242502.html,pared to ELISA,the immunochromatographic strip technique has a tendency to detect wide range of samples without pretreatment,low sam-ple volume requirement,long-term preservation,inexpensive and no need of high technology machinery.The critical role of the immunochromatographic strip technique is in point-of-care testing(POCT)[14]which is a test designed to be used at or near the site where the patient is located.This does not require permanent dedicated space and are performed outside the physical facilities of the clinical laboratories.In the immunochromatographic strip,colloidal gold nanoparticles are the most commonly used signal reporters due to their stability,controllable particles size,and good compatibility with biological molecules,i.e.,antibodies,antigens,DNA,and RNA[15].Chou[16]developed a manual self-assembled colloidal gold nanoparticle-immunochromatographic

strip Electronic supplementary material The online version of this article

(doi:10.1007/s00604-014-1362-y)contains supplementary material,

which is available to authorized users.

Y.Man

:X.Lv(*):J.Iqbal:G.Peng:D.Song:C.Zhang:

Y.Deng(*)

School of Life Science,Beijing Institute of Technology,

Beijing100081,China

e-mail:xuefeilv@https://www.360docs.net/doc/7f19242502.html,

e-mail:deng@https://www.360docs.net/doc/7f19242502.html,

Microchim Acta

DOI10.1007/s00604-014-1362-y

for detection of interferon-γup to very low concentrations (ng mL?1level).This strip can be used for homecare and point-of-care testing in health evaluation and preventive medi-cine.Tripathi et al.[17]developed a rapid colloidal gold nano-particles based competitive immunochromatographic strip assay for detection of17-α-hydroxy progesterone(17-α-OH-P)in serum.The detection limit of the test strip was2.5ng mL?1by visual observation.The detection limit is the lowest quantity of a substance that can be distinguished from the absence of that substance(a blank value)within a stated confidence limit[18]. Therefore,this test could work better in screening congenital adrenal hyperplasia(CAH)patients with generally high17-α-OH-P values.

In2009,Guo et al.[19]designed two formats of gold-labeled antibody lateral-flow strips for simultaneous detection of carbofuran and triazophos in water samples within a short time(8–10min)without any equipment.The results indicated that the detection limits for carbofuran and triazophos were32 and4ng mL?1using the preferable strip,respectively.

For simultaneous detection of IL-6and TNF-α,Worsley et al.[20]prepared a lateral flow assay format utilizing fluo-rescent microspheres with8.5pg mL?1and55.5pg mL?1 limits of detection respectively in hydrogel samples and 7.15pg mL?1and10.7pg mL?1respectively in plasma.Abe et al.[21]recognized IL-6and TNF-αin the independent microchannels on a microchip by the sandwich ELISA with the detection limit of0.28pg mL?1and0.46pg mL?1,respec-tively.Above discussion shows that immunochromatographic strip provided better alternate of ELISA by reducing the problems related to ELISA and combined the advantages of microchip.This system is very quick and efficient that needs less sample volume and is applicable to POCT.

Present study is specifically designed to develop a colloidal gold immunochromatographic strip for simultaneous,rapid, and visual detection of IL-6and TNF-α.Furthermore,com-bining the advantages of microchip,achieved the visual and simultaneous detection of IL-6and TNF-αon the fabricated immunochromatographic microchip.

Experimental

Materials and chemicals

Human IL-6was ordered from Cell Signaling Technology,Inc (Boston,USA,https://www.360docs.net/doc/7f19242502.html,/).Human TNF-α, TNF-αmonoclonal antibodies(Anti-TNF-αmAbs),TNF-αpolyclonal antibodies(Anti-TNF-αpAbs),IL-6monoclonal antibodies(Anti-IL-6mAbs),IL-6polyclonal antibodies (Anti-IL-6pAbs),and Goat anti-mouse IgG were ordered from Abcam Inc.(Cambridge,USA,http://www.abcam. com/).Gold chloride(HAuCl4·3H2O)was purchased from Sigma-Aldrich(Shanghai,China,https://www.360docs.net/doc/7f19242502.html,)and was directly used without further purification. Bovine serum albumin(BSA),sodium azide,sodium citrate, and Tween-20were ordered from AMRESCO(OH,USA, https://www.360docs.net/doc/7f19242502.html,/).PEG20000was bought from Alfa Aesar(China)Chemical Co.,Ltd.(Shanghai,China, https://www.360docs.net/doc/7f19242502.html,).Skim milk from Becton, Dickinson and Company(BD,NJ,USA,http://www.bd. com/).Sucrose was ordered from Beijing Chemical Works (Beijing,China,https://www.360docs.net/doc/7f19242502.html,/com-17148/). Water was purified and deionized with a Milli-Q system (Millipore,America).Norland optical adhesive81(NOA 81)was bought from Norland Products Inc.(Cranbury,NJ, USA).All of the chemicals used in current studies were of analytical grade.

Conjugation of monoclonal antibodies with colloidal gold nanoparticles

Colloidal gold nanoparticles were prepared according to the standard procedures in literatures[22,23]with little modification(See Electronic Supplementary Material, ESM).In order to achieve the optimal conjugation effi-ciency of monoclonal antibodies with colloidal gold nanoparticles,the conjugation conditions including the concentration of monoclonal antibodies and the pH val-ue of colloidal gold solution were determined prior to conjugation(See EMS).

The conjugation of monoclonal antibodies with colloidal gold was conducted according to the method in the literature [24].Briefly,5mL of colloidal gold was mixed with40μg of monoclonal antibodies in50mL centrifugation tube with continuous magnetic stirring.This mixture was incubated for 4h at room temperature.Following incubation,the remaining binding sites of the colloidal gold were blocked by adding 10%(m/V)BSA(Tris–HCl,pH8.0)to the final concentration of0.5%(m/V)and continuous stirring for30min at room temperatures.The reaction was further stabilized with10% PEG20000(Tris–HCl,pH8.0),the final concentration of PEG20000was0.2%.The mixture was further incubated at room temperature for20min while stirring.After incu-bation,mixture was centrifuged at8,000rpm for30min to separate unbound monoclonal antibodies.Colorless super-natant was pumped out carefully with a syringe and was discarded.Subsequently,the precipitate of dark red gold-labeled antibody conjugate was resuspended in0.01M Tris–HCl(pH8.0)containing1%BSA and centrifuged at8,000rpm for30min.This procedure was repeated three times to ensure cleanup of the unlabeled monoclonal anti-bodies.Finally,the precipitate of the monoclonal antibodies -colloidal gold conjugate was resuspended in0.5mL of 20mM Tris–HCl buffer(pH8.0)containing1%BSA, 5%sucrose,0.05%PEG20,000,and0.1%Tween-20, and stored at4°C for further use.

M.Yan et al.

Preparation of immunochromatographic test strips

Three drops(0.5μL each)of polyclonal antibody solution (1mg mL?1)and anti-mouse IgG(1mg mL?1)were put on the nitrocellulose(NC)membrane as test and control dots,and dried at37°C for2h.An absorption pad,antibody-immobilized NC membrane,sample pad,and the conjugate pad were assembled as the test strip(50mm in length and 4mm in width),which was attached to polyvinyl chloride (P V C)s h e e t.A s c h e m a t i c d i a g r a m o f t h e immunochromatography test strip is shown in Fig.S1(See ESM).Firstly,the target proteins were captured by the gold nanoparticles immobilized with monoclonal antibody,and then these nanoparticles were captured by polyclonal antibody in the test dots.On the other hand,the monoclonal antibody immobilized gold nanoparticle without target proteins were captured by the anti-mouse IgG in the control dots.The positive test showed red color both in the test dots and control dots on the NC membrane.Weak positive showed light red color in the test dots and red color in the control dots.Negative test showed red color only in the control dots.The invalid test showed red only in the test dots or did not show red color both in the control dots and test dots.

Chip fabrication

The microscope slides were taken,cleaned by alcohol follow-ed by deionized water,and then dried with nitrogen(N2).The structure of chip channel in the photomask was designed using the software of Adobe Illustrator CS5.Following method was adopted for the preparation of chip.First of all,0.4mL of NOA81was placed on the microscope slides,and limited in a given area by a batardeau made by photomask frame,and then the photomask with channel structure was placed on the NOA81layer.The thickness of this NOA81layer was con-trolled by the height of photomask frame.The microscope slides with NOA81were exposed to UV light of Mask Aligner (JKG3,Shanghai,China,https://www.360docs.net/doc/7f19242502.html,/).The NOA81was cured by UV light except the chip channel.The NOA81in the channel can be washed off completely by the mixture of acetone and ethanol(V:V=8:2).In this way,the designed channel structure was fabricated on the microscope slides.In this way,the NOA81substrate with channel structure was bonded with microscope slides.Thirdly,the colloidal gold pad and NC membranes were placed gently in the chip channel by using tweezers.Then,a NOA81substrate with a fluid inlet and a fluid outlet was made on the glass wafers coated with a Cr layer(chrome plate)as described above.The NOA81substrate can be torn off from the chrome plate after UV curing.Finally,this NOA81substrate was bonded with the NOA81channel structure substrate prepared in the first step using the UV curing gun.Hence, an airtight microfluidic channel with a fluid inlet and a fluid outlet was formed,and the immunochromatographic chip was fabricated completely.

Results and discussion

Characterization of the colloidal gold

The TEM image of the colloidal gold nanoparticles revealed that the colloidal gold nanoparticles were of nice round shape and homogeneous in size.Average diameter of the colloidal gold particles was24.42±1.78nm(n=100)(Fig.1a).The maximum absorbance of the colloidal gold solution was at 521nm(Fig.1b).From the absorbance spectrum,it can also be found that the colloidal gold solution was at a stable state, and the prepared reproducibility was good.

Conjugation of monoclonal antibodies with colloidal gold nanoparticles

Colloidal gold nanoparticles are negatively charged having electrical layers around them.As a result,its stable state is due to the repulsive interactions among the colloidal gold nano-particles.The conjugation of protein(monoclonal antibodies herein)with colloidal gold nanoparticles predominantly de-pends on electrostatic interactions.When the pH value of the colloidal gold solution is slightly lower than the pI value of the protein,the protein is partially positively charged,which leads to the formation of the ionic bonds with the colloidal gold nanoparticles.When the colloidal gold particles are tremen-dously coated with the protein,NaCl is unable to induce the aggregation of colloidal gold nanoparticles.So,the optimal pH value and concentration of monoclonal antibodies concen-tration is crucial for the conjugation of monoclonal antibodies with colloidal gold.

Optimal concentration of monoclonal antibodies

for conjugation with colloidal gold

The concentration of monoclonal antibodies for conjugation with colloidal gold particles was optimized under the constant volume of colloidal gold particles and by increasing mono-clonal antibodies concentrations.The results are shown in Table S3and Fig.S2(See ESM).It was found that the color of colloidal gold was blue-black in the absence of monoclonal antibodies.By increasing the concentration of monoclonal antibodies from0.5to2μg,the color of mixture gradually changed from blue-black to blue-violet.When the monoclonal antibodies concentration was4μg,the color of mixture changed to purple red,however,when the concentration of monoclonal antibodies increased from6to12μg,the color of the mixture changed to red,and after that it remained almost

Gold nanoparticles-labeled immunochromatographic microchip

unchanged.So,6μg of monoclonal antibodies per 1mL of colloidal gold was selected as the minimal amount.The opti-mal concentration of monoclonal antibodies was 7.8μg mL ?1colloidal gold,i.e.,20%more than the minimal amount.Optimal pH value for conjugation of monoclonal antibodies with colloidal gold particles

The pH value for conjugation of monoclonal antibodies with colloidal gold was optimized under the constant volume of colloidal gold and the constant amount of monoclonal anti-bodies.The results are shown in Table S4and Fig.S3(see ESM)indicated that when pH value increased from 7.0to 8.0,the color of mixture turns gradually into brilliant red,and did not change further.However,the color of mixture changed from brilliant red to purplish red when pH value increased from 8.5to 9.5.As is clear,the optimal pH value for conju-gation of monoclonal antibodies with colloidal gold is the highest pH value for keeping the colloidal gold at a stable state.So,pH value of 8.0was selected as the optimal value for conjugation of monoclonal antibodies with colloidal gold particles.

Characterization of colloidal gold and monoclonal antibodies conjugate

The UV-visible absorption spectrum of colloidal gold parti-cles,colloidal gold -IL-6mAbs conjugate and colloidal gold particles -TNF-αmAbs conjugate was scanned by UV-visible spectrophotometer at a wavelength of 400to 600nm.As shown in Fig.S4(See ESM),the uncoated gold nanoparticles has a UV absorption wavelength at 521nm.When it was covered by the protein (antibody),the UV absorption wave-length will be changed.So,the results indicated that the

monoclonal antibodies were successfully conjugated with colloidal gold particles.

Determination of human IL-6with immunochromatographic strip

Standard solutions of IL-6with concentrations of 2,000,1,000,250,125,62.5and 0ng mL ?1were prepared by diluting IL-6with PB buffer.120μL of different concentra-t i o n s o f I L -6s a m p l e s w e r e u s e d i n t h e immunochromatographic strip detection assay.After 30min,various shades of red color on the test dots could be visually observed by naked eyes,the intensity of red color increased with the increase of IL-6concentration (Fig.2).When the concentration of IL-6was 62.5ng mL ?1,the red color on the test dots became almost invisible.The results indicated that the detection limit for IL-6is 62.5ng mL ?1.

Determination of TNF-αwith immunochromatographic strip Standard solutions of TNF-αat concentrations of 500,300,100,50,25,10,0ng mL ?1were prepared by diluting TNF-αwith PB buffer.120μL of different concentrations o f T N F -αs a m p l e s w e r e u s e d i n t h e immunochromatographic strip detection.As shown in Fig.3a ,the intensity of red color was elevated with the increase of concentration of TNF-α.It can be observed that with 50–100ng mL ?1concentration of TNF-αis,the test dots still showed clear red color.So,another group of TNF-αstandard solution at concentrations of 100,60,40,30,and 15ng mL ?1were prepared and detected by the strip.The results shown in Fig.3b demonstrated that when the concentrations of TNF-αis 30ng mL ?1,the red color on the test dots is almost invisible.Moreover,below

this

Fig.1TEM image of colloidal gold nanoparticles (a )and UV-visible spectrum of the colloidal gold solution (b ).Three lines in (b)represented that the colloidal gold nanoparticles were repeatedly prepared three times

M.Yan et al.

concentration,no red color was observed on the test dots.So,the detection limit of TNF-αfor the test strip was found to be 30ng mL ?1.The whole analysis process using immunochromatographic strip test for determination of TNF-αwas completed in 10min.

Similar results were also found for some macromolecular proteins detection using the same typical method.For exam-ple,the detection limits of 30,50,and 50ng mL ?1were obtained for troponin I [25],ricin [26],and human IgG (HIgG)[27],respectively with the naked

eyes.

Fig.2Determination of human IL-6with different concentrations using the prepared IL-6colloidal gold immunochromatographic

strip

Fig.3Determination of human TNF-αwith different

concentrations using the prepared TNF-αcolloidal gold

immunochromatographic strip

Gold nanoparticles-labeled immunochromatographic microchip

In addition,in order to study the stability of antibody-gold conjugates in a complex media such as serum and if the prepared strip can be appropriate for detecting TNF-αin serum,the TNF-αconcentrations of 160,80,and 0ng mL ?1were prepared by diluting TNF-αwith human serum,then the different concentrations of TNF-αwere detected using the prepared strips.As shown in Fig.S5(See ESM),the color

of testing dots was in accordance with the Fig.3.So the serum sample can be detected by the prepared strip and the serum has no influence on the stability of antibody-gold conjugates.Simultaneous detection of IL-6and TNF-αusing the immunochromatographic strip

For the simultaneous detection of IL-6and TNF-α,IL-6test dots,TNF-αtest dots and control dots were prepared on one NC membrane simultaneously.The antibody concentration of the dots is 1mg mL ?1.Rests of the steps are the same as mentioned above.The concentrations of IL-6and TNF-αsamples used in the prepared strip can be seen in Table S5(See ESM),and test results are shown in Fig.4.Strip No.1showed red color both at IL-6and TNF-αtest dots;strip No.2showed only red color at IL-6test dots,and strip No.3showed red color only at TNF-αtest https://www.360docs.net/doc/7f19242502.html,pared to strip No.1,strip No.2and strip No.3can be selected as a control test.From the control test,it was found that there is no false positive in the test strip.

Simultaneous detection of IL-6and TNF-αusing the prepared colloidal gold immunochromatographic microchip by visual observation

T h e 3D s t r u c t u r a l d i a g r a m o f t h e p r e p a r e d immunochromatographic chip is shown in Fig.5a .A tube (1mm O.D.×0.5mm I.D.)was inserted in the inlet and connected to the peristaltic pump.The sample flowed into the chip under the drive of peristaltic pump,and reached the conju-gate pad first.The colloidal gold-monoclonal antibody on

the

Fig.4Simultaneous detection of IL-6and TNF-αusing the prepared colloidal gold immunochromatographic

strip

Fig.5Simultaneous detection of IL-6and TNF-αusing the prepared colloidal gold immunochromatographic

microchip.a shows 3D structural diagram of the prepared

immunochromatographic chip.The sample containing 1,000ng mL ?1of IL-6and 500ng mL ?1of TNF-αwas detected in (b ).The sample only containing 500ng mL ?1of TNF-αwas detected in c

M.Yan et al.

conjugate pad will capture the target in the sample. Then the colloidal gold-monoclonal antibody-target complex flowed into the channel,and would be cap-tured by the polyclonal antibody on the NC membrane. The unbound colloidal gold nanoparticles will be cap-tured by the second antibody.So,when the sample contains the target,both the test dots and control dots will show red color.On contrary,when the sample does not contain target,only control dots will show the red color.

As for the colloidal gold immunochromatographic microchip for simultaneous detection of IL-6and TNF-α,the concentration of the polyclonal antibody and the second antibody concentration of IL-6and TNF-αwas1mg mL?1each on the NC membrane. The samples containing1,000ng mL?1of IL-6and 500ng mL?1of TNF-αwere injected in the chip as shown in Fig.5b using a peristaltic pump(TS-2A/ L0107-2A,China,https://www.360docs.net/doc/7f19242502.html,/)with the flow rate of15μL min?1.At the same time,the sample containing only500ng mL?1of TNF-αwas injected in the chip as shown in Fig.5c.

As shown in Fig.5b,two test dots and one control dot altogether showed red color due to injection of the sample containing IL-6and TNF-α,while only the TNF-αtest dot and control dot showed red color in Fig.5c as a result of injection of the sample only containing TNF-α.It can be concluded that simultaneous detection of IL-6and TNF-αw a s p e r f o r m e d b y u s i n g t h e p r e p a r e d immunochromatographic chip based on the colloidal gold particles.

Conclusions

Simultaneous and visual detection of IL-6and TNF-αwas successfully achieved by using the immunochromatographic strip.Moreover,combining the colloidal gold immunochromatographic strip with microchip,IL-6and TNF-αwere simultaneously and visually detected on micro-chip as well.The developed immunochromatographic strip and microchip offered considerable advantages,i.e.,high specificity,required no pretreatment,less sample amount,easy to operate,rapid determination,no cross-reaction,and long-term preservation.With further development,the immunochromatographic strip and microchip might be cho-sen as a potential analytical tool in real time detection. Acknowledgments This work was supported by the National Nature Science Foundation of China(21275019),the National Key Technology R&D Program of China(2009BAK59B01)and National Key Scientific Instrument and Equipment Development Projects of China (2012YQ040140).References

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