Gold Nanoclusters as Signal Amplification Labels for Optical Immunosensors
ARTICLE
https://www.wendangku.net/doc/2213728905.html,/JPCC Gold Nanoclusters as Signal Amplification Labels for
Optical Immunosensors
Hongying Liu,?,?Ximei Wu,?Xiang Zhang,?Clemens Burda,*,?and Jun-Jie Zhu*,?
?State Laboratory of Analytical Chemistry for Life Science,School of Chemistry and Chemical Engineering,Nanjing University, Nanjing210093,P.R.China
?Center for Chemical Dynamics and Nanomaterials Research,Department of Chemistry,Case Western Reserve University,Cleveland, Ohio44106,United States
low-cost optical immunosensor has been developed using environment-friendly gold nanoclusters
The stable and robust?lm,poly(dopamine),was used to immobilize biomolecules on an indium construction of biosensor,as con?rmed by scanning electron microscopy,contact angle,and electrochemical Human IgG served as a model to demonstrate the performance of the proposed sensor.Through
IgG could be qualitatively and quantitatively determined by colorimetry,?uorescence method,
1.INTRODUCTION
Immunoassay is a signi?cant research area because it provides the opportunity for understanding fundamental biological pro-cesses involving in disease progression and monitoring patient responses to therapy methods.1Therefore,developing sensitive, portable,and low-cost methods for the detection of disease-related biomarkers has attracted considerable attention.Many methods have been developed to improve the sensitivity of immunosensors.Some of them have already been implemented in clinical diagnosis.2à4However,increasing demands for early and ultrasensitive monitoring of disease-related biomarkers are pushing the milestone for sensitive detection of biomarkers by signal ampli?cation.5,6Among them,nanoparticle-based signal ampli?cation has attracted wide interest due to its unique optical, electronic,and biocompatible properties.For example,an im-munosensor with CdTe quantum dots(QDs)as the?uorescent and electrochemical label was reported.7Considering the toxicity of QDs,it is important to develop nontoxic?uorescence materials to replace the QDs.Recently,gold nanoparticles (AuNPs)have been used as labels in the bioassay because of their inherent advantages,such as easy preparation and good biocompatibility.8,9Moreover,the technique using AuNPs probes with silver enhancement has been used in immunoassays to improve the sensitivity of the immunosensor.10à14In the presence of silver ions and reducing reagents,the AuNPs could be used as catalysts to reduce silver ions to metallic silver covered on the surface of AuNPs cores,which can typically be quanti?ed for the target with colorimetry or semiquanti?ed with eye observation.15
Recently,optical techniques have been widely used in the immunosensors.16Among these optical techniques,the gel imaging system17,18is an e?ective candidate for the determina-tion of biomolecules.Concurrently,colorimetry also has been used in the biosensing because of its rapid signal generation, portability,and low cost.19à21Using this method,protein could be quanti?ably or semiquanti?ably assessed in real time without
Received:July2,2011
Revised:October31,2011
Published:December21,2011
any advanced instruments.Recent studies demonstrated that gold nanoclusters (AuNCs)were supposed to be attractive labels due to their low toxicity,good biocompatibility,stability,?uor-escence,and AuNP-related intrinsically properties.22à25It could be used in the immunosensing by combining the gel imaging system and colorimetry,which pushed the milestone for sensitive detection of biomarkers.
Immobilization of biomolecules on the substrate surface is a crucial step in the fabrication of sandwich-type immunosensors.Inspired by adhesive proteins secreted by marine mussels,Lee reported the preparation of the multifunctional biopolymer poly-(dopamine)(P-DA)layer on a wide range of inorganic and organic materials through self-polymerizing dopamine in an aqueous solution.26Furthermore,the P-DA layer could be used to immobilize biomolecules onto the substrate surface.27,28
Herein,we report the fabrication of a portable,low-cost optical immunosensor for sensitive detection of protein using AuNCs as labels for signal ampli ?cation as shown in Scheme 1.The antibody was immobilized on an ITO chip by the multi-functional biopolymer P-DA monolayer.The formation process of the immunosensor was characterized by scanning electron microscopy (SEM),electrochemical impedance spectroscopy (EIS),contact angle,and ?uorescence microscopy image.The protein could be qualitatively and quantitatively determined by colorimetry,?uorescence,as well as eye observation according to the clinical requirement.
2.EXPERIMENTAL SECTION
2.1.Materials.1-Ethyl-3-(3-dimethylaminopropyl)carbodii-
mide hydrochloride (EDC),goat antihuman IgG,rabbit antihuman IgG,human IgG,and bovine serum albumin (BSA)were purchased from Sigma-Aldrich.Chloroauric acid (HAuCl 4)and silver-staining solution (A and B)were obtained from Shanghai Reagent (Shanghai,China).ITO-coated glass slides with surface resistance of 30à60Ω/cm 2were purchased from Condue Optics and Electronics Technol-ogy (Jintan,China).The phosphate buffer solution (PBS,50mM)
with various pH values was prepared by mixing NaH 2PO 4and Na 2HPO 4stock solution and was then adjusted with NaOH and H 3PO 4(0.05M).All other reagents were of analytical reagent grade and used without further purification.
2.2.Characterizations.Imaging Software Quantity One was from BIO-RAD Company.The UV àvis absorption spectra were carried on a Shimadzu 3600UV àvis spectrometer (Shimadzu,Japan).The fluorescence measurements were carried out on a NF920fluorescence spectrometer.Fluorescence microscopy images were taken by a Nikon TE2000-U inverted optical micro-scope.The high-resolution transmission electron microscopy (HRTEM)image was observed by JEOL JEM-2100.Scanning electron microscopy (SEM)image was obtained from JEOL JSM-6340F.X-ray photoelectron spectroscopy (XPS)was carried out on an ESCALAB MK II X-ray photoelectron spectrometer.All Fourier-transform infrared (FTIR)spectroscopic measurements were performed on a Bruker model VECTOR22Fourier-transform spectrometer using KBr pressed disks.The static contact angles were measured by a contact angle meter (Rame-Hart-100)employ-ing drops of double-distilled water.The readings were stabilized and taken within 120s.Electrochemical impedance spectroscopy (EIS)were performed with an Autolab electrochemical analyzer (Eco Chemie,The Netherlands)in 0.1M KNO 3containing 5mM Fe(CN)63àand 5mM Fe(CN)64à.
2.3.Synthesis of AuNCs.AuNCs were synthesized according to the literature with mild modification.29In brief,HAuCl 4solution (20mL,10mM,37°C)was added to BSA solution (20mL,50mg/mL,37°C)under vigorous stirring.After 2min,2mL of NaOH solution (1M)was introduced,and the mixture was incubated at 37°C for 12h.The solution color changed from light-yellow to light-brown and finally to deep brown.The obtained AuNCs were further purified by ultrafiltration.30The first step was carried out on 30000MW filter and centrifugated at 3000g for 15min at 4°C to remove unreacted BSA.The AuNCs were washed three times with 50mM PBS (pH 7.4).The upper level was decanted,dissolved in PBS,and subjected additionally to a second ultrafiltration step on 50000MW filter.After the
Scheme 1.Schematic Illustration of the Optical Immunosensor
Using Environment-Friendly Luminescent Gold Nanoclusters As Labels
second ultrafiltration step,the lower level was concentrated on 3000MW filter to get the final AuNCs.The purified water-soluble AuNCs possessed bright fluorescence and good stability in the PBS.
2.4.Bioconjugation of AuNCs with Antibody.The con-jugation procedure of AuNCs with rabbit antihuman IgG (Ab 2)was similar to previous reports.31,32We added 100μL of freshly prepared EDC solution (4.2mg/mL)to 1mL of purified AuNCs (dissolved in 10mM PBS,pH 7.4)and vortexed the solution for 20min.Then,20μL of rabbit antihuman IgG (0.5mg/mL)was added to the mixture.The solution was incubated at room temperature (RT)and shaken in the dark for 24h.Then,the unreacted AuNCs as well as the byproduct of the conjugation were removed by ultrafiltration.The above mixture was sub-jected to ultrafiltration using 50000MW filter.After the lower level was removed,the upper level containing AuNCs-antibody was decanted;then,50mM PBS with 1%BSA was added.The solution was stored at 4°C.
2.5.Preparation of Immunosensor.In brief,2mg/mL of dopamine was dissolved in 10mM Tris-HCl (pH 8.5),and the ITO chip was dipped into the solution at RT for 12h after cleaning with acetone,ethanol,and distilled water in sequence.The coated surface was rinsed with distilled water and dried under a stream of nitrogen,and a 3-mm-thick poly(dimethylsiloxane)(PDMS)with a circular opening of 4-mm diameter was bound to ITO surface to form specific reaction area.Then,10μL of goat antihuman IgG solution (Ab1,25μg/mL)was spread on the chip surface and incubated at 4°C in a moisture chamber for 12h.After incubation,the chip was rinsed with PBST (PBS +0.05%Tween-20)to remove physically adsorbed Ab 1.Then,the chip was incubated in the 2%BSA (PBS)at 37°C for 1h to block excess active group and nonspecific binding site.The chip was washed with PBST before use and incubated with several different concentrations (from lower to higher)of human IgG antigen (10μL)at 37°C for 50min,followed by washing with PBST to remove the nonspecific adsorption.Next,10μL of AuNCs-Ab 2was added to the reaction area of the chip and incubated at 37°C for another 50min.To decrease background response,the chip was washed with double-distilled water to remove nonspecifically bound AuNCs-Ab 2.
2.6.Optical Immunosensor.Fluorescence Detection.Fluo-rescence immunoassay was performed by using the gel imaging system (Bio-Rad).The relative intensity of each dot was scored by using a quantitative analysis software program Quantity One (Bio-Rad).33,34The intensity of fluorescence was named after lightness density (LD).Performing the fluorescence properties of AuNCs,when more AuNCs are immobilized on the surface of the modified electrode,the LD become larger.
Colorimetric Detection.For colorimetric detection,the chip was covered with 10μL of silver enhancement solution for 10min,which was prepared by mixing the silver-staining solutions A and B with the ratio of 1:1;then,the picture was taken by the gel image system.The relative intensity of each dot was scored by the quantitative analysis software program-quantity one (Bio-Rad).The color intensity was named after darkness density (DD).When little silver ion was reduced,the DD value was low.
Figure 1.(A)Fluorescence emission and UV àvisible absorption spectra of the AuNCs solution.Insert:photographs under ultraviolet light (left)and visible light (right)for the AuNCs solution.(B)Fluorescence intensity of the AuNCs (λmax =630nm)in PBS (50mM)at di ?erent pH.(C)Fourier-transform infrared (FTIR)spectra of
BSA and BSA-AuNCs.(D)HRTEM image of the AuNCs.
More silver metal precipitated on the surface of the film can make the color turn darker,which results in the increased DD value.
3.RESULTS AND DISCUSSION
3.1.Characterization of AuNCs.The AuNCs were synthe-
sized through the reduction of HAuCl 4with BSA to form covalent Au àS bonds and characterized by UV àvis absorption spectrum (UV àvis),fluorescence spectrum,Fourier-transform infrared spectrum (FTIR),high-resolution transmission electron microscopy (HRTEM),and X-ray photoelectron spectroscopy (XPS).As shown in Figure 1A,the AuNCs emitted an intense red light under ultraviolet light and deep brown light under visible light.They exhibited a broad absorption in the range with an onset at 500nm and the strong emission peak at 630nm with an excitation maximum around 517nm.The quantum yield of AuNCs is 6%,which is calculated by using rhodamine B in ethanol as a reference.The relationship between the fluorescence intensity and pH was also studied,as shown in Figure 1B.The pH value could not obviously affect the fluorescence intensity of AuNCs.Furthermore,considering that the value of physiological pH is 7.4,PBS of pH 7.4was selected in the following experi-ments.In addition,the FTIR curve of the AuNCs is similar to that of BSA,as shown in Figure 1C.The peaks in the spectra can be assigned as follows:1654cm à1corresponds to a protein with a high proportion of α-helix,which is the characteristic amide band of BSA;1545cm à1is attributed to strong primary amine scissoring while 3435cm à1is attributed to primary amines;2958cm à1is ascribed to C àH vibration;and 701cm à1is attributed to àNH 2and àNH wagging.The disappearance of S àH stretching band (2525cm à1)of AuNCs is also observed,suggesting the formation of covalent bonds between S àH and AuNCs.This result indicates that BSA is modified on the surface of AuNCs.Moreover,the HRTEM image in Figure 1D indicates that the particles are well-dispersed.XPS result was also used to measure the binding energy of the AuNCs.The observed values for Au 4f 7/2and 4f 5/2were 83.98and 87.58eV,respectively,which is consistent with the metallic Au (0).35
3.2.Characterization of the P-DA/ITO.The immobilization of antibody on ITO chip is an important factor for the fabrication of immunosensor.Recently,Lee reported an effective method to immobilize biomolecules through self-polymerization of dopa-mine.This film is suitable for our requirement because it could provide good adhesion to immobilize antibody.According to ref 36,the ITO chip was immersed in 2mg/mL of dopamine solution (dissolved in 10mM Tris-HCl buffer solution (pH 8.5))
at 4°C for 24h.Then,SEM and contact angle experiments were carried out to characterize this process.As shown in Figure 2A,the P-DA film by spontaneous oxidative polymerization of dopamine was modified to the ITO surface.Moreover,the biocompatibility for loading biomolecules and preserving their bioactivity could be characterized by the hydrophilicity,which could be measured by the contact angle of the bare ITO and P-DA film-modified ITO.As shown in Figure 2B,C,the contact angles of the bare ITO and P-DA film-modified ITO were 28°and 76°,respectively.The P-DA film-modified ITO showed lower contact angle,showing better hydrophilicity.37,38There-fore,the P-DA film-modified ITO could provide a biocompatible surface,which was beneficial to antibody immobilization.
3.3.Characterization of the Immunosensor.EIS was used to study the stepwise assembly process of the immunosensor because it is an effective tool to characterize interface properties of surface-modified film.39,40In EIS,the impedance spectrum includes a semicircle portion at high frequencies corresponding to an electron transfer limited process,and a linear portion at low frequencies represents a diffusion-limited electrochemical pro-cess.The electron transfer resistance (Ret)of the modified layer is calculated by semicircle diameter,which is used to show its blocking behavior of electrode.The modified process is char-acterized by the Ret change,41,42as shown in Figure 3A.It exhibits the Nyquist plots of EIS for the bare ITO (a),P-DA/ITO (b),and Ab 1/P-DA/ITO (c)https://www.wendangku.net/doc/2213728905.html,pared with the bare ITO,P-DA modified ITO shows a larger semicircle diameter.The reason is that P-DA is a nonconductive polymer,which blocks the electron transfer of the redox probe [Fe(CN)6]3?/4?.Additionally,after Ab 1was modified on the P-DA/ITO chip,the semicircle diameter of EIS increased again as compared with the P-DA/ITO chip.This indicated that P-DA film and Ab 1were successfully modified on the bare ITO and P-DA/ITO in sequence.As reported,dopamine could self-polymerize to form thin and surface-adherent P-DA film.The P-DA film is easily adapted for a variety of materials without surface pretreatment.It can offer selectivity of reaction with amine or imidazole func-tional groups of biomolecules.Consequently,the P-DA film could be modified on the ITO surface by dip-coating of objects in dopamine solution,and Ab 1could be immobilized on the P-DA/ITO surface with high stability and bioactivity by immersing the P-DA/ITO into Ab 1solution (pH 7.4).
Fluorescence microscopy was further used to image the bio-activity and speci ?city of AuNCs-Ab 2,which resulted from the recognition reaction between Ag and AuNCs-Ab 2conjugates.After Ab 1was immobilized on the ITO chip with P-DA ?lm,
Figure 2.(A)SEM image of the poly(dopamine)on the surface of the ITO chip.Contact angle
of the bare ITO (B)and poly(dopamine)/ITO (C)chip.
HIgG was immobilized through the speci ?c reaction between Ag and Ab.After blocking with BSA,AuNCs-Ab 2conjugates were covered on the surface of HIgG-modi ?ed ITO chip by the reac-tion of Ag and Ab.The binding reaction continued for 50min at 37°C.As shown in Figure 3C,a strong red ?uorescence was observed at the HIgG-coated area after being incubated with the AuNCs-Ab 2,indicating that AuNCs-Ab 2was successfully at-tached to the surface of HIgG-modi ?ed ITO chip.However,after AuNCs were replaced with AuNCs-Ab 2to incubate HIgG-modi ?ed ITO,as shown in Figure 3B,no ?uorescence was ob-served,which con ?rmed that nonspeci ?c adsorption was negli-gible.The above results proved that the AuNCs-Ab 2conjugates could be used as labels in bioassay.
3.4.Immunoassay on the ITO Chip.Because of the attractive properties of AuNCs,such as nontoxic,red emission,and good biocompatibility,the AuNCs can be selected as the signal amplification labels for the detection of protein in gel imaging system by fluorescence and colorimetry.The protein could be quantifiably measured by colorimetry and fluorescence or qua-litatively determined by eye observation within 10min to match the clinical requirement.
Fluorescence Detection.First,fluorescence immunoassay in the gel imaging system was fabricated to determine the concentration
of HIgG.AuNCs are extremely efficient in emitting highly stable fluorescence.Protein could be determined with the gel imaging system in 5min by immunoreaction.Figure 4showed the fluorescence image for the ITO chip in gel imaging system.The relative amount of each result was scored by quantity one software (Bio-Rad).The fluorescence intensity of Ab-spotted area increased with the HIgG concentration from 0.1to 600ng/mL (B àF),which showed good quantitative results with the detection limit as low as 0.04ng/mL (n =3).The control experiment was conducted through the same procedure without exposure to Ag,as shown in the insert of Figure 4,which indicated that the nonspecific adsorption could be negligible.To obtain good precision,five repetitive measurements were carried out in 0.1,100,and 500ng/mL HIgG.The RSD was 5.6%,6.0%,and 5.0%,respectively.The interassay precision or the fabrication reproducibility was estimated by determining the HIgG level with five immunosensors fabricated at the same ITO independently.
Colorimetrical Detection.Although fluorescence is suitable for the determination of protein,the sensitivity needs improve-ment for clinical requirement.Colorimetry is convenient and attractive for its simplicity in monitoring disease.On the basis of dual signal amplification of AuNCs and silver enhancement,
Figure 3.(A)Nyquist diagrams for the electrochemical impedance measurements of ITO chips in 0.1M KNO 3solution containing 5mM Fe(CN)63àand 5mM Fe(CN)64à:(a)bare ITO chip,(b)P-DA/ITO chip,and (c)Ab 1/P-DA/ITO chip.(B)Fluorescence microscopy image of the HIgG-coated ITO chip incubated with AuNCs under UV illumination.(C)Fluorescence microscopy image of HIgG-coated ITO chip incubated with the AuNCs-Ab 2under UV illumination.
Figure 4.Lightness density versus the concentration of HIgG.Insert:?uorescence images of gel imaging system on the ITO chips for di ?erent HIgG concentration (from A to F:0,0.1,1.0,10,100,and 600ng/mL HIgG,respectively).
Figure 5.Darkness density versus the concentration of HIgG.Insert:colorimetric images on the ITO chips for di ?erent HIgG concentration (from A to F:0,0.01,
0.1,
1,
10,and 100ng/mL HIgG,respectively).
sensitivity of the immunosensor could be improved.In this way, the concentration of HIgG could be observed by the change of solution color.The colorimetrical signal was captured by a regular camera or observed by eye in an appropriate illumination, which proceeded after AuNCs-Ab2incubation and silver en-hancement process.The amount of metallic gold depended on the detection of antibody bounded to the target antigen.The color intensity,taken from the reaction wells,visually estimates the concentration of target antigen.The relative amount of each result was also measured with quantity one(Bio-Rad).Theore-tically,the more target antigens were immobilized,the more antibodies were detected;as a result,the deeper color could be observed.As shown in the insert of Figure5,the DD value increased gradually with the increase in HIgG concentration from0.01to100ng/mL.The plot in Figure5showed that the DD level was related to the logarithm of the HIgG concentration in the range from0.01to100ng/mL.Control experiments were conducted through the full procedure without exposure to Ag,as shown in Figure5.It was observed that the DD value of control well was higher than that of other wells,which showed that the nonspecific adsorption was negligible.To evaluate the precision of this method,we carried out five repetitive measurements with the concentration of0.1and10ng/mL.The RSDs are7.8%and 6.5%,respectively,which are yielded by the above experiments. In addition,the concentration of HIgG could be detected by eye within10min without any advanced equipment.It is a portable and highly sensitive method for the determination of antibody with good stability and reproducibility.
3.5.Evaluation of the Immunosensor.Specificity,regenera-tion,and stability are three important factors to evaluate sensors. First,the specificity was evaluated by the potential interference from coexisting species toward HIgG detection,such as carci-noembryonic antigen and C-reactive protein.Each potential interference at a concentration of10ng/mL in the presence of HIgG(10ng/mL)was used to evaluate its selectivity in comparison with HIgG alone.It was determined by fluorescence and colorimetry,respectively.No obvious significant difference of lightness intensity(RSD5.6%)and darkness intensity(RSD 6.0%)was observed by comparing with the result given by HIgG only.No interference was observed in the experiment. Furthermore,the regeneration was obtained by rinsing the sensor with glycine-HCl solution to dissociate antigenàantibody https://www.wendangku.net/doc/2213728905.html,pared with the initial value after?ve assay runs,the renewed immunosensor-restored?uorescence and darkness inten-sity with95.2and92%,respectively,showing accepted reusability. When the sensor was not in use,it was stored at4°C.No obvious signi?cant change was observed in1month of storage at 4°C,which indicated that the immunosensor was of acceptable stability.
3.6.Application of the Optical Immunosensor in Human Serum.The result tested by the proposed optical method for human serum sample was compared with that of ELISA.These serum samples were diluted with PBS(7.4).The comparison is shown in Table1.The relative deviation of these methods is from 3.4%to11.2%,suggesting no significant difference for fluores-cence and colorimetry.Moreover,no obvious difference between our method and ELISA is observed;that is,the proposed immunosensor can be reasonably and satisfactorily applied in the clinical determination of HIgG levels in human plasma.
4.CONCLUSIONS
In summary,a sensitive optical immunoassay for the determi-nation of HIgG was developed by using environmentally friendly AuNCs as labels for signal ampli?cation.This is the?rst example of?uorescence AuNCs as probes in the immunoassay.Protein could be determined by colorimetry,?uorescence,and eye observation according to the clinical requirement.This sensor is portable,low-cost,and visualization.In addition,this method is environmentally friendly,simple,and precise with high sensitiv-ity and excellent selectivity toward HIgG.This method has the potential for reliable point-of-care diagnostics of cancer and other diseases and will expand readily in detecting other relevant biomarkers.
’ACKNOWLEDGMENT
We greatly appreciate the support of the National Natural Science Foundation of China(21020102038and21121091). This work is also supported by National Basic Research Program of China(2011CB933502)and the Fundamental Research Funds for the Central Universities(1112020504).
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