Triple Signal Amplification of Graphene Film, Polybead Carried Gold

Article pubs.acs.org/ac

Triple Signal Amplification of Graphene Film, Polybead Carried Gold Nanoparticles as Tracing Tag and Silver Deposition for Ultrasensitive Electrochemical Immunosensing Dajie Lin,† Jie Wu,*,† Min Wang,† Feng Yan,‡ and Huangxian Ju*,† †

State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093, P.R. China ‡ Jiangsu Institute of Cancer Prevention and Cure, Nanjing 210009, P.R. China ABSTRACT: A triple signal amplification strategy was designed for ultrasensitive immunosensing of cancer biomarker. This strategy was achieved using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead (PSA) carried gold nanoparticles (AuNPs) as tracing tag to label signal antibody (Ab2) and AuNPs induced silver deposition for anodic stripping analysis. The immunosensor was constructed by covalently immobilizing capture antibody on chitosan/electrochemically reduced graphene oxide film modified glass carbon electrode. The in situ synthesis of AuNPs led to the loading of numerous AuNPs on PSA surface and convenient labeling of the tag to Ab2. With a sandwich-type immunoreaction, the AuNPs/PSA labeled Ab2 was captured on the surface of an immunosensor to further induce a silver deposition process. The electrochemical stripping signal of the deposited silver nanoparticles in KCl was used to monitor the immunoreaction. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect carcinoembryonic antigen with a linear range of 0.5 pg mL−1 to 0.5 ng mL−1 and a detection limit down to 0.12 pg mL−1. The immunosensor exhibited good stability and acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.

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been used as electroactive labels to develop enzyme-free immunosensing strategies. For example, QDs have been used as trace labels to develop sensitive electrochemical bioassay of DNA and proteins by measuring the metallic component of the QDs. Unfortunately, the QDs-based detection requires harsh detection conditions, including nanocrystals dissolution, high potential accumulation, and deoxygenation, which is not suitable for clinical application. Gold nanoparticles (AuNPs) are the most used metal nanoparticles in electrochemical immunosensors due to its unique abilities of good biocompatibility and easy functionalization with proteins. Although AuNPs tags can be directly detected by anodic stripping analysis in HCl, the electrochemical oxidization occurs at a relatively positive potential.19 Compared with AuNPs, silver nanoparticles (AgNPs) can be oxidized at more negative potential with a relatively sharp peak, which is favorable to obviating the interference of reducing species and improving the detection precision and sensitivity. Our previous work designed a streptavidin-functionalized AgNPs-enriched CNT tag to label the signal antibody and develop an ultrasensitive

ighly sensitive and selective immunoassay capable of detecting protein biomarker is essential for disease diagnosis, drug screening, and biodefense applications.1,2 Among various immunoassay methods, electrochemical immunoassay has been well developed because of the intrinsic advantages of good portability, low cost, and high detection sensitivity,3−5 resulting in great progress in the field of electrochemical immunosensors. Recently, great efforts have been made to develop ultrasensitive immunosensors for the detection of low-abundance biomarkers. Signal amplification is the most popular strategy that has been employed for the development of ultrasensitive immunoassay methods. Normally, the signal amplification can be achieved using multienzyme report probes, which are prepared by bioconjugating large amount of enzymes, including alkaline phosphatase,6,7 horseradish peroxidase,8−12 and glucose oxidase,3 on nanocarriers, such as nanoparticles, 8,10,12 carbon nanotubes (CNTs),3,6,11 and magnetic beads,9,13 to amplify the detection signals. However, the practical applications of these nanomaterial-based multienzyme probes are limited due to ease of denaturation, leakage, and the time-consuming and costly preparation and purification process of enzymes.14−16 To overcome these limitations, nanomaterials, such as quantum dots (QDs)17,18 and metal nanoparticles,19−21 have © 2012 American Chemical Society

Received: January 14, 2012 Accepted: March 21, 2012 Published: March 21, 2012 3662

dx.doi.org/10.1021/ac3001435 | Anal. Chem. 2012, 84, 3662−3668

Analytical Chemistry

Article

Scheme 1. Schematic Representation of (A) the Preparation of Tracing Tag and Labeled Ab2, and (B) Immunosensor Fabrication and Sandwich-Type Immunoassay Procedure

multiplexed electrochemical immunoassay method.21 By combinination with AgNP-promoted Ag deposition, this method could detect biomarkers down to sub pg mL−1. However, this method needed a complicated synthesis and labeling procedure, and the AgNP probe was unstable. As AuNPs can in situ catalyze the deposition of silver22,23 and be conveniently labeled to the signal antibody (Ab2), the immunoassay using AuNPs-catalyzed Ag deposition to amplify the anodic stripping signal have been quickly developed.24,25 This work further combined the AuNPs-catalyzed Ag deposition with graphene accelerated electron transfer and microbead carried AuNPs to design a triple signal amplification strategy with which an ultrasensitive electrochemical immunosensing method was developed. The microbead carried AuNPs prepared by a convenient in situ synthesis of AuNPs on a poly(styrene-co-acrylic acid) microbead (PSA) surface showed uniform size distribution and good stability, and could easily serve as a tracing tag to label Ab2 (Scheme 1A). The graphene was immobilized on a immunosensor surface using chitosan and electrochemically reduced to accelerated the electron transfer, and the amino group on chitosan film was used to covalently link the capture antibody (Ab1) (Scheme 1B). After sandwich-type immunoreaction, the anchored AuNPs further induced the chemical deposition of silver for electrochemical stripping analysis of target antigen. This method avoided the deoxygenation procedure for usual electrochemical detection and harmful solution for dissolution of tracing tag, and showed a detection limit down to sub pg mL−1 level, corresponding to nanomaterial-based multienzyme amplification strategies (pg mL−1).3,9 The assay approach had acceptable stability, precision, and accuracy, showing potential applications in clinical diagnostics.

Zhenglong Biochem. Lab (Chengdu, China). Bovine serum albumin (BSA), poly(diallyldimethylammonium chloride) (PDDA, 20%, w/w in water, MW: 200 000−350 000), silver enhancer solutions A and B, and chitosan were purchased from Sigma-Aldrich (USA). Styrene and acrylic acid were purified by distillation under vacuum and stored at −25 °C. CEA standard solutions from 0 to 75 ng mL−1 were from a CEA ELISA kit, which was supplied by Fujirebio Diagnostics AB (Göteborg, Sweden). Chloroauric acid (HAuCl4·4H2O) and trisodium citrate were obtained from Shanghai Reagent Company (Shanghai, China). Sodium borohydride (NaBH 4) was obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Ultrapure water obtained from a Millipore water purification system (≥18 MΩ, Milli-Q, Millipore) was used in all assays. The clinical serum samples were from Jiangsu Institute of Cancer Research. All other reagents were of analytical grade and used as received. Tris-HNO3 buffer (0.1 M, pH 7.4) containing 0.05% (w/v) Tween-20 was used as the washing solution. Tris-HNO3 (0.1 M) containing 5% or 10% (w/v) BSA was used as blocking solution for preparation of immunosensor or labeled Ab2, respectively. The mixture solution of silver enhancer solutions A and B was freshly diluted at 20 times for silver-deposition enhancement. Preparation of Tracing Tag and Labeled Ab2. The PSA was prepared according to the method reported previously.26 Briefly, 40 mL of ultrapure water was filled into a three-necked flask submerged in a water bath and purged with nitrogen for 1 h. Styrene (5.25 g) and acrylic acid (0.18 g) were added into the three-necked flask under stirring at 350 rpm at 70 °C and purging with N2, respectively. The emulsifier-free emulsion copolymerization of the styrene and acrylic acid was started by adding 10 mL of 0.01 g mL−1 ammonium persulfate solution into the mixture. After refluxing for 8 h, the resulting PSA suspension was centrifuged three times with ultrapure water at 13 000 rpm for 20 min, and stored at 4 °C.

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EXPERIMENTAL SECTION Materials and Reagents. Mouse monoclonal capture and signal anticarcinoembryonic antigen (CEA) antibodies (clone no. 27D6 and 28E4) were purchased from Shuangliu 3663



Article

Figure 1. (A) SEM image of PSA cores; (B) TEM image of tracing tag; (C) XPS spectra of PSA (curve a) and tracing tag (curve b); (D) XPS spectra of tracing tag (curve a) and labeled Ab2 (curve b).

for further reaction for 2 h. Water (2.8 L) and 30% H2O2 (50 mL) were finally added to produce a brilliant yellow color along with bubbling. The mixture was suspended in water and subjected to dialysis for 1 week to remove the residual salts. After the mixture was dried at 50 °C overnight, the obtained powder was exfoliated into GO by ultrasonicating a 0.05 wt % aqueous dispersion for 30 min, and the unexfoliated graphite oxide was removed by ultrafiltration at 2000 rpm for 5 min. The glassy carbon electrode (GCE) with 3 mm diameter was polished to a mirror using 1.0, 0.3, and 0.05 μm alumina slurry (Buehler) followed by rinsing thoroughly with deionized water. After successive sonication in 1:1 nitric acid, acetone, and deionized water, the electrode was rinsed with water and allowed to dry at room temperature. As shown in Scheme 1B, 5 μL of 0.5 mg mL−1 GO solution was dropped on the pretreated GCE, which was dried in air. Then 3 μL of 0.05% chitosan solution was dropped on the GO film and dried in air. After electrochemical reduction of GO at −1.0 V in pH 8.0 PBS, the modified electrode was washed with water and incubated with 5 μL of 2.5% glutaraldehyde (in 50 mM PBS, pH 7.4) for 2 h, followed by washing with water. Five microliters of 0.2 mg mL−1 anti-CEA Ab1 was then dropped onto the surface and incubating sequentially at room temperature for 60 min and 4 °C overnight in a 100% moisture-saturated environment. Subsequently, excess antibody was removed with washing buffer and pH 7.4 Tris-HNO3, respectively. Finally, 5 μL of blocking solution was dropped on the electrode surface and incubated for 60 min at room temperature to block possible remaining active sites against nonspecific adsorption. After another wash with washing buffer and pH 7.4 Tris-HNO3, the immunosensor was obtained. Apparatus and Measurement Procedure. The morphology of the polybeads was examined using a JEM 2100 highresolution TEM (Japan). Scanning electron microscopic (SEM) images were obtained using a Hitachi S-4800 scanning electron microscope (Japan). X-ray photoelectron spectroscopic (XPS) measurements were performed using a PHI5000 Versa Probe spectrometer (ULVAC-PHI, Japan) with an ultra high vacuum generator. All electrochemical immunoassays were

The AuNPs-loaded PSA was prepared according to the previous report27 with minor modification. The self-assembled three-layer film was first formed on PSA by incubating sequentially a PSA dispersion (0.2 mL, 2.5 wt %) with PDDA, PSS, and PDDA solutions (1 mL, 1 mg mL−1 in 0.5 M NaCl) for 30 min for each polyelectrolyte layer. The resulting particles underwent centrifugation three times (13400 rpm) with ultrapure water to remove excess polyelectrolyte and diluted by 18 mL of ultrapure water. Then 0.1 mL of both 25 mM HAuCl4 and 1% citrate were added to the polyelectrolyte functionalized polybeads under vigorous stirring in an ice− water bath for 30 min, followed by addition of 0.3 mL of freshly prepared ice-cold NaBH4 (0.1 M) and vigorous stirring for 2 min. Afterward, the unattached AuNPs were isolated by centrifugation, repeated 3 times (6000 rpm) with ultrapure water. The obtained AuNPs/PSA as the tracing tag was dispersed and stored at 4 °C. Anti-CEA signal antibody (20 μg mL−1) was added into 1 mL of AuNPs/PSA suspension, which was previously adjusted to pH 9.2 with 0.1 M NaOH, followed by incubation at 4 °C for 4 h with slow stirring. Afterward, 0.1 mL of blocking buffer was dropped into the suspension and the resulting solution gently stirred at 4 °C for 2 h. The final product was acquired by centrifugation at 7000 rpm for 15 min. After the final product was washed twice with pH 7.4 Tris-HNO3, labeled Ab2 was obtained and resuspended in 1 mL of pH 7.4 Tris-HNO3 containing 0.1% BSA. Fabrication of Immunosensor. The graphene oxide (GO) was synthesized from graphite by the modified Hummers method.28,29 Twelve grams of graphite powder was added to a 50 mL mixture of concentrated H2SO4, 10 g of K2S2O8, and 10 g of P2O5 at 80 °C. The mixture was reacted for 6 h and then diluted with 2 L of water, filtered, washed using a 0.2 μm nylon Millipore filter, and dried in air overnight. The oxidized graphite was added to 460 mL of concentrated H2SO4 chilled to 0 °C using an ice bath. Then 60 g of KMnO4 was slowly added with temperature controlled below 10 °C. After this mixture was allowed to react at 35 °C for 2 h, 920 mL of distilled water was slowly added at a temperature below 50 °C 3664



Article

Figure 2. (A) Electrochemical impedance spectroscopy of chitosan/GCE (a), chitosan/GO/GCE (b), and chitosan/electrochemically reduced GO/ GCE (c) in 0.1 M KCl containing 5 mM Fe(CN)62−/3−; (B) linear sweep stripping voltammetric curves of AgNPs deposited at chitosan/GCE (a), chitosan/GO/GCE (b), and chitosan/electrochemically reduced GO/GCE (c) after a sandwich immunoreaction with 0.5 ng mL−1 CEA and labeled Ab2 as tracing tag to induce silver deposition.

Figure 3. TEM image of GO (A) and SEM images of GO/GCE (B), chitosan/GO/GCE (C), and Ab1-chitosan/electrochemically reduced GO/ GCE (D).

finally rinsed with water to carry out the anodic stripping analysis from −0.1 to 0.2 V at 50 mV s−1 in 1.0 M KCl solution.

performed on a CHI 660D electrochemical workstation (Chenhua, Shanghai, China). The reference levels of CEA in the human serum samples were detected with an automation electrochemiluminescent analyzer (Elecsys 2010, Roche). To carry out the immunoreaction and electrochemical measurements, the immunosensor was first incubated with 5 μL of CEA standard solution or serum sample with certain concentration for 50 min at 37 °C. After being washed with washing buffer and pH 7.4 Tris-HNO3, the immunosensor was further incubated with 5 μL of labeled Ab2 for 50 min at 37 °C. After the immunosensor was washed with washing buffer and pH 7.4 Tris-HNO3, silver-deposition enhancement was performed using a 10 μL mixture of enhancer solutions for 4 min at 37 °C in a dark incubator. The immunosensor was

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RESULTS AND DISCUSSION Characterization of Tracing Tag and Labeled Ab2. The entire assembly process of the preparation for tracing tag and labeled Ab2 is illustrated in Scheme 1A. An SEM image shows that the obtained PSA cores, with an average diameter of 323 nm, were uniform (Figure 1A). After AuNPs loading, a TEM image displays the homogeneous distribution of numerous, individual, and dark “Au islands” on the surface of PSA (Figure 1B), indicating the successful synthesis of tracing tag. The synthesis of tracing tag was further confirmed by XPS characterization. In contrast to the XPS spectrum of PSA (curve a, Figure 1C), obvious Au 4f peaks at 82.34 and 85.97 3665



Article

Figure 4. Ratios of signal to noise at (A) different dilution times of silver enhancer solutions and (B) silver deposition times, and effects of (C) Ab2 concentration for preparation of labeled Ab2 and (D) incubation time on the stripping peak current at 50 pg mL−1 CEA.

modified GCE by glutaraldehyde cross-linking, an obvious aggregation of the trapped biomolecules could be clearly observed (Figure 3D), indicating the successful assembly of Ab1 on the immunosensor surface. Optimization of Detection Conditions. The deposited AgNPs on the immunosensor could be easily detected by anodic stripping voltammetric analysis. During a linear potential sweep, the deposited AgNPs showed a well-defined anodic stripping peak with a peak potential of +0.057 V, which was more negative than that reported previously due to the presence of Cl−. The sharp stripping peak was favorable for obtaining high detection precision. The peak current increased with the increasing concentration of antigen used in the sandwich immunoreaction, and could thus be used for immunoassay. Apparently, the amount of deposited silver at the electrode surface increased with the increasing concentration of silver enhancer solutions and the deposition time. However, higher concentrations of silver enhancer solutions and longer deposition time produced higher background current, which was measured in absence of the tracing tag due to the direct reduction of Ag+ by the reducing reagent in silver enhancer solutions. To obtain higher sensitivity, this work used the ratio of signal to noise to optimize the deposition conditions. Upon the successive dilution of silver enhancer solutions, the signalto-noise ratio quickly increased and trended to a maximum value at 20-fold dilution (Figure 4A). Thus, the enhancer solutions with 20-fold dilution were used for silver deposition, at which the signal-to-noise ratio could be kept at a high level within the deposition time of 4 min (Figure 4B). Considering the high detection signal obtained at longer deposition time, 4 min was selected as the optimal deposition time for immunoassay. Because of the intrinsic property of high surface-to-volume ratio of PSA, numerous AuNPs could be loaded on the surface of PSA to perform the catalytically silver deposition amplification. For immunosensing, AuNPs-loaded PSA was used as tracing tag to label Ab2. As shown in Figure 4C, the

eV were observed on the spectrum of tracing tag (curve b, Figure 1C). Through the interaction between amine or SH groups in protein and AuNPs, antibody could be easily loaded on the surface of AuNPs to form labeled Ab2.30 As shown in Figure 1D, the XPS spectrum of labeled Ab2 showed an obvious N 1s peak at 399.0 eV (curve b), whereas no N 1s peak was observed in the XPS spectrum of tracing tag (curve a). This result confirms the successful modification of antibody on the tracing tag. Characterization of Immunosensor. Graphene, especially electrochemically reduced GO, exhibits high conductivity and mediate electron transfer at its edge planes. It has been widely used in biosensor to improve the detection sensitivity.31−33 This work used reduced GO to construct a sensitive immunosensor for CEA. After GO was doped in chitosan, the resulting chitosan/GO/GCE showed much lower electron transfer resistance, Ret (Figure 2A, curves a and b). Upon the electrochemical reduction of GO, the Ret further decreased (Figure 2A, curve c). The linear sweep stripping voltammetric curve of AgNPs formed at Ab1-chitosan modified electrode after incubation with 0.5 ng mL−1 CEA and the labeled Ab2 showed a very small response in 1.0 M KCl solution (Figure 2B, curve a), whereas Ab1-chitosan/GO modified electrode with the same incubation processes showed a sharp stripping peak (Figure 2B, curve b), indicating that the graphene efficiently improved the detection sensitivity due to its ability to accelerate the electron transfer between deposited AgNPs and electrode. After electrochemical reduction of the GO, the resulting immunosensor showed further enhanced stripping peak (Figure 2B, curve c), leading to higher sensitivity. The prepared GO showed silk veil-like waves with a very stable nature under the electron beam (Figure 3A). After the GO was coated on the surface of GCE, the SEM photo displayed crumpled sheets-like waves (Figure 3B). The coating of chitosan on the GO film led to a smoother and more uniform surface topography (Figure 3C). When Ab1 was immobilized on the chitosan/electrochemically reduced GO 3666



Article

Figure 5. Linear sweep stripping voltammetric curves of AgNPs (A) and calibration curve (B) for detection of CEA from 0 to 0.5 ng mL−1.

Table 1. Assay Results of Clinical Serum Samples Using the Proposed and Reference Methods sample no.

1 −1

proposed method (pg mL ) reference method (ng mL−1) relative error (%) a

4.46 4.03 10.7

a,b,c

1 a,b,c

37.4 4.03 −7.20

1

2

a,b,c

a,b,c

421 4.03 4.47

223 20.5 8.78

3 128a,b,c 13.6 −5.88

The serum samples were diluted at 1000 times. bThe serum samples were diluted at 100 times. cThe serum samples were diluted at 10 times.

Meanwhile, these results also highlighted that PSA−AuNPs− Ab2 as tracing tag had good stability. Application in Detection of Serum Tumor Marker. The analytical reliability and application potential of the proposed method was evaluated by comparing the assay results of clinical serum samples using the proposed immunosensor with the reference values obtained by commercial electrochemiluminescent tests. Because of the high sensitivity, serum samples were appropriately diluted with 0.02 M pH 7.4 PBS prior to assay. The results showed an acceptable agreement with relative errors less than 10.7% (Table 1), indicating acceptable accuracy of the proposed method for the detection of CEA in clinical samples.

stripping voltammetric response increased with the increasing Ab2 concentration until the concentration of 20 μg mL−1; after that, the response slightly decreased. Therefore, 20 μg mL−1 Ab2 was used for the preparation of labeled Ab2. The incubation time is an important parameter affecting the analytical performance of immunoassay. With the increasing incubation time, the stripping peak current for CEA increased and trended to a constant value after an incubation time of 50 min (Figure 4D), which showed a saturated binding between the analyte and the antibodies. Therefore, the incubation time of 50 min was selected for the sandwich-type immunoassay. Analytical Performance. Under optimum conditions, the stripping peak current of AgNPs deposited on the immunosensor increased with increasing concentration of CEA in the incubation solution (Figure 5). The same peak potential of these stripping peaks indicated good reproducibility of the immunosensor topography. The calibration plot showed a good linear relationship between the stripping peak current and the logarithm of the analyte concentration in the range from 0.5 pg mL−1 to 0.5 ng mL−1 with a correlation coefficient of 0.9994 (Figure 5B). The limit of detection at a signal-to-noise ratio of 3σ (where σ is the standard deviation of signal in a blank solution) was 0.12 pg mL−1, which was much lower than those reported previously.3,7,9 Compared with traditional enzyme labels, the PSA carried AuNPs had unique advantages of easier operation and better stability. More importantly, the detection of AgNPs could be performed with a one-step stripping analysis in KCl solution without pretreatment, for example, harmful dissolution solution, high stripping potential, addition of substrates, and deoxygenation procedure, was needed, which greatly simplified the analytical procedure. Reproducibility and Precision of Immunosensor. Both the intra-assay and interassay precisions of the immunosensor were examined with 50 pg mL−1 CEA five times. The relative standard deviations (RSD) were 4.8% and 6.5%, respectively, showing good precision and acceptable fabrication reproducibility. In addition, when the immunosensor was stored in dry conditions at 4 °C, over 90% of the initial response remained after a storage period of 2 weeks. These results indicated that the immunosensor had acceptable reliability and stability, and was suitable for the clinical diagnosis of protein markers.

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CONCLUSIONS An ultrasensitive electrochemical immunosensing method for cancer biomarker was proposed by using a triple signal amplification strategy based on nanobiotechnology. The introduction of graphene on immunosensor surface efficiently accelerated the electron transfer and enhanced the detection signal. The second signal amplification came from the loading of numerous AuNPs on PSA as nanocarrier by in situ synthesis of the tracing tag. After immunoreaction, the AuNPs-induced silver metallization produced a mass of AgNPs on the surface of PSA, which could be conveniently detected by the anodic stripping analysis. The electrochemical immunosensing method excluded specific detection conditions, such as pretreatment of the NPs, high stripping potential, and deoxygenation procedure. The proposed immunosensor showed excellent analytical performance for CEA with high sensitivity, convenient operability, and acceptable reproducibility, stability, and accuracy, thus providing great promise in clinical application.

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +86-25-83593593; e-mail: [email protected] (H.X.J.). E-mail: [email protected] (J.W.). Notes

The authors declare no competing financial interest. 3667



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ACKNOWLEDGMENTS We gratefully acknowledge the National Basic Research Program of China (2010CB732400), the National Science Fund for Creative Research Groups (21121091) and the projects (21075055, 21135002) from National Natural Science Foundation of China, and the Leading Medical Talents Program from the Department of Health of Jiangsu Province.

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Triple Signal Amplification of Graphene Film, Polybead Carried Gold

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