Ultrasensitive Immunoassay Based on Electrochemical Measurement

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Ultrasensitive Immunoassay Based on Electrochemical Measurement of Enzymatically Produced Polyaniline Guosong Lai,*,†,‡ Haili Zhang,† Tasnuva Tamanna,‡ and Aimin Yu*,†,‡ †

Hubei Key Laboratory of Pollutant Analysis and Reuse Technology, Department of Chemistry, Hubei Normal University, Huangshi 435002, PR China ‡ Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn VIC 3122, Australia ABSTRACT: A novel ultrasensitive immunoassay method was developed based on the electrochemical measurement of polyaniline, which was catalytically produced by horseradish peroxidase-functionalized gold nanoparticle (HRP-Au NP) probe at an immunosensor. The immunosensor was prepared step-wise by first modifying the electrode with reduced graphene oxide (rGO)/Au NPs nanocomposite followed by the immobilization of capture antibodies on its surface. After performing a sandwich immunoreaction, the quantitatively captured HRP-Au NP nanoprobes could catalyze oxidation of aniline to produce electroactive polyaniline on the immunosensor surface. The electrochemical measurement of polyaniline enabled a novel detection strategy for HRP-based immunoassay. Both the signal amplification of the HRP-Au NP nanoprobe and the electron transfer acceleration of rGO/Au NPs on the immunosensor surface greatly improved the detection sensitivity of the immunoassay method. With the use of human IgG as a model analyte, this method showed a wide linear range over 4 orders of magnitude with a detection limit of 9.7 pg/mL. In addition, the immunosensor had low cost, satisfactory reproducibility and stability, and acceptable reliability. The relatively positive potential range for the polyaniline measurement completely excluded the conventional interference from dissolved oxygen. Thus, this method provides a promising potential for practical applications.

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cally deposited Ag NPs on the immunosensor surface could be electrochemically measured in an oxygen interference-free potential range, the high cost of ALP and its substrates also limits its wider application. Polyaniline (PAn) is a useful conductive polymer, which is generally prepared by chemical or electrochemical oxidation of aniline in a strong acid condition.16,17 Due to its excellent conductivity and redox property, PAn has been extensively studied in the fields of electrocatalysis and electrochemical sensors.18−20 Recent research has shown that the electroactive PAn can be alternatively prepared through the HRP-catalytic oxidation of aniline in a weak acid condition.21−24 Herein, we report the development of a new detection strategy for the sensitive immunoassay based on the electrochemical measurement of PAn catalytically deposited by HRP labels at a immunosensor. In this work, a HRP functionalized Au NP (HRP-Au NP) probe was prepared and used for the sandwich immunoassay at a reduced graphene oxide (rGO)/Au NPs based immunosensor (Scheme 1). After the sandwich immunoreaction and subsequent incubation in aniline solution, PAn was catalytically deposited on the immunosensor surface by the quantitatively captured HRP-Au NP nanoprobes. This enzymatically produced PAn could be electrochemically measured for the

he sensitive detection of protein biomarkers shows great importance for the clinical cancer screening, disease diagnosis, and monitoring.1,2 Compared with the conventional methods such as enzyme-linked immunosorbent assay, radioimmunoassay, and fluorescence method, electrochemical immunosensors have drawn considerable attention because of their simple instrumentation, low cost, and good portability.3−5 As early cancer diagnosis requires highly sensitive methods to accurately determine specific protein biomarkers at ultralow levels, great efforts are currently focusing on the development of effective signal amplification strategies for achieving ultrasensitive immunoassays.6,7 Recently, various nanomaterials including gold nanoparticles (Au NPs) and carbon nanotubes have been widely used to load high amounts of enzyme labels and prepare different signal tracing nanoprobe for sandwich immunoassay.8−12 Due to the dual signal amplification from the enzymatically catalytic cycle and multiple signal labels on the nanoprobes corresponding to each immuno-recognition event, the analytical sensitivity has been enhanced greatly in these methods. Among these enzyme labels, horseradish peroxidase (HRP) is the cheapest one, which has been commercially used in many bioassays. However, the HRP-based electrochemical immunoassays often encounter the deoxygenation problem for excluding the interference from dissolved oxygen in the detection process,13,14 which greatly limits their practical application. Our previous work attempted to solve this problem by designing an alkaline phosphatase (ALP)-Au NP nanoprobecatalyzed silver deposition method.15 Although the enzymati© XXXX American Chemical Society

Received: November 15, 2013 Accepted: January 7, 2014

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with a FEI Quanta 400F scanning electron microscope at an acceleration voltage of 3 kV. The FTIR spectra were recorded at a Thermo Scientific Nicolet iD5 spectrometer. Preparation of HRP-Au NP Nanoprobe. First, the colloidal Au NPs with the average diameter of 13 nm were prepared by a typical citrate reduction method according to the previous report.15 Then, 2 μg anti-HIgG and 20 μg HRP were added to 1.0 mL colloidal Au NPs which were adjusted to pH 9.0 by 0.1 M K2CO3 and gently mixed at room temperature for 60 min. After 30 min centrifugation at 6000 rpm and washing the soft red sediment with 0.1 M pH 7.4 PBS, the resulted HRP-Au NP nanoprobe was finally resuspended in 1.0 mL of pH 7.4 PBS containing 0.1% BSA and stored at 4 °C prior to use. Preparation of Immunosensor. The screen-printed carbon electrode (SPCE) system containing a carbon working electrode (2 mm in diameter), a carbon auxiliary electrode, and an Ag/AgCl reference electrode was fabricated with screenprinting technology, according to our previous report.25 The insulating layer printed around the working area constituted an electrochemical microcell. The rGO/PDDA nanocomposite was prepared by the chemical reduction of GO with hydrazine under the protection of PDDA.26 The preparation process of the immunosensor was illustrated in Scheme 1. First, in order to obtain rGO/Au NPs nanocomposite with good dispersibility, excess (2.5 mL) 13nm Au NPs were added into 0.50 mL of 1.0 mg/mL rGO/ PDDA and mixed up to 30 min. After centrifugation and washing thrice by water to remove the excess nanoparticles, the rGO/Au NPs nanocomposite was obtained and redispersed in 0.5 mL water. Then, 1.5 μL of the as-prepared rGO/Au NPs nanocomposite was dropped onto the surface of the working electrode and dried at room temperature. Subsequently, 1.0 μL of 0.5 mg/mL anti-HIgG was cast to the rGO/Au NPs modified electrode and incubated in a 100% moisture-saturated environment overnight at 4 °C. The high density of Au NPs decoration on the rGO/Au NPs nanocomposite provided high amounts of sites for the antibody immobilization, thus leading to large signal response for the sensitive immunoassay. After washing thrice with PBST and PBS to remove the loosely adsorbed antibodies, the resulting electrode was further incubated with blocking solution for 60 min at room temperature in order to block the possible remaining active sites against nonspecific adsorption. After washing again with PBST and pH 7.4 PBS, the desired immunosensor was finally obtained and stored at 4 °C in a dry environment prior to use. Measurement Procedure. To carry out the immunoreaction and electrochemical measurement, the immunosensor was first incubated with a 12 μL drop of HIgG standard solutions or serum samples for 50 min at room temperature, followed by washing with PBST and PBS. It was then incubated with 12 μL of HRP-Au NP nanoprobe dispersion for another 50 min. After rewashing with PBST and PBS, 30 μL of 0.1 M HAc−NaAc solution (pH 4.3) containing 30 mM aniline and 2 mM H2O2 was delivered to the electrochemical microcell and kept for 10 min reaction. After rinsing with pH 4.3 HAc−NaAc, differential pulse voltammetry (DPV) was performed in pH 4.3 HAc−NaAc solution to record the peak currents of PAn deposited at the immunosensor for quantitative analysis.

Scheme 1. Schematic Representation of the Preparation of Immunosensor and Sandwich Immunoassay Based on the Electrochemical Measurement of PAn Catalytically Deposited by a HRP-Au NP Nanoprobe

quantitative immunoassay. Due to the multiple labels signal amplification of HRP-Au NP nanoprobe and the excellent electron transfer acceleration of rGO/Au NPs nanocomposite, the sensitivity of this method was enhanced greatly, resulting in ultrasensitive electrochemical immunoassay. Due to the relatively positive potential range for the PAn measurement, this method was free from the conventional interference of dissolved oxygen. When used for the determination of human IgG, this method showed excellent analytical performance, including wide linear range and low detection limit, thus indicating great potentials for practical applications.

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EXPERIMENTAL SECTION Materials and Chemicals. Human IgG (HIgG), mouse IgG (MIgG), and polyclonal rabbit antihuman IgG (anti-HIgG) were purchased from Wuhan Boster Biological Technology Ltd. Horseradish peroxidase (HRP), human serum albumin (HSA), bovine serum albumin (BSA), PDDA (20%, w/w in water, Mw: 100000−200000), aniline, and NaBH4 were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). Chloroauric acid (HAuCl4·4H2O) was obtained from Shanghai Reagent Company (Shanghai, China). Graphene oxide (GO) prepared through the modified Hummer’s method was provided by Jicang Nano Company (Nanjing, China). The bovine serum sample was obtained from Beijing Solarbio Science & Technology Ltd. All other reagents were of analytical grade. Aniline was distilled under reduced pressure prior to use and the others were used as received. Ultrapure water obtained from a Millipore water purification system (Milli-Q) was used in all assays. Phosphate-buffered solution (PBS) of pH 7.4 was prepared by mixing the stock solutions of 50 mM NaH2PO4 and Na2HPO4 and used as a working solution. A 50 mM pH 7.4 PBS containing 0.05% (w/v) Tween-20 (PBST) was used as a washing buffer and a 50 mM pH 7.4 PBS containing 2% (w/v) BSA was used as a blocking solution. An acetic acid−sodium acetate (HAc−NaAc) buffer (0.1 M, pH 4.3) was prepared and used as a detection solution. A 0.1 M pH 4.3 HAc−NaAc solution containing 30 mM aniline and 2 mM H2O2 were prepared daily and used as the enzymatic deposition solution. Apparatus. All electrochemical experiments were performed on a CHI 430 electrochemical workstation. The scanning electron microscopy (SEM) images were recorded

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RESULTS AND DISCUSSION Electrochemical Immunoassay at the Immunosensor. With consideration of the excellent conductivity and bioB

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Then, the electrochemical behavior of the deposited PAn was investigated in a pH 4.3 HAc−NaAc solution by cyclic voltammetry. From Figure 3A, we can find that the

compatibility of graphene and Au NPs, an rGO-Au NPs nanocomposite was prepared for the electrode modification and immunosensor construction in this work.27−29 After sandwich immunoreaction with 100 ng/mL HIgG and HRP-Au NP nanoprobe followed by 10 min incubation in pH 4.3 HAc− NaAc containing 30 mM aniline and 2 mM H2O2, SEM was used to characterize the morphology change of the immunosensor (Figure 1). By comparison with the immuno-

Figure 3. (A) Cyclic voltammograms recorded at the immunosensor (a) before and after sandwich immunoreaction followed by (b) enzymatic deposition reaction. (B) DPV responses of enzymatically deposited PAn toward 100 ng/mL HIgG at (c) a bare SPCE based immunosensor and (d) the rGO/Au NPs based immunosensor. Electrolyte, 0.1 M pH 4.3 HAc-NaAc buffer.

Figure 1. SEM images of the rGO/Au NP-based immunosensor (A) before and after sandwich immunoreaction followed by (B) enzymatic deposition reaction.

immunosensor did not show any obvious redox peak in the potential range from 0.4 to −0.3 V. However, a pair of welldefined redox peaks appeared at 0.028 and 0.086 V after sandwich immunoreaction followed by enzymatic reaction at the immunosensor. This redox behavior is similar with the previous reports,24,31 which further confirmed the successful enzymatic deposition of PAn on the immunosensor surface. In order to obtain an excellent electrochemical signal for immunoassay, the more sensitive electroanalytical method of DPV was used for the quantitative measurements of PAn. From Figure 3B, we can observe that the enzymatically deposited PAn corresponding to 100 ng/mL of HIgG immunoassay showed a well-defined DPV oxidation peak at 0.044 V. Moreover, the peak current at this immunosensor was about seven fold higher than that at a bare SPCE-based immunosensor prepared with the previous method.32 This current enhancement should be attributed to the modification of rGO/Au NPs nanocomposite on the immunosensor surface, which greatly promoted the electron transfer rate of the deposited PAn and also increased the effective surface area of the electrode. In addition, compared with the conventional signal HRP label, the high contents of HRP on the HRP-Au NP probes greatly amplified the signal response, corresponding to each immuno-recognition event. Hence, a sensitive immunoassay method could be developed by combining the catalytic deposition of PAn at an rGO/Au NP-based immunosensor by HRP-Au NP nanoprobe and the sensitive measurement of PAn with the DPV method. Optimization of Detection Conditions. The catalytic efficiency of HRP-Au NP nanoprobe toward the aniline oxidation is the key problem affecting the performance of the immunoassay. In accordance with literature,21 a low pH (4.0− 4.5) is required to enzymatically produce electrically conducting PAn, whereas too lower pHs will decrease the activity of HRP. We have studied the pH effect of enzymatic deposition solution on the current response of the enzymatically produced PAn. Same with the HRP-catalyzed PAn deposition reported previously,23,24 the maximum current response was obtained at pH 4.3, indicating that the catalytic activity of the HRP-Au NP nanoprobe could be sufficiently maintained to produce electrically conducting PAn for sensitive immunoassay. In addition, enough H2O2 is required to saturate the enzymatic

sensor surface, we can find obvious density increase of nanoparticles distributed on the wrinkled rGO nanosheets as well as some increase in their average diameter upon the sandwich immunoreaction and enzymatic reaction. This should be attributed to the quantitatively captured HRP-Au NP nanoprobes through sandwich immunoreaction and their catalytic production of PAn at the immunosensor. Both the HRP-Au NP probes and the catalytically produced PAn increased the density of nanoparticles on the electrode surface. This enzymatic reaction product was further characterized by FT-IR spectroscopy. By comparison with the spectrum of the immunosensor, intense CN stretching vibration of the quinoid form at 1710 cm−1, obvious C−N stretching vibrations at 1334 and 1238 cm−1 as well as out-of-plane C−H bending vibrations of the benzene ring at 1018 and 793 cm−1 were clearly observed21,30 on the electrode surface after enzymatic reaction (Figure 2). These results demonstrated the successful catalytic deposition of PAn polymer on the immunosensor surface by the HRP-Au NP nanoprobe.

Figure 2. FT-IR spectra of the rGO/Au NP-based immunosensor (a) before and after sandwich immunoreaction followed by (b) enzymatic deposition reaction. C

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reaction, whereas higher concentrations of H2O2 can also render the HRP inactive.22 The experiments showed that the current response of the enzymatically produced PAn increased with the concentration of H2O2 in the pH 4.3 HAc−NaAc solution containing 30 mM aniline (saturated concentration) when it increased from 0.5 to 2 mM, and then began to decrease slowly over 2 mM. This result indicated that 2 mM of H2O2 could saturate the enzymatic reaction without inactivation of the HRP-Au NP nanoprobe. Therefore, a pH 4.3 HAc− NaAc solution containing 30 mM aniline and 2 mM H2O2 was used as the enzymatic deposition solution in this work. As the amount of PAn enzymatically deposited on the immunosensor surface was depended on the reaction time, the DPV response of the immunosensor toward 100 ng/mL HIgG at different PAn deposition time was investigated. As shown in Figure 4A, after sandwich immunoreaction and further

Figure 5. DPV responses of the immunosensor toward different concentrations of (A) HIgG and the (B) calibration curve using the designed detection strategy. Curves a−g correspond to HIgG at the HIgG concentrations from 0.01 to 500 ng/mL.

electrochemical immunoassays, which is more favorable for its practical applications. Specificity, Reproducibility, Stability, and Reliability. MIgG and HSA were used as noncognate proteins to investigate the specificity of the immunosensor. As shown in Figure 6, no obvious current response over the blank control

Figure 4. Effects of the (A) enzymatic reaction time and (B) incubation time on the DPV responses of the immunosensor toward 100 ng/mL HIgG.

incubation in a pH 4.3 HAc−NaAc solution containing 30 mM aniline and 2 mM H2O2, the DPV response of the immunosensor increased sharply with the reaction time up to 10 min. Although the DPV current still increased slowly after 10 min, longer deposition time also produced a higher background current, which led to the decrease of the signal− background ratio. Thus, 10 min of PAn deposition was adopted as the optimal condition in this work. In order to achieve excellent analytical performance of the immunoassay method, the effect of the incubation time was also investigated. At room temperature, the DPV response of the immunosensor toward 100 ng/mL HIgG increased with the increasing incubation time used in sandwich immunoassay until a constant value was reached after 50 min (Figure 4B), which showed the saturated binding of the sandwich immunoreaction. Therefore, an incubation time of 50 min was selected for the sandwich immunoassay. Analytical Performance. Under the optimal conditions, the DPV responses of the immunosensor for HIgG measurement increased with increasing concentrations of analyte (Figure 5). The calibration plot showed a good linear relationship between the peak currents and the logarithm values of the analyte concentrations in the range from 0.02 to 500 ng/mL. The linear regression equation was expressed as I (μA) = 0.3862 + 0.1348 lg C (ng/mL) with a correlation coefficient of 0.9914. The limit of detection at a signal-to-noise ratio of 3 was estimated to be 9.7 pg/mL, which is much lower than many HRP-catalyzed substrate-based electrochemical immunoassay methods reported previously.27,33−35 Compared with other electrochemical measurement methods, this detection process is low in cost and free of the interference from dissolved oxygen involved in the conventional HRP-based

Figure 6. DPV responses of the immunosensor toward blank control, 1% HAS, 100 ng/mL MIgG, and HIgG.

was observed for MIgG and HSA samples. However, the immunosensor showed obvious current response toward the target protein of HIgG. These results indicated that the crossreactivity of the immunosensor toward noncognate proteins was negligible. Five immunosensors were prepared for the repeated measurements of two different concentrations of HIgG. The coefficients of variation were 4.6% and 3.1% for the measurements of 0.1 and 100 ng/mL HIgG, respectively. In addition, the immunosensor could retain 93% of the initial response for 100 ng/mL HIgG after a storage period of two weeks in dry air at 4 °C. These results indicated that the immunosensor had satisfactory reproducibility and stability. To evaluate the reliability of the designed immunosensing method, different amounts of HIgG were added into bovine serum for recovery tests. The test results from three repeated experiments were listed in Table 1, which showed acceptable results with the recovery of the standard addition experiments Table 1. Recovery Tests of HIgG in Bovine Serum Sample

D

no.

added (ng/mL)

found (ng/mL)

RSD (%)

recovery (%)

1 2 3

0.5 5 50

0.48 5.15 54.51

6.5 4.8 5.1

96 103 109

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between 96% and 109% and relative standard deviation (RSD) lower than 6.5%. These results indicated acceptable reliability of the method for practical applications.

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CONCLUSIONS A novel ultrasensitive immunoassay method was developed by combining the rGO/Au NP-based immunosensor with HRPAu NP-catalyzed deposition of PAn. The sensitive electrochemical measurement of the enzymatically produced PAn enables a new detection strategy for the HRP-based immunoassay. Both the signal amplification of HRP-Au NP nanoprobe and the electron transfer acceleration of rGO/Au NPs greatly improve the sensitivity of the immunoassay method. The relatively positive potential range for the PAn measurement completely excludes the interference from dissolved oxygen. In consideration of the excellent analytical performance including wide linear range, ultrahigh sensitivity, low cost and satisfactory stability, reproducibility, and accuracy for HIgG measurement, this method provides a promising potential in protein biomarker determination for clinical application.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86 714 6515602. *E-mail: [email protected]. Tel: +61 3 9214 8161. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant 21205031), Science and Technology Foundation for Creative Research Group of HBDE (Grant T201311), and ANZ Trustees Medical Research & Technology in Victoria program (Grant CT20692).

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dx.doi.org/10.1021/ac4037119 | Anal. Chem. XXXX, XXX, XXX−XXX