In Situ Amplified Electrochemical Immunoassay for

Anal. Chem. 2008, 80, 8064–8070

In Situ Amplified Electrochemical Immunoassay for Carcinoembryonic Antigen Using Horseradish Peroxidase-Encapsulated Nanogold Hollow Microspheres as Labels Dianping Tang*,† and Jingjing Ren Key Laboratory of Analytical Chemistry (Chongqing), College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China Methods based on sandwich-type electrochemical enzyme immunoassay protocol have been extensively developed for the detection of biomolecules, but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. In this study, we initially synthesized specially horseradish peroxidase-encapsulated nanogold hollow microspheres (HRP-GHS), and then the prepared HRP-GHS was conjugated to the secondary carcinoembryonic antibody (HRP-GHS-anti-CEA). Carcinoembryonic antigen (CEA), as a model protein, was monitored by using the electrochemical sandwich-type enzyme immunoassay format. Under optimized conditions, the linear range of the immunoassay by using single HRP-labeled anti-CEA (HRP-anti-CEA) as secondary antibodies is 2.5-120 ng/mL with a detection limit of 1.5 ng/mL CEA, while the assay sensitivity by using HRPGHS-anti-CEA as secondary antibodies is further increased from 0.01 to 200 ng/mL with a lower detection limit of 1.5 pg/mL CEA. The intra- and interassay reproducibility is acceptable. The CEA concentrations of the clinical serum specimens assayed by the developed immunoassay show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay. This immunoassay system has many desirable merits including sensitivity, accuracy, and little required instrumentation. Significantly, the new protocol may be quite promising, with potentially broad applications for clinical immunoassays. Electrochemical immunoassays, with simple instrumentation and easy signal quantification, have become the predominant analytical techniques for quantitative detection of biomolecules.1-4 * To whom correspondence should be addressed. Tel./Fax: +86 23 6825 4000. E-mail: [email protected]. † Alexander von Homboldt Fellow Chair for Analytical Chemistry (Institute of Hydrochemistry, Technische Universita¨t Mu ¨ nchen, Marchioninistrasse 17, 81377 Mu ¨ nchen, Germany) on leave from College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China. (1) Wu, J.; Fu, Z.; Yan, F.; Ju, H. TrAC, Trends Anal. Chem. 2007, 26, 679– 688. (2) Lequin, R. M. Clin. Chem. 2005, 51, 2415–2418. (3) Jiang, Z.; Huang, W.; Li, J.; Li, M.; Liang, A.; Zhang, S.; Chen, B. Clin. Chem. 2008, 54, 116–123. (4) Dong, H.; Li, C.; Zhang, Y.; Cao, X.; Gan, Y. Lab Chip 2007, 7, 1752–1758.

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For sandwich-type immunoassays, signal amplification and noise reduction are crucial for obtaining low detection limits and high sensitivity in clinical immunoassays.5 Much attention has been focused on signal amplification by using an enzyme, such as horseradish peroxidase (HRP) and alkaline phosphatase. Rusling6 used bioconjugates featuring HRP labels and secondary antibodies linked to carbon nanotubes for electrochemical PSA immunoassay with a detection limit of 5 pg/mL. Wilson and Nie7,8 reported a series of electrochemical enzyme immunoassays by covalently binding or physically adsorbing analytes on iridium oxide electrodes to recognize alkaline phosphatase-labeled antibodies. Ju and coauthors9 introduced a disposable multianalyte electrochemical immunosensor assay for automated simultaneous determination of tumor markers with the detection limits of 1.1 ng/mL, 1.7 ng/mL, 1.7 kilounits/L, and 1.2 units/L for carcinoembryonic antigen (CEA), R-AFP, CA 125, and β-hCG by using the HRPlabeled antibodies. Recently, gold nanoparticles have received much attention as bioelectrocatalysts in electroanalytical chemistry.10 The electrocatalytic properties of nanoparticle labels have been used for signal amplification in clinical immunoassays.11 One major merit of using nanoparticles is that the nanoparticles can provide unique chemical and physical properties to enable new and advanced functions, such as high surface-to-volume ratio, and surface free energy, in comparison with bulk materials.12 Yang13 reported an ultrasensitive and simple electrochemical sandwich-type immunoassay by catalytic action of p-nitrophenol to p-aminophenol using goldnanocatalyst labels for IgG detection with a very low detection limit of 1.0 fg/mL. Li14 and Fan15 used gold nanoparticles as labels for the IgG detection in human with low detection limits. (5) Tang, H.; Haisall, H.; Heineman, W. Clin. Chem. 1991, 37, 24524–24528. (6) Yu, X.; Munge, B.; Patel, V.; Jensen, G.; Bhirde, A.; Gong, J.; Kim, S. N.; Gillespie, J.; Gutkind, J. S.; Papadimitrakopoulos, F.; Rusling, J. F. J. Am. Chem. Soc. 2006, 128, 11199–11205. (7) Wilson, M.; Nie, W. Anal. Chem. 2006, 78, 6476–6483. (8) Wilson, M. S.; Nie, W. Y. Anal. Chem. 2006, 78, 2507–2513. (9) Wu, J.; Yan, F.; Tang, J.; Zhai, C.; Ju, H. Clin. Chem. 2007, 53, 1495–1502. (10) Georganopoulou, D.; Chang, L.; Nam, J.; Thaxton, C.; Mufson, E.; Klein, W.; Mirkn, C. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 2273–2276. (11) Tang, H.; Chen, J.; Nie, L.; Kuang, Y.; Yao, S. Biosens. Bioelectron. 2007, 22, 1061–1067. (12) Tang, D. P.; Yuan, R.; Chai, Y.; An, H. Adv. Funct. Mater. 2007, 17, 976– 982. (13) Das, J.; Aziz, M.; Yang, H. J. Am. Chem. Soc. 2006, 128, 16022–16023. (14) Wang, Z.; Hu, J.; Jin, Y.; Yao, X.; Li, J. Clin. Chem. 2006, 52, 1958–1961. (15) Fan, A.; Lau, C.; Lu, J. Anal. Chem. 2005, 77, 3238–3242. 10.1021/ac801091j CCC: $40.75  2008 American Chemical Society Published on Web 09/25/2008

Significantly, Merkoci et al.16 introduced a new double-codified label, consisting of gold nanoparticles conjugated to an HRPlabeled anti-IgG, for human IgG detection by using an electrochemical sandwich-type immunoassay. The presence of gold nanoparticles enhanced the immobilized amount of HRP-anti-IgG, which improved the sensitivity of the immunoassay when used as the secondary antibodies. Compared with classical ELISAs, the double-codified, label-based immunoassay exhibited better analytical properties. In conventional sandwich-type enzyme immunoassays, the detection signal mainly derives from the labeled enzyme on the secondary antibodies toward the catalytic reaction of the corresponding substrate. However, Yang and coauthors13 introduced the highly sensitive electrochemical immunoassay method without the use of enzyme. The signal was amplified mainly by using the catalytic properties of gold nanoparticles. The aim of this study is to simultaneously combine the bioelectrocatalytic properties of enzyme with nanogold particles for the electrochemical signal amplification of the antigen-antibody reaction. HRP molecules were encapsulated into nanogold hollow microspheres, which were used as the label of secondary anti-CEA antibodies (horseradish peroxidase-encapsulated nanogold hollow microspheres (HRP-GHS)-anti-CEA) for the detection of CEA in a sandwichtype immunoassay format. The focus of the present work is to investigate the double-amplified properties of the HRP-GHS-antiCEA. The objective of this survey is to exploit some advanced HRP-GHS-based probes for molecular biology, genomics, proteomics, and diagnosis and therapy of infectious disease and cancer. EXPERIMENTAL SECTION Materials. CEA, and mouse monoclonal anti-CEA (clone no. 10) were purchased from Sigma. Thionine, gultaraldehyde, HRP (EC 1.11.1.7, RZ > 3.0, A > 250 U/mg), bovine serum albumin (BSA, 96%-99%), bis(2-ethylhexyl) sodium sulfosuccinate (AOT), and 16-nm gold colloids were obtained from Sigma. All other reagents were of analytical grade and were used without further purification, unless specified otherwise. Deionized and distilled water was used throughout the study. 0.1 M acetic acid buffer solution (ABS) with various pH values were prepared by using HAc, NaAc, and K2HPO4. Clinical serum samples were gifts from Chongqing City Tumor Hospital. Preparation of HRP-GHS and HRP-GHS-anti-CEA. The HRP-encapsulated nanogold hollow microspheres were synthesized consulting the literature with a little modification.17 Two solutions were initially prepared as follows: solution A including 200 µL of 0.05 mol/L CaCl2, 100 µL of 1.5 × 10-4 mol/L HRP aqueous solution, and 15 mL of 0.1 mol/L AOT isooctane suspension; and solution B containing 200 µL of 0.05 mol/L Na2CO3, 100 µL of 1.5 × 10-4 mol/L HRP aqueous solution, and 15 mL of 0.1 mol/L AOT isooctane suspension. After being adequately stirred, solution B was added dropwise into solution A with violent stirring until the formation of the translucent HRPdoped CaCO3 colloids. Following that, 100 µL of 5% HAuCl4 solution was added into the reverse micellar solution drop by drop. (16) Ambrosi, A.; Castaneda, M.; Killard, A.; Smyth, M.; Alegret, S.; Merkoci, A. Anal. Chem. 2007, 79, 5232–5240. (17) Kumar, R.; Maitra, A.; Patanjali, P.; Sharma, R. Biomaterials 2005, 26, 6743– 6753.

After being stirred further for 2 h at room temperature, 100 µL of 0.05 mol/L aqueous hydrazine hydrate was injected into the prepared suspension. With the aid of NH2OH, the Au3+ ions coating the CaCO3 surface were reduced to metallic Au0. With the progressive addition of hydrazine hydrate to reverse micelles, the solution acquired a fawn to red color due to the formation of gold colloids. After being stirred for 1 h at room temperature, 3 mL of absolute ethanol was added and stirred for 10 min, which resulted in the complete breakdown of reverse micelles with the formation of two immiscible layers of aqueous ethanol and isooctane. The ethanol was carefully removed using a separating funnel. The particles thus obtained were washed four times with isooctane and centrifuged to remove any residual AOT. The pelleted particles were then dispersed in 10 mL of water by vigorous stirring for 30 min, and the dispersed system was dialysed against distilled water for 2 h using a 12-kDa cutoff dialysis bag. The hollow microspheres of nanogold particles were finally formed via the addition of 0.1 mol/L HCl into the nanogoldcoated HRP-doped-CaCO3 suspension. The synthesized procedure of the HRP-GHS is illustrated in the top of Figure 1. The synthesized HRP-GHS was used for the anti-CEA label via the conjugation between nanogold particles and thiols or alkylamines of the antibody. A 500-µL aliquot of 500 ng/mL antiCEA was added into 5 mL of a 2% (w/w) HRP-GHS suspension with constant stirring for 4 h. The mixture was centrifugated at 4000 rpm for 15 min. Following that, the precipitation was added into a BSA solution to block possible remaining active sites of the nanogold particles and avoid the nonspecific adsorption. The prepared HRP-GHS-anti-CEA was then stored in pH 7.4 PBS at 4 °C until use. The as-synthesized HRP-GHS-anti-CEA was characterized by using tunnel electron microscopy (TEM) (H600, Hitachi Instrument Co.) and an 8500 UV-vis spectrophotometer. Preparation of the Immunosensor. Figure 1a (bottom) shows the fabrication procedure of the base electrodes. A gold electrode (4 mm in diameter) was polished repeatedly with 1.0, 0.3-µm alumina slurry, followed by successive sonication in bidistilled water and ethanol for 5 min and dried in air. Prior to the experiment, the gold electrode was cleaned with hot piranha solution for 10 min and then continuously scanned within the potential range of -0.3 to 1.5 V in freshly prepared deoxygenated 0.5 M H2SO4 until a voltammogram characteristic of the clean gold electrode was established. After the cleaned electrode was thoroughly rinsed with water and absolute ethanol, an electropolymerized thionine film was deposited onto a gold electrode to form a multitude of amino groups on the base surface using voltammetric cycles between 0 and 1.5 V at 50 mV/s in 0.1 M thionine aqueous solution.18 Following that, the thionine-modified electrode was placed into 16-nm gold colloids and 500 µL of 1 mg/mL protein A solution for 6 h at 4 °C in turn. After being rinsed with water, the formed protein A/nanogold/thioninemodified gold electrode was submerged into 500 ng/mL anti-CEA solution. After being stored for ∼12 h at 4 °C, the formed immunosensor was incubated in 0.25 wt % BSA for 60 min at 37 °C to eliminate nonspecific binding effect and block the remaining active groups. The finished immunosensor was stored at 4 °C when not in use. (18) Tang, D. P.; Yuan, R.; Chai, Y Electroanalysis 2006, 18, 259–266.


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Figure 1. Synthesized process of hollow gold microspheres with the encapsulated HRP (TOP) and schematic representation of the electrochemical sandwich-type enzyme immunoassay (BOTTOM): (a) the fabrication process of probe; (b) HRP-GHS and HRP-GHS-labeled anti-CEA; and (c) schematic view of electrochemical sandwich-type enzyme assay procedure.

Figure 2. Measurement procedure of the electrochemical sandwich-type enzyme immunoassay.

Measurement Method. The measurement process and detection principle of the developed electrochemical sandwich-type immunoassay (ESEIA) are illustrated in Figure 2 and Figure 1c (bottom). Electrochemical measurements were performed with an AutoLab system. A three-electrode system comprising a prepared working electrode, a platinum wire as auxiliary electrode, and an Ag/AgCl as reference electrode was employed for all 8066


electrochemical experiments. After incubating the proposed immunosensor with various CEA concentrations for 30 min at room temperature, the resulting substrates were submerged into HRPGHS-anti-CEA solution for 30 min at room temperature. After rinsing thoroughly with pH 7.4 PBS to remove the unbound secondary antibodies, amperometric responses of the immunosensor were recorded by using cyclic voltammetry at 50 mV/s

Figure 3. (a) TEM image of HRP-GHS and (b) UV-vis spectra of HRP-GHS, HRP, and nanogold colloids.

in pH 6.8 ABS containing 0.8 mmol/L H2O2 due to the catalytic reduction of the bound HRP toward H2O2. After each immunoassay run, the immunosensors were regenerated by immersing into 0.1 M glycine-HCl (pH 2.8) for 5 min, washed with pH 7.4 PBS, and then carried out the following run cycle. All electrochemical measurements were done in an unstirred electrochemical cell at 25 ± 0.5 °C. Commercial ELISA for CEA. A commercially available ELISA assay was utilized for method comparison studies. In sandwich ELISA with standard polystyrene 96-well plates, 50 µL of serum sample suspension was incubated at 37 °C for 30 min, and the wells were rinsed 3 times (3 min each) with 0.1 mol/L PBS (pH 7.4) containing 0.5 mol/L NaCl and 1 mL/L Tween 20. Then we added 50 µL of conjugate solution and incubation continued for 1 h. The wells were again rinsed and 50 µL of 3,3′,5,5′-tetramethylbenzidine reagent was added and incubated at 37 °C for 10 min. The enzymatic reaction was stopped by adding 50 µL of 2.0 mol/L H2SO4 to each well. The results of ELISA were measured by a spectrophotometric ELISA reader at a wavelength of 450 nm. RESULTS AND DISCUSSION Characteristics of HRP-GHS. In this study, the amplification of the electrochemical signal was mainly produced by the encapsulated HRP toward the reduction of H2O2. The presence of the nanogold hollow microspheres provided a larger room for the encapsulation of the inner HRP and immobilization of the external secondary antibody. The TEM image of HRP-encapsulated nanogold hollow microspheres is shown in Figure 3a, which displays a well dispersion with a mean diameter of 45 nm. The scraggy structure provided a larger room for the immobilization of anti-CEA antibody than that of a slippery sphere surface due to the strong interaction between gold colloids and proteins as a result of high surface to volume ratio and high surface energy of nanogold particles.19 Moreover, the H2O2 molecules in the solution was easily diffused and accessed into the encapsulated HRP via the interstices between nanoparticles. Furthermore, gold nanoparticles could enhance the electron-transfer rate due to the quantum size effect of nanogold particles.19-21 (19) Jia, J. B.; Wang, B. Q.; Wu, A. G.; Cheng, G. J.; Li, Z.; Dong, S. J. Anal. Chem. 2002, 74, 2217–2223. (20) Okahata, Y.; Kawase, M.; Niilura, K.; Ohtake, F.; Furusawa, H.; Ebara, Y. Anal. Chem. 1998, 70, 1288–1296. (21) Ebato, H.; Gentry, C. A.; Herton, J. N.; Muller, W.; Okahata, Y.; Ringsdorf, H.; Suci, P. A. Anal. Chem. 1994, 66, 1683–1689.

In this study, when the HRP-GHS-anti-CEA probe was used, a biomolecular recognition event could be signaled by one nanogold hollow microsphere, in which hundreds to thousands of HRP molecules were integrated to greatly enhance the response signal. When applied appropriately in bioanalysis, HRP-GHS-anti-CEA provided a great improvement for analytical sensitivity. To further monitor the formation of HRP-GHS, we studied the UV-vis absorption spectra of the pure HRP, pure gold colloids, and HRPGHS, respectively (Figure 3b). As shown in Figure 3b, the pure HRP shows an absorption peak at 398 nm, while there is a peak at 520 nm for the pure gold colloids. When HRP molecules were encapsulated into the nanogold hollow microspheres, two absorption peaks were simultaneously observed. The peak at 378 nm is mainly ascribed to the HRP molecules, while the peak at 550 nm is attributed to the gold nanoparticles. Compared with those obtained with pure HRP solution and pure gold colloids, the slight deviation between absorption wave numbers was due to the interaction between gold nanoparticles and HRP molecules.12 Thus, we might make a conclusion that HRP molecules and colloidal gold could be doped into the synthesized nanocomposites via the reverse micellar method. Comparison of Variously Labeled Probes. Signal amplification and noise reduction are critical for a sandwich-type electrochemical enzyme immunoassay. To investigate the effect of the HRP-filled nanogold hollow microspheres on the sensitivity of the assay, we prepared three differently labeled probes as secondary antibodies: HRP-bound anti-CEA (HRP-anti-CEA), HRP-anti-CEAconjugated nanogold particle, and anti-CEA-conjugated HRP-GHS (inset of Figure 4). During the measurement process, we initially used the same batch immunosensors with the reaction of the same CEA concentration, and then variously labeled probes were utilized as secondary antibodies. The judgment is based on the amperometric change of the immunosensor before and after the antigen-antibody interaction. As shown in Figure 4, the use of HRP-GHS-anti-CEA exhibits much greater current shift than those obtained at other two labeled probes. The reason might be the fact that thousands of HRP molecules were entrapped into the nanogold hollow microsphere. When one antibody molecule bound onto the HRP-GHS surface was reacted with the corresponding antigen (analyte), the carried HRP molecules exhibited higher catalytic efficiency relative to the H2O2 system than that of directly using HRP-labeled secondary antibody. Meanwhile, the high surface-to-volume ratio of the nanogold hollow microsphere Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

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Figure 4. Cyclic voltammograms of the electrochemical sandwich-type enzyme immunosensors before (solid line) and after (dotted line) addition of 0.8 mmol/L H2O2 into pH 6.8 ABS by using various labels: (a) HRP, (b) nanogold particles, and (c) HRP-GHS.

Figure 5. Effects of (a) pH of ABS, (b) incubation temperature, and (c) incubation time on amperometric response of the ESEIA toward 50 ng/mL CEA under optimal conditions. Table 1. Comparison of the Sensitivity Data of the Developed Immunoassay with Those of Other CEA Immunoassays immunoassay method

Figure 6. Calibration curves of the ESEIA for CEA determination in 0.8 mmol/L H2O2 ABS (pH 6.8). Inset: amplification of calibration curve in the low CEA concentration.

greatly enhanced the immobilization amount of HRP and antiCEA.22 Therefore, we chose HRP-GHS-anti-CEA as second labeled antibodies for the following experiments. Optimization of Experimental Conditions. Figure 5a shows the effect of pH of the detection solution on the current responses of the immunoassay in the presence of 0.8 mmol/L H2O2 toward 50 ng/mL CEA. As shown in Figure 5a, the current change was increased with the increment of pH value from pH 3.5 to 6.8 and then decreased. The optimal amperometric response was achieved at pH 6.8. The reason is that highly acidic or alkaline surroundings would damage the immobilized protein, especially in alkalinity.23 (22) Tang, D. P.; Yuan, R.; Chai, Y. J. Phys. Chem. B 2006, 110, 11640–11646. (23) Tang, D. P.; Yuan, R.; Chai, Y.; Zhong, X.; Liu, Y.; Dai, J. Langmuir 2004, 20, 7240–7245.

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chemiluminescent multiplex immunoassay multianalyte electrochemical immunoassay multiplexed electrical detection time-resolved fluoroimmunoassay immunofluorescence assay amperometric immunoassay piezoelectric immunoassay chemiluminescence enzyme immunoassay potentiometric immunoassay ESEIA immunoassay

linear range

detection limit

ref

1.0-70 ng/mL

0.65 ng/mL 24

e188 ng/mL

1.1 ng/mL

9

e0.9 pg/mL 25 e70 pg/mL 26 0.5-1000 pmol/L 0.01-160 ng/mL 1.5-30 µg/mL 2-162 ng/mL

1.31 pmol/L 5.0 pg/mL 1.5 µg/mL 0.69 ng/mL

1.5-200 ng/mL 0.5 ng/mL 0.01-200 ng/mL 1.5 pg/mL

27 28 29 30 22 this work

So pH 6.8 of the acetic acid buffer solution was selected as the electrolyte for CEA detection. (24) Fu, Z.; Yang, Z.; Tang, J.; Liu, H.; Yan, F.; Ju, H. Anal. Chem. 2007, 79, 7376–7382. (25) Zheng, G.; Patolsky, F.; Cui, Y.; Wang, W.; Lieber, C. Nat. Biotechnol. 2005, 23, 1294–1301. (26) Yuan, J.; Wang, G.; Majima, K.; Matsumoto, K. Anal. Chem. 2001, 73, 1869–1876. (27) Honda, N.; Lindberg, U.; Persson, P.; Hoffmann, S.; Takei, H. Clin. Chem. 2005, 51, 1955–1961. (28) Tang, D. P.; Yuan, R.; Chai, Y. Anal. Chem. 2008, 80, 1582–1588. (29) Zhang, B.; Zhang, X.; Yan, H.; Xu, S.; Tang, D.; Fu, W. Biosens. Bioelectron. 2007, 23, 19–25. (30) Dungchai, W.; Siangproh, W.; Lin, J.; Chailapakul, O.; Lin, S.; Ying, X. Anal. Bioanal. Chem. 2007, 387, 1965–1971.

Table 2. Interference Degree or Crossing Recognition Level of the Developed ESEIAs C[interfering agents] (ng/mL or kilounits/L); current change (µA)b

a

crossing reagentsa

0

CEA + HBsAg CEA + CA 125 CEA + CA 19-9 Normal serum

11.6 11.9 12.7 11.6 12.3 13.7 11.6 11.1 13.6 CV < 4.5% (as control test)

5

50

100

200

mean ± SD (µA)

CV (%)

13.1 14.9 13.7

12.1 13.8 14.2

12.3 ± 0.55 13.3 ± 1.17 12.8 ± 1.24

4.4 8.8 9.7

Containing 50 ng/mL CEA and various concentrations of interfering agents. b The average value of three assays.

The time and temperature of the antigen-antibody reaction also greatly affected the analytical performance of the proposed immunoassay. Seen from Figure 5b, the amperometric response increased with an increment of incubation temperature from 10 to 50 °C and reached a plateau at 35 °C. To simplify the analytical process, all the experiments were carried out at room temperature. At this temperature, the amperometric response of the immunoassay increased with the incubation time increase and leveled off after 30 min (Figure 5c). Longer incubation time did not improve the response. Therefore, 30 min was selected as the incubation time for the determination of CEA antigen in this study. Electrochemical Response of the Immunoassay. To assess the sensitive and the quantitative range of the proposed immunoassay, we assayed routine samples of different CEA concentrations by using the developed ESEIA format with HRP-GHS-antiCEA as tracer and H2O2 as enzyme substrate under optimal conditions. The current responses increased with the increment of CEA concentration in the sample solution after the antigenantibody interaction in pH 6.8 ABS. The curve was not a linear one, as is commonly observed for immunoassays, and we used curve-fitting for the calibration procedure. The relationships between the current response and CEA concentration, however, could be fitted to the experimental points from 0.01 to 200 ng/ mL CEA. Figure 6 shows the plots of amperometric response versus CEA concentration, and linear regression equation is as follows: ∆i (µA) ) 1.0034 + 0.2121C[CEA] (ng/mL) (R2 ) 0.9978). In the direct assay of corresponding serum samples, the proposed ESEIA exhibits a low detection limit of 1.5 pg/mL CEA (estimated to be 3× the standard deviation of zero-dose response). The reason for the low detection limit is that quite a number of anti-CEA molecules conjugated onto the HRP-GHS could enhance the access chance of the antigen-antibody interaction, especially when the analyte (antigen) concentration in the sample is too low. Thus, the developed ESEIA can allow for the directly quantitative detection of CEA in clinical samples. For comparison, we also investigated the analytical performance by using HRP-anti-CEA and HRP-anti-CEA-bound nanogold particle as secondarily labeled antibodies, respectively. The regression equations, linear ranges and detection limits are as follows: (i) Using HRP-anti-CEA as secondary antibodies, ∆i (µA) ) 0.903 + 0.1192C[CEA] (ng/mL), 2.5-120 ng/mL, 1.5 ng/mL CEA; (ii) using HRP-anti-CEA-bound nanogold particle as secondary antibodies, ∆i (µA) ) -0.762 + 0.1653C[CEA] (ng/mL), 1.0-160 ng/mL, 0.5 ng/mL CEA. These results further demonstrated the amplified properties of the HRP-GHS-anti-CEA as labels for the proposed ESEIAs.

To further clarify the amplification of the developed immunoassay toward the antigen-antibody reaction, the analytical performance of the proposed ESEIAs have been compared with those of other CEA immunoassays. Characteristics such as the linear range and detection limit are summarized for all of them in Table 1. As can be observed, the detection limit of the developed ESEIAs exhibited higher sensitivity and lower detection limit than those of other CEA immunoassays. The reason might be the fact that the nanogold hollow microspheres contained a large quantity of HRP molecules, and they gave a higher catalytic efficiency than that of a single HRP molecule. Importantly, the high catalytic efficiency could make them especially suitable for ultrasensitive bioanalysis without the need for additional reagents or signal amplification steps. Reproducibility, Selectivity and Stability. In this study, the repeatability and reproducibility of the proposed immunoassay were assessed by the variation coefficients (CVs) of intra- and interassays. The intra-assay precision of the developed immunoassay was assessed by assaying four CEA levels five times per run in 10 h. The CVs of intra-assay were 3.7, 5.6, 6.3, and 5.5% at 0.05, 5.0, 50, and 150 ng/mL CEA, respectively. Similarly, the interassay CVs on five immunosensors were 6.7, 5.9, 8.2, and 6.43% at 0.05, 5.0, 50, and 150 ng/mL CEA, respectively. Thus, the precision and reproducibility of the proposed immunoassay was acceptable. Since selectivity is a very important characteristic, it was necessary to check it for the developed immunoassay method here. HBsAg, CA 125, vitamin C, and normal (negative) serum samples were used in this study. Amperometic responses of the proposed immunosensor in 0.05, 5.0, 50, and 150 ng/mL CEA solutions containing interfering substances of different concentrations were assayed, and the CV values were 3.5-9.7, 2.7-8.9, 4.4-9.7, and 3.1-9.6% for HBsAg, CA 125, vitamin C, and normal (negative) serum samples, respectively. Table 2 shows the experimental data in 50 ng/mL (as an example) CEA solutions containing various interfering substrates. So the selectivity of the as-prepared immunosensor was acceptable. The stability of HRPGHS was examined. When the HRP-GHS-labeled anti-CEA was not in use, it was stored in PBS (pH 7.4) at 4 °C. It retained 90.1% of its initial response after a storage period of 21 days. The slow decrease of response might due to the destructibility of the HRPGHS. Regeneration of the Developed Immunoassay. In this experiment, 0.2 M glycine-hydrochloric acid buffer solution (pH 2.8) was chosen to break the antibody-antigen linkage. After detecting 50 ng/mL CEA, the immunosensor was dipped into Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

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glycine-hydrochloric acid buffer solution for 5 min, removed to detect 50 ng/mL CEA, and repeated 15 times continuously. The immunosensor retained 90.2% of the original amperometric response for the initial nine times; the relative standard deviation (RSD) was 4.8% for nine successive assays. The reason is that the extreme conditions degraded or chemically deactivated the antibody surface. The electrode-to-electrode reproducibility was estimated from the response to 50 ng/mL CEA with six different immunosensors. This series yielded a mean amperometric response of 11.3 µA and a RSD of 5.3%. Good reproducibility may be explained by the fact the HRP molecules encapsulating the nanogold hollow microspheres could maintain native bioactivity and anti-CEA molecules could firmly attach on the surface of nanogold microspheres. Analysis of Clinical Specimens. To monitor the effect of composite substrate on analytical performance of the developed immunoassay method, routine samples of various concentrations of CEA were added into normal serum and analyzed. The recovery for CEA was from 87.3 to 121.7%. To further investigate the technique’s application for clinical analysis, we examined 113 serum specimens by using the proposed immunoassay and standard CEA ELISA. The regression equation (linear) for these data is as follows (x-axis, ESEIA; y-axis, ELISA): y ) 0.98 (±0.101)x -0.671 (±3.72) (R2 ) 0.9932) for CEA. These data show no significant difference between the results of the two methods.

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CONCLUSIONS This article describes an electrochemical immunoassay for CEA measurement. Nanogold encapsulated HRP label, conjugated to the secondary anti-CEA antibody in a sandwich enzyme immunoassay format, was used to increase the analytical sensitivity of the assay. Although the present assay system is focused on the determination of the target antigen molecules, it can be easily extended to the detection of other antigens or biocompounds. Moreover, the potential of this method for application is a simple and efficient diagnostic strategy for immunoassays. Importantly, this approach does not require sophisticated fabrication and is well suited for high-throughput biomedical sensing and application in both clinical and biodefense areas. ACKNOWLEDGMENT This project was supported by the Postgraduate Science and Technology Innovation Program, Foundation of Excellent Ph.D. Dissertation of SWU (Grant 200602) to D.T. We thank Chongqing Institute of Cancer Prevention and Cure for providing the serum samples for the method-comparison study.

Received for review May 29, 2008. Accepted August 27, 2008. AC801091J

In Situ Amplified Electrochemical Immunoassay for

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