An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label

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1 Biosensors and Bioelectronics 20 (2005) An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label Xia Chu a,b, Xin Fu a, Ke Chen a, Guo-Li Shen b,, Ru-Qin Yu b a Chemistry and Chemical Engineering College, Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), Hunan Normal University, Changsha , PR China b State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha , PR China Received 4 July 2004; accepted 9 July 2004 Available online 25 August 2004 Abstract A novel, sensitive electrochemical immunoassay has been developed based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) as a model. The influence of relevant experimental variables, including the reaction time of antigen with antibody, the dilution ratio of the colloidal gold-labeled antibody and the parameters of the anodic stripping operation, upon the peak current was examined and optimized. The anodic stripping peak current depended linearly on the IgG concentration over the range of 1.66 ng ml 1 to gml 1 in a logarithmic plot. A detection limit as low as 1ngml 1 (i.e., M) human IgG was achieved, which is competitive with colorimetric enzyme linked immuno-sorbent assay (ELISA) or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is attributed to the sensitive ASV determination of silver (I) at a glassy-carbon electrode (detection limit of M) and to the catalytic precipitation of a large number of silver on the colloidal gold-labeled antibody Elsevier B.V. All rights reserved. Keywords: Gold nanoparticle; Metalloimmunoassay; Electrochemical stripping analysis; Silver enhancement 1. Introduction Immunoassays are based on the specific reaction of antibodies with the target substances (antigens) to be detected and have been widely used for the measurement of targets of low concentration in clinical biofluid specimen such as urine and blood and the detection of the trace amounts of drugs and chemicals such as pesticides in biological and environmental samples. According to the nature of a label, immunoassay can be classified as label-free immunoassay, radio-immunoassay, enzyme immunoassay, fluorescent immunoassay, chemiluminescent immunoassay, bioluminescent immunoassay and so on. Since metalloimmunoassay, i.e., immunoassay involving metal-based labels, was developed, great progresses have Corresponding author. Tel.: ; fax: address: glshen@hnu.cn (G.-L. Shen). been made along this direction with the use of a variety of metal-based labels such as colloidal metal particles (Tu et al., 1993; Kimura et al., 1996; Sato et al., 2000; Lyon et al., 1998; Storhoff et al., 1998; Ni et al., 1999), metal ions (Doyle et al., 1982; Hayes et al., 1994; Wang et al., 1998), organometallics (Limoges et al., 1993; Rapicault et al., 1996; Bordes et al., 1997), or coordination complexes (Yuan et al., 1998; Blackburn et al., 1991). Although many analytical methods for example, atomic or colorimetric absorption spectrophotometry, infrared or Raman spectroscopy, time-resolved fluorescence and so on, are suitable for the quantitative determination of the metalloimmunoassay, the electrochemical detection holds great promise for metalloimmunoassays owning to its unique advantages such as rapidity, simplicity, inexpensive instrumentation and field-portability. Nevertheless, the sensitivities of the metalloimmunoassay based on organometallic compounds (Limoges et al., 1993; /$ see front matter 2004 Elsevier B.V. All rights reserved. doi: /j.bios

2 1806 X. Chu et al. / Biosensors and Bioelectronics 20 (2005) Rapicault et al., 1996; Bordes et al., 1997) or metal ions (Doyle et al., 1982; Hayes et al., 1994; Wang et al., 1998) remained insufficient compared with fluorescent europium chelate labels for which picomolar levels could be determined. Recently, a new electrochemical metalloimmunoassay based on a colloidal gold label was reported which pushed the sensitivity of the metalloimmunoassay to the picomolar domain (Dequaire et al., 2000). In this work, a new silver-enhanced colloidal gold electrochemical stripping detection strategy is presented, which should further improve the sensitivity of the metalloimmunoassay through the catalytic precipitation of silver on the gold nanoparticles. Silver deposition on gold nanoparticles is commonly used in histochemical microscopy to visualize the distribution of an antigen over a cell surface (Xu, 1997). Mirkin and co-workers have developed a scanometric DNA array (Taton et al., 2000), a electrical detection-based DNA array (Park et al., 2002) and the Raman spectroscopic fingerprints for DNA and RNA detection (Cao et al., 2002) based on silver deposition on gold nanoparticles. Silver enhancement was also used for detecting the DNA hybridization event by the scanning electrochemical microscopy (Wang et al., 2002) and the electrochemical stripping metal analysis (Wang et al., 2001a). Inspired by such similar use of gold nanoparticle labeling and subsequent silver enhancement, the present work aims at developing an analogous metalloimmunoassay. Such electrochemical metalloimmunoassay based on the use of gold nanoparticle labels and silver enhancement has not been reported. The present protocol uses the electrochemical stripping technique for detecting the silver deposited on the gold nanoparticles. Stripping analysis is a powerful electroanalytical technique for trace metal measurements. Its remarkable sensitivity is attributed to the preconcentration step, during which the target metals are accumulated onto the working electrode. The highly sensitive electrochemical stripping analysis was used earlier for determining the colloidal gold tags in DNA hybridization event (Authier et al., 2001; Wang et al., 2001b) but not in connection with the metalloimmunoassay. The new silver-enhanced colloidal gold electrochemical stripping metalloimmunoassay combines the inherent high sensitivity of stripping metal analysis with the remarkable signal amplification resulting from the catalytic precipitation of silver on the gold nanoparticle tags, which should push the sensitivity of the metalloimmunoassay to the picomolar domain. The analytical procedure consists of the immunoreaction of the antigen (analyte) with the primary antibody adsorbed on the walls of a polystyrene microwell, followed by binding a secondary colloidal gold-labeled antibody, silver enhancement and an acid dissolution and stripping detection of the silver at a glassy-carbon electrode. The detailed optimization and attractive performance characteristics of the developed metalloimmunoassay are reported in the following sections. 2. Experimental 2.1. Materials Human IgG, goat anti-human IgG, horseradish peroxidase (HRP)-labeled goat anti-human IgG and bovine serum albumin (BSA) were purchased from Shanghai Huamei Biochemical Reagents (Shanghai, China). Chloroauric acid (HAuCl 4 ), trisodium citrate, hydroquinone, silver nitrate (AgNO 3 ) and tetramethylbenzidine (TMB) were obtained from Shanghai Chemical Reagents (Shanghai, China). All of the solutions were prepared with doubly distilled water Buffers and solutions The following buffers were used in this study: (a) carbonate buffer (15 mm Na 2 CO 3 and 35 mm NaHCO 3,pH 9.6); (b) phosphate-buffered saline (PBS; 137 mm NaCl, 1.7 mm KH 2 PO 4, 8.3 mm Na 2 HPO 4 and 3.0 mm KCl, ph 7.4); (c) Tris-buffered saline (TBS; 20 mm Tris and 150 mm NaCl, adjusting ph to 8.2 with concentrated HCl); (d) TBS containing 0.1% BSA (TBS-BSA); (e) citrate buffer (0.243 M C 6 H 8 O 7 H 2 O and M Na 3 C 6 H 5 O 7 2H 2 O, ph 3.5). Human IgG standard solutions were diluted from a stock solution (10.9 mg ml 1 ) with PBS. Primary goat anti-human IgG (300 gml 1 ) was prepared by dilution of a stock solution (3 mg ml 1 ) with carbonate buffer. The colloidal gold-labeled goat anti-human IgG antibody solutions were prepared and diluted with TBS-BSA. The silver-enhancer solution was composed of 1.0 g hydroquinone, 35 mg AgNO 3, 50 ml citrate buffer and 50 ml doubly distilled water (Xu, 1997), which was prepared fresh as needed Au colloid preparation Colloidal gold particles of average diameter 20 nm ± 4.7 nm were prepared according to Natan (Grabar et al., 1995) with slight modifications. All glassware used in this preparation was thoroughly cleaned in aqua regia (three parts HCl, one part HNO 3 ), rinsed in doubly distilled water, and oven-dried prior to use. In a 500-ml round-bottom flask, 250 ml of 0.01% HAuCl 4 in doubly distilled water was brought to a boil with vigorous stirring. To this solution was added 3.75 ml of 1% trisodium citrate. The solution turned deep blue within 20 s and the final color change to wine-red occurred 60 s later. Boiling was pursued for an additional 10 min, the heating source was removed, and the colloid was stirred for another 15 min. The colloidal solution was stored in dark bottles at 4 C and was used to prepare antibodycolloidal gold conjugate as soon as possible. The resulting solution of colloidal particles was characterized by an absorption maximum at 520 nm. Transmission electron microscopy (TEM) indicated a particle size of 20 ± 4.7 nm (100 particles sampled).

3 X. Chu et al. / Biosensors and Bioelectronics 20 (2005) Preparation of the antibody-colloidal gold conjugate Determination of the amount of coating protein A curve was constructed for (goat anti-human IgG)- colloidal gold conjugate to determine the amount of protein that was necessary to coat the exterior of the gold particles (Lyon et al., 1998; Xu, 1997). The solutions were prepared from 1 mg ml 1 stock solution aliquots (0 50 g) of goat anti-human IgG and were added in 5- g increments to cuvettes containing 1.0 ml of 20-nm diameter colloidal solution adjusted to ph 9.0 using 0.1 M NaOH. The volumes of these samples were corrected to ml with doubly distilled water and 100 l of 10% NaCl was added to each. The solutions were agitated and then placed for 10 min. The absorbances at 520 nm of these samples were recorded and plotted versus the amount of coating protein. Then the optimum amount of coating protein can be determined as that where the decrease in absorbances starts to be insignificant. For 20-nm gold colloid, the optimum amount of goat anti-human IgG for coating the gold nanoparticles is 30 g per 1 ml colloidal gold solution and is effective to prevent aggregation Preparation of the antibody-colloidal gold conjugate The antibody-colloidal gold conjugate was prepared by addition of the goat anti-human IgG antibody to 20 ml of ph-adjusted colloidal gold solution followed by incubation at room temperature with periodic gentle mixing for 1 h, during which the goat anti-human IgG antibodies adsorbed onto the gold nanoparticles through a combination of ionic and hydrophobic interactions. The conjugate was then divided into 1-ml fractions in 1.5-ml microcentrifuge tubes and centrifuged at g for 10 min. Two phases can be obtained: a clear to pink supernatant of unbound antibody and a dark red, loosely packed sediment of the antibody-labeled immunogold. The supernatant was discarded and the soft sediment of immunogold was rinsed by resuspending in 1ml of TBS-BSA and collected after a second centrifugation at g for 10 min. Finally, the conjugate was resuspended in 250 l of 20 mm TBS with 0.1% BSA added to increase stability of immunogold colloid and minimize nonspecific adsorption during the assays. Conjugates can be stored at 4 C for more than 1 month without loss of activity Immunoassay procedure Primary goat anti-human IgG antibody (200 l, 300 gml 1 ) was added to the polystyrene microwells and incubated at 4 C for over night. After removing the solution, the wells were rinsed with 0.5 M NaCl and doubly distilled water three times each for 3 min, and human IgG standard solutions (200 l) were added and incubated in the wells at 37 C for 1 h. Next, the microwells were drained and rinsed as described above. Following this step, 200 lof colloidal gold-labeled goat anti-human IgG was added and incubated at 37 C for 1 h. A last washing cycle was then performed as mentioned above. After removing the rinsing solution, 200 l of silver-enhancer solution was pipetted into the microwells and incubated at room temperature for 30 min in the dark. The wells were then washed with doubly distilled water three times. Finally, 300 l of 1.5 M HNO 3 was added to the microwells and incubated at room temperature for 30 min to dissolve the metal silver deposited on the walls of the microwells Electrochemical measurement The glassy-carbon electrode (Jiangsu Electroanalytical Instruments, Jiangsu, China) and platinum wire electrode (Shanghai Exact Scientific Instrument Ltd., Shanghai, China) were used as the working electrode and the counter electrode, respectively. A saturated calomel electrode (SCE) (Shanghai Dianguang Device Factory, Shanghai, China) was employed as a reference electrode, which was separated from the electrolyte solution by a double electrolytic salt bridge filled with saturated KNO 3 in order to avoid determination interference caused by the continuous leaching of chloride anion that lead to AgCl precipitation. The solutions of silver (I) ions (300 l) were transferred from the microwells into a 10-ml beaker containing 3 ml of 0.6 M KNO 3 and 0.1 M HNO 3 as electrolyte solution, and the released silver (I) ions were then quantified by ASV under the following instrumental conditions: 10-min deposition at 0.5 V versus SCE reference and the potential scan at 100 mv s 1. All electrochemical experiments were conducted at a CHI 660 A electrochemical analyzer (Shanghai Chenhua Instruments, Shanghai, China). The glassy-carbon electrode was cleaned by preconditioning at +1.0 V versus SCE for 1 min between each measurement Enzyme linked immuno-sorbent assay (ELISA) protocol Primary goat anti-human IgG antibody (100 l, 300 gml 1 ) was added to the polystyrene microwells and incubated at 4 C for over night. The wells were washed three times with PBST (10 mm PBS containing 0.05% Tween 20, ph 7.4) and incubated with 100 l per well of the diluted human serum samples at 37 C for 1 h. After another washing step, 100 l of the HRP-labeled goat anti-human IgG antibody was added to the wells and incubated at 37 C for 1 h. After a final washing step, 100 l per well of TMB solution (400 l of 0.6% TMB-DMSO and 100 l of1% H 2 O 2 diluted with 25 ml of citrate-acetate buffer, ph 5.5) was added. The reaction was stopped after an appropriate time by adding 50 l of2mh 2 SO 4, and absorbance was read at 450 nm. 3. Results and discussion The principle of the heterogeneous electrochemical immunoassay based on silver-enhanced colloidal gold is

4 1808 X. Chu et al. / Biosensors and Bioelectronics 20 (2005) Fig. 1. Schematic representation of the analytical procedure of the heterogeneous electrochemical immunoassay based on silver-enhanced gold nanoparticle label. depicted in Fig. 1, and it was applied to human IgG analyte. Primary antibodies specific for human IgG are adsorbed passively on the walls of a polystyrene microwell. The human IgG analyte is first captured by the primary antibody and then sandwiched by a secondary colloidal gold-labeled antibody. After removal of the unbound labeled antibody, the silver-enhancer solution is added and incubated in the dark. As the silver ions in the silver-enhancer solution can only be catalytically reduced exclusively on the gold colloids, a large amount of specific silver deposition is produced at the walls of the polystyrene microwell through the catalytic reduction of the silver ions on the antibody-colloidal gold conjugate. The silver metal thus deposited is then dissolved in an acidic solution and the silver ions (Ag I ) released in solution are quantitatively determined at a glassy-carbon electrode by ASV. The electrochemical signal is directly proportional to the amount of analyte (human IgG) in the standard solution or sample Determination of silver (I) at a glassy-carbon electrode Anodic stripping voltammetry (ASV) has been proved to be a very sensitive method for trace determination of metal ions (Dequaire et al., 2000; Authier et al., 2001). In this analytical technique, the metal is cathodically electrodeposited onto the surface of an electrode during a preconcentration period, and it is then stripped from the electrode by anodic oxidation. The analytical performance of the glassy-carbon electrode for the detection of Ag I by ASV was firstly investigated. The study was carried out in a 0.6 M potassium nitrate solution containing 0.1 M HNO 3 (0.6 M KNO 3 /0.1 MHNO 3 ), since HNO 3 is required for the efficient dissolution of silver in the final step of the electrochemical Fig. 2. CV curve (v = 100 mv s 1 ) recorded at a glassy-carbon electrode immersed in 3 ml of 0.6 M KNO 3 /0.1 M HNO 3 containing 10 MAg I after electrodeposition at 0.5 V vs. SCE during 10 min under magnetic stirring. Inset: CV curve (100 mv s 1 ) at a glassy-carbon electrode in the solution of Ag I in 0.6 M KNO 3 /0.1 M HNO 3 without preliminary electrodeposition. immunoassay. The CV curve (Fig. 2) recorded at a glassycarbon electrode after cathodic polarization at 0.5 V versus SCE for 10 min in a magnetically stirred solution containing 10 MAg I shows a well-defined anodic peak at 0.4 V (peak potential E p,a ) which is characteristic of the oxidation of electrodeposited silver. During the scan reversal, a small cathodic peak located at 0.3 V is visible. Based on the finding of a cathodic peak near 0.3 V in the CV curve of Ag I ions without electrodeposition (Fig. 2, inset), it seems this small cathodic peak corresponds to the reduction of Ag I ions anodically released and still present in the diffusion layer. The shift of reduction peak of Ag I towards a little higher potential might arise from the catalytic effect of silver particles deposited on the electrode surface, which cannot be totally oxidized in the stripping step. According to the electrochemical theory, the anodic stripping peak current (i p,a ) and the integration of the stripping peak current are directly proportional to the concentration of Ag I ions over a certain range. Several parameters were investigated in order to establish optimal conditions for the detection of Ag I. The influence of the electrodeposition potential (E d ) upon the stripping response was tested (Fig. 3). As E d decreased from 0.1 to 0.9 V, the anodic peak current (i p,a ) resulting from the oxidation of electrodeposited silver increased rapidly between 0.1and 0.5 V and then decreased relatively slowly below 0.5 V. A deposition potential of 0.5 V versus SCE was selected for the further studies. The effect of the deposition time upon the stripping response was also examined in Fig. 4. The anodic peak current (i p,a ) increased in a nearly linear fashion up to 10 min and then reached a constant value. Consequently, an electrodeposition time of 10 min was chosen for all of the experiments. Under the optimal conditions chosen as above, a good linear dependence of the anodic stripping peak current (i p,a ) with the silver (I) concentration over the Mto5

5 X. Chu et al. / Biosensors and Bioelectronics 20 (2005) Fig. 3. Effect of the deposition potential upon the silver stripping peak current. Deposition time, 10 min; electrolyte, 0.6 M KNO 3 /0.1 M HNO 3 ; concentration of Ag I, M M range was obtained, and the linear correlation coefficient was (Fig. 5). It is to be noted that the same glassy-carbon electrode was used to obtain all of the data plotted in Fig. 5, which could be achieved by preconditioning the electrode at +1.0 V versus SCE for 1 min between each measurement. The standard deviation (S.D.) of five measurements (n = 5) of the background noise was 2.5 A and the detection limit calculated from three times of the standard deviation was M. These results further showed that ASV is a very sensitive and effective method for trace determination of metal ions and hence can be applied to the detection of silver (I) ions produced by the electrochemical immunoassay based on silver-enhanced gold nanoparticle label Optimization of immunoassay conditions The electrochemical immunoassay of human IgG was performed as depicted in Fig. 1 using colloidal gold-labeled goat Fig. 5. Calibration plots of Ag I recorded by ASV at a glassy-carbon electrode. Electrodeposition at 0.5 V vs. SCE for 10 min under magnetic stirring in 3 ml of 0.6 M KNO 3 /0.1 M HNO 3. Error bars represent S.D., n =4. anti-human IgG antibody in connection with the silver enhancement, and the detailed optimization of each of these steps was reported below. The effect of the antigen antibody reaction time upon the anodic stripping peak current was firstly investigated in Fig. 6. The response increased nearly linearly with the reaction time between 20 and 60 min and then leveled off above 60 min. This indicates that the interaction of antigen with antibody has reached equilibrium after 60 min, and hence a reaction time of 60 min was selected for all of the experiments. The quality of the colloidal gold-labeled goat anti-human IgG antibody affects strongly the response of the anodic stripping voltammetry, and hence its preparation should be performed strictly according to the method described in Section 2. In order to avoid the aggregation between the gold nanoparticles, the colloidal gold solution should be used to synthesize the antibody-colloidal gold conjugate as soon as possible after its preparation. In addition, a fit amount of Fig. 4. Effect of the deposition time upon the silver stripping peak current. Deposition potential, 0.5 V vs. SCE; electrolyte, 0.6 M KNO 3 /0.1 M HNO 3 ; concentration of Ag I, M. Fig. 6. Effect of the antigen antibody immunoreaction time upon the anodic stripping peak current. Concentration of human IgG, gml 1 ; dilution ratio of the antibody-colloidal gold conjugate, 1:4; silver staining time, 30 min. Other conditions, as in Fig. 5.

6 1810 X. Chu et al. / Biosensors and Bioelectronics 20 (2005) Fig. 7. Effect of the dilution ratio of antibody-colloidal gold conjugate upon the anodic stripping peak current. Concentration of human IgG, gml 1 ; antigen antibody immunoreaction time, 60 min; silver staining time, 30 min. Other conditions, as in Fig. 5. BSA was added to the TBS buffer solution used to resuspend the sediment of the antibody-colloidal gold conjugate, since it was beneficial to retain the stability of the colloidal gold-labeled antibody during the long store. Moreover, the influence of the concentration of the colloidal gold-labeled antibody upon the response of the anodic stripping voltammetry was also investigated in Fig. 7. The anodic stripping peak current increased in a nearly linear fashion by decreasing the dilution ratio (i.e., increasing the concentration) of the colloidal gold-labeled antibody between 1:64 and 1:4, and then reached a constant value at more concentrated solutions. A dilution ratio of 1:4 was consequently selected for the further studies. Under these optimal conditions described above, the red color of the antibody-colloidal gold conjugate can be observed at the walls of a polystyrene microwell after the incubation and washing steps, which indicates that the colloidal gold-labeled antibody has been adsorbed on the walls of a polystyrene microwell by the sandwich immunoassay format illustrated in Fig. 1. Further amplification of the sensitivity of the electrochemical immunoassay based on colloidal gold-labeled antibody can be achieved by catalytic precipitation of silver on the gold nanoparticle tags, which can produce relatively large particles. This procedure is called as silver enhancement or silver staining procedure. Apparently, when the concentration of each component of the silver-enhancer solution is fixed, the amount of silver produced by catalytic precipitation on the gold nanoparticle tags would be strongly influenced by the silver staining time. Indeed, it was observed that the anodic peak current resulting from the oxidation of deposited silver increased nearly linearly with the silver staining time (not shown). However, increased silver staining time, while offering very favorable signal enhancement, leads to an increase in the background response. In contrast to the analytical signal generated by the silver deposited exclusively on the gold nanoparticle tags, such a background response might result from the nonspecific binding of silver ions onto the walls of the polystyrene microwell or the immobilized proteins, which also increase with the silver staining time. This back- Fig. 8. Calibration plot (A) and log-log calibration data (B) of the anodic stripping peak current vs. human IgG concentration. Reaction time, 60 min; dilution ratio of the antibody-colloidal gold conjugate, 1:4; silver staining time, 30 min. Other conditions, as in Fig. 5. Error bars represent S.D., n =4. ground contribution would limit the detectability. With the two factors (the high signal response and the low detection limit) taken into account, 30 min of the silver staining time was selected for the further studies Analytical performance Fig. 8 displays the dependence of the ASV response upon the concentration of the human IgG. The analytical response resulting from the integration of the stripping peak current (Q p ) was chosen because it has a little more sensitive than the i p,a response. The signal increased rapidly with the human IgG concentration at first (up to 1.7 gml 1 ), then more slowly, and started to level off above gml 1 (Fig. 8A). Such curvature can be addressed by using a logarithmic scale, which resulted in a highly linear response up to gml 1 and the linear correlation coefficient was (Fig. 8B). The dynamic range for the assay extended between 1.66 ng ml 1 and gml 1. The signal saturated above gml 1 human IgG, owing to

7 X. Chu et al. / Biosensors and Bioelectronics 20 (2005) the limited amount of antibody available on the surface of microwells. Similar logarithmic scales were employed in analogous electrochemical immunoassay (Dequaire et al., 2000) and electrochemical detection of DNA hybridization (Authier et al., 2001; Wang et al., 2001b). The detection limit was estimated to be 1.0 ng ml 1, i.e., M human IgG (according to 3S.D., where S.D. is the standard deviation of five measurements of a blank solution, S.D. = 1.4 C, n = 5). The sensitivity of the method is competitive with another gold nanoparticle-based electrochemical immunoassay recently reported for goat IgG (detection limit of 0.5 ng ml 1 )(Dequaire et al., 2000) and superior to the previous electrochemical immunoassay of IgG based on a bismuth chelate label (detection limit of 600 ng ml 1 )(Hayes et al., 1994). A series of eight repetitive measurements of the gml 1 human IgG solution was used to estimate the precision. This series yielded a mean integration of the stripping peak current of 163 C and a relative standard deviation of 7.2%. Such signal variations reflect the good reproducibility of the protocol of the immunoassay and electrochemical detection. Theoretically, the silver-enhanced colloidal gold electrochemical immunoassay developed in the present work will have a lower detection limit than the electrochemical immunoassay based on colloidal gold tags recently reported for goat IgG (Dequaire et al., 2000), since each colloidal gold particle can act as a catalytic site and result in a large amount of silver deposition on the colloidal gold tags. Nevertheless, the detection limits obtained in the present study is almost the same as that reported for colloidal gold labels (Dequaire et al., 2000). This is due to the fact that the present study only simply released the silver precipitated on gold nanoparticle tags in a beaker containing 3 ml electrolyte solution for ASV analysis, while the reported colloidal gold-based immunoassay had to use screen-printed microband electrodes to reduce the electrochemical detection volume to a 35- l droplet so as to improve the detection limit in subsequent ASV determination. Then, it might be reckoned that the silver enhancement step increases the amount of metal tags by about 100 times. As a result, it is expected that the detection limit of the present immunoassay method could still be substantially lowered by dissolving the silver precipitates in an electrochemical microcell and avoiding the dilution of the silver ions solution Analytical application To demonstrate the applicability of proposed electrochemical immunoassay to clinical diagnostics, four human serum samples provided by Xiangya Medical College, Central South University were analyzed. The results are shown in Table 1. The results obtained by the proposed technique were in good agreement with those obtained by ELISA method, which indicates that it is feasible to apply the developed electrochemical immunoassay to detect human IgG in serum samples. Table 1 IgG concentration in human serum samples tested by proposed electrochemical immunoassay format and ELISA method a Serum sample IgG concentration (ng ml 1 ) Electrochemical immunoassay ELISA ± ± ± ± a The data are given as average value ± S.D. (n = 3). 4. Conclusion We have demonstrated for the first time the feasibility of the electrochemical stripping metalloimmunoassay based on the precipitation of silver onto gold nanoparticle tags. In the case of human IgG, the dynamic range and the detection limit of the proposed method are competitive with or better than other electrochemical immunoassays based on the colloidal gold label (Dequaire et al., 2000) or the bismuth chelate label (Hayes et al., 1994). The new silver-enhanced colloidal gold electrochemical immunoassay combines the inherent high sensitivity of stripping metal analysis with the dramatic signal amplification of the silver precipitation on gold nanoparticle tags and, hence, offers great promise for the ultrasensitive immunoassay. The new approach possesses the attractive performance such as simplicity and high sensitivity and can be extended to a large variety of bioaffinity assays of analytes of environmental or clinical significance even the DNA hybridization detection. Moreover, the colloidal gold label is more stable than the radioistopic or enzyme labels, and the gold colloid labeling procedure is very simple and does not affect generally the biochemical activity of the labeled compound. We now envisage the simultaneous detection of several analytes by using different colloidal metal labels with distinct anodic stripping potentials. Acknowledgement Financial support from the National Natural Science Foundation of China (Grant No ) is gratefully acknowledged. References Authier, L., Grossiord, C., Brossier, P., Limoges, B., Gold nanoparticle-based quantitative electrochemical detection of amplified human cytomegalovirus DNA using disposable microband electrodes. Anal. Chem. 73, Blackburn, G.F., Shah, H.P., Kenten, J.H., Leland, J., Kamin, R.A., Link, J., Peterman, J., Powell, M.J., Shah, A., Talley, D.B., Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. Clin. Chem. 37,

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