Silver-enhanced colloidal gold metalloimmunoassay for Schistosoma japonicum antibody detection

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1 Journal of Immunological Methods 31 (25) Research paper Silver-enhanced colloidal gold metalloimmunoassay for Schistosoma japonicum antibody detection Xia Chu a,b, T, Zhi-Feng Xiang a, Xin Fu a, Shi-Ping Wang c, Guo-Li Shen b, T, 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 481, PR China b State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 482, PR China c Department of Parasitology, Xiangya Medical College, Central South University, Changsha 478, PR China Received 11 October 24; received in revised form 28 February 25; accepted 19 March 25 Available online 8 April 25 Abstract A silver-enhanced colloidal gold metalloimmunoassay has been proposed for the determination of Schistosoma japonicum antibody (SjAb) in rabbit serum. The adult worm antigen of S. japonicum (SjAg) was adsorbed passively on the walls of a polystyrene microwell and then reacted with the desired SjAb. The colloidal gold-labeled goat anti-rabbit IgG secondary antibody was adsorbed on the walls of the polystyrene microwells through the reaction with SjAb, followed by the silver enhancement process, dissolution of silver metal atoms in an acidic solution, and determination of dissolved silver ions by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. Assay conditions were optimized, including the reaction time of SjAg with SjAb, the interaction of SjAb with the colloidal gold-labeled secondary antibody, the dilution ratio of the colloidal gold-labeled secondary antibody and the silver enhancement time. The integration of the anodic stripping peak current depended linearly on the SjAb logarithmic concentration over the range of 6.4 ng/ml to Ag/ml. A detection limit as low as 3. ng/ml SjAb was achieved, which was better than the piezoelectric body acoustic wave sensor (detection limit of 7.2 Ag/ml) and the renewable amperometric immunosensor (detection limit of.36 Ag/ml). Rabbit serum samples with various degrees of infection were analyzed, and the results demonstrate that the proposed method meets the requirements of clinical analysis. D 25 Elsevier B.V. All rights reserved. Keywords: Metalloimmunoassay; Colloidal gold label; Anodic stripping voltammetry; Silver enhancement; Schistosoma japonicum antibody Abbreviations: ASV, anodic stripping voltammetry; BSA, bovine serum albumin; GCE, glassy-carbon electrode; IgG, immunoglobulin G; PBS, phosphate-buffered saline; Q p, integration of anodic stripping peak current; SCE, saturated calomel electrode; SD, standard deviation; SjAb, Schistosoma japonicum antibody; SjAg, Schistosoma japonicum antigen; TBS, Tris-buffered saline. T Corresponding authors. State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 482, PR China. Tel.: ; fax: addresses: xiachu@hnu.cn (X. Chu), glshen@hnu.cn (G.-L. Shen) /$ - see front matter D 25 Elsevier B.V. All rights reserved. doi:1.116/j.jim

2 78 X. Chu et al. / Journal of Immunological Methods 31 (25) Introduction Schistosoma japonicum (Sj) is a widespread parasitic disease, mainly found in some parts of Africa, Asia and Latin America (Liu et al., 1991) and is widely regarded as a threat to human health. According to the statistics of the World Health Organization (WHO), about 1.6 billion people in 76 countries have been infected with this parasite. Some conventional diagnostic methods, such as enzyme-linked immunosorbent assay (ELISA), radio immunoassays (RIA), counter immunoelectrophoresis (CIE), indirect fluorescence antibody test (IFAT) (Liu et al., 1991), etc., have been adapted to clinical analyses. However, these methods usually require tedious assay time, radioactive chemicals, or expensive instruments. Furthermore, these methods have been used mainly for qualitative and semiquantitative analysis for SjAb. In recent years, a piezoelectric body acoustic wave sensor (Wu et al., 1999) and a renewable amperometric immunosensor (Liu et al., 21) have been developed for the determination of SjAb. Unfortunately, the detection limits of these two sensors are relatively high so that some weakly positive samples escape the screening test. According to clinical experience only ~9% of infected samples tested show positive results. Therefore, searching for rapid, sensitive diagnostic methods with real-time output and low cost is still of considerable significance. Metalloimmunoassay is an immunoassay involving metal-based labels. Since it was developed, great progress has been made 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., 2; 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 technique holds great promise for metalloimmunoassays owing to its unique advantages, such as rapidity, simplicity, inexpensive instrumentation and field-portability. Nevertheless, the sensitivity of the conventional electrochemical metalloimmunoassays is very limited because only one (or several) organometallic or metal ion is labeled in every protein molecule. Recently, a new electrochemical metalloimmunoassay based on a colloidal gold label was reported (Dequaire et al., 2), in which each colloidal gold particle contained thousands of atoms, and consequently, a lower detection limit (picomolar level) could be attained compared with conventional methods (nanomolar level). In recent years, there has been growing interest in the development of metal nanoparticle-based detection techniques for chemical and biological analysis due to their unique optical and electrical properties. Electrochemical stripping detection of DNA hybridization based on gold nanoparticles as DNA labels have been investigated (Authier et al., 21; Wang et al., 21b). The amount of bound nanoparticle labels was determined by acidic dissolution of the nanoparticle, followed by a stripping analysis of the dissolved metal ions. 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., 2), an electrical detection-based DNA array (Park et al., 22) and Raman spectroscopic fingerprints for DNA and RNA detection (Cao et al., 22) based on silverenhanced gold nanoparticle labels for sensitivity improvement. Silver enhancement was also used for detecting the DNA hybridization event by scanning electrochemical microscopy (Wang et al., 22) and electrochemical stripping metal analysis (Wang et al., 21a). Some other groups have successfully incorporated the silver enhancement technique with gold nanoparticle labels for enhanced electrochemical hybridization detection (Lee et al., 23; Cai et al., 22), in which the silver depositing on the gold nanoparticle label was measured directly by its oxidation current response during voltammetric scans. Efforts have also been made to utilize gold nanoparticle-based electrocatalytic silver deposition as an alternative so that the background noise arising from the non-optimized silver enhancement conditions could be minimized (Lee et al., 24). In this work, a new silver-enhanced colloidal gold metalloimmunoassay is presented. Inspired by similar use of gold nanoparticle labeling and subsequent

3 X. Chu et al. / Journal of Immunological Methods 31 (25) silver enhancement, the present work aims to develop an analogous metalloimmunoassay; an electrochemical metalloimmunoassay based on the use of gold nanoparticle labels and silver enhancement has not been reported by other groups. The new silverenhanced colloidal gold electrochemical stripping metalloimmunoassay combines the inherent high sensitivity of stripping metal analysis with the remarkable signal amplification resulting from 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 goldlabeled antibody, silver enhancement and acid dissolution, and stripping detection of the silver at a glassy-carbon electrode. The detailed optimization and attractive performance characteristics of the metalloimmunoassay are reported in the following sections. 2. Materials and methods 2.1. Reagents An adult worm antigen (AWA) of S. japonicum was prepared according to the reported method (Wang et al., 1992). The concentration of SjAg was 12 mg/ml. S. japonicum antibody used as the calibration standard was prepared by immunizing rabbits for 32 days with adult worms. The antibody in the infected rabbit serum was then purified by precipitation from saturated ammonium sulfate solution, as described in the literature (Liu et al., 1991). The actual concentration of SjAb was determined using the ELISA method. The goat anti-rabbit immunoglobulin G (IgG) secondary antibody (1 mg/ml) and bovine serum albumin (BSA) were purchased from Sino-Americal Biotechnology Company (Shanghai, China). Chloroauric acid (HAuCl 4 ), trisodium citrate, hydroquinone, silver nitrate (AgNO 3 ) were of analytical reagent grade and obtained from Shanghai Chemical Reagents (Shanghai, China). Double-distilled water was used throughout Apparatus Electrochemical measurements were performed on a CHI 66A electrochemical analyzer (Shanghai Chenhua Instruments, Shanghai, China) with a threeelectrode system comprising a platinum wire as auxiliary electrode, a saturated calomel electrode (SCE) as reference and a glassy-carbon electrode (GCE, 3 mm in diameter) as working electrode. The saturated calomel electrode was separated from the electrolyte solution by a double electrolytic salt bridge filled with saturated KNO 3 in order to avoid interference caused by the continuous leaching of chloride anion that leads to AgCl precipitation. A model CSS51 thermostat (Chongqing, China) was used to control the incubating temperature. A TGL-16A highspeed freezing centrifuge (Changsha, China) was used for the isolation and purification of the colloidal goldlabeled goat anti-rabbit IgG secondary antibody 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. mm KCl, ph 7.4); (c) Tris-buffered saline (TBS; 2 mm Tris and 15 mm NaCl, ph adjusted to 8.2 with concentrated HCl); (d) TBS containing.1% BSA (TBS BSA); (e) citrate buffer (.243 M C 6 H 8 O 7 d H 2 O and.163 M Na 3 C 6 H 5 O 7 d 2H 2 O, ph 3.5). Standard rabbit SjAb solutions were prepared by diluting a stock SjAb solution (1 mg/ml) with PBS. The SjAg solution (12 Ag/ml) was prepared by diluting a stock SjAg solution (12 mg/ml) with carbonate buffer. The colloidal gold-labeled goat antirabbit IgG secondary antibody solutions were prepared and diluted with TBS BSA. The silver-enhancer solution was composed of 1. g hydroquinone, 35 mg AgNO 3, 5 ml citrate buffer and 5 ml double-distilled water (Xu, 1997); it was prepared fresh as needed Preparation of colloidal gold-labeled goat antirabbit IgG secondary antibody Preparation of colloidal gold particles Colloidal gold particles were prepared according to the literature (Grabar et al., 1995) with slight

4 8 X. Chu et al. / Journal of Immunological Methods 31 (25) modifications. In a 5-ml round-bottom flask, 25 ml of.1% HAuCl 4 in double-distilled water was brought to the boil with vigorous stirring. To this solution was added 3.75 ml of 1% trisodium citrate. Boiling was pursued for an additional 1 min, the heating source was removed, and the colloid was stirred for another 15 min. The resulting solution of colloidal particles was characterized by an absorption maximum at 52 nm. Transmission electron microscopy (TEM) indicated a particle size of 2F4 nm ( particles sampled) Determination of optimum amount of coating protein A curve was constructed for colloidal gold-labeled secondary antibody to determine the optimum 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 stock solution aliquots ( 5 Ag) of goat anti-rabbit IgG secondary antibody and were added in 5-Ag increments to cuvettes containing 1. ml of 2-nm diameter colloidal solution adjusted to ph 9. using.1 M NaOH. The volumes of these samples were corrected to 1.15 ml with double-distilled water and Al of 1% NaCl was added to each. The solutions were agitated and then allowed to stand for 1 min at room temperature. The absorbance values at 52 nm of these samples were recorded and plotted vs. the amount of coating protein. The optimum amount of coating protein can then be determined as that where the decrease in absorbance starts to be insignificant. For 2-nm gold colloid, the optimum amount of goat anti-rabbit IgG secondary antibody for coating the gold nanoparticles is 3 Ag/ml colloidal gold solution and is effective to prevent aggregation. Optimization of the amount of coating protein is beneficial for decreased non-specific adsorption of proteins and improves the sensitivity of the immunoassay Preparation of colloidal gold-labeled goat antirabbit IgG secondary antibody The colloidal gold-labeled secondary antibody was prepared by the addition of the goat anti-rabbit IgG secondary antibody to 2 ml of ph-adjusted colloidal gold solution followed by incubation for 1 h at room temperature with periodic gentle mixing. The solution was then divided into 1-ml fractions in 1.5-ml microcentrifuge tubes and centrifuged at 17,39g for 1 min. Two phases can be obtained: a clear to pink supernatant of unbound antibody and a dark red, loosely packed sediment of the colloidal gold-labeled secondary antibody. The supernatant was discarded and the soft sediment was rinsed by resuspending in 1 ml of TBS BSA and collected after a second centrifugation at 17,39g for 1 min. Finally, the colloidal gold-labeled secondary antibody was resuspended in 25 Al of 2 mm TBS with.1% BSA added to increase the stability of colloidal goldlabeled secondary antibody and minimize non-specific adsorption during the assays. The colloidal goldlabeled secondary antibody can be stored at 4 8C for more than 1 month without loss of activity Immunoassay procedure An indirect immunoassay format was used in this study to detect S. japonicum antibody. The SjAg solution (2 Al, 12 Ag/ml) was added to the polystyrene microwells and incubated at 4 8C overnight. After removing the solution, the wells were rinsed with.5 M NaCl and double-distilled water three times each for 3 min, and rabbit primary SjAb standard solutions (2 Al) were added and incubated in the wells at 37 8C for 6 min. Next, the microwells were drained and rinsed as described above. Following this step, 2 Al of 1/4 colloidal gold-labeled goat anti-rabbit IgG secondary antibody was added and incubated at 37 8C for 7 min. A last washing cycle was then performed as mentioned above. After removing the rinsing solution, 2 Al of silverenhancer solution was pipetted into the microwells and incubated at room temperature for 3 min in the dark. The wells were then washed with doubledistilled water three times. Finally, 3 Al of 1.5 M HNO 3 was added to the microwells and incubated at room temperature for 3 min to dissolve the metal silver deposited on the walls of the microwells Anodic stripping voltammetric (ASV) measurement The solutions of silver(i) ions (3 Al) were transferred from the microwells into a 1-ml beaker containing 3 ml of.6 M KNO 3 and.1 M HNO 3 as electrolyte solution, and the released silver(i) ions were then quantified by ASV under the following

5 X. Chu et al. / Journal of Immunological Methods 31 (25) conditions: 1-min deposition at.5 V vs. SCE reference and the potential scan from +.2 to +.7 V at mv/s scan rate. The integration of the stripping peak current ( Q p ) was recorded as the analytical signal. The glassy-carbon electrode was cleaned by preconditioning at +1. V vs. SCE for 1 min between each measurement. 3. Results and discussion The principle of the silver-enhanced colloidal gold metalloimmunoassay for S. japonicum antibody detection is depicted in Fig. 1. An indirect immunoassay format was used in this study, consisting of six steps: (a) passive adsorption of the adult worm antigen of S. japonicum (SjAg) on the walls of a polystyrene microwell; (b) immunoreaction of the SjAg with rabbit primary antibody specific for S. japonicum (SjAb); (c) immunoreaction of the SjAb with colloidal goldlabeled goat anti-rabbit IgG secondary antibody; (d) silver enhancement process; (e) silver metal dissolution in an acidic solution; and (f) ASV detection of the released Ag I ions at a glassy-carbon electrode. The silver enhancement process results in a large amount of silver deposition on the walls of the polystyrene microwell due to the catalytic reduction of the silver ions by the gold nanoparticle tags. The electrochemical signal is directly proportional to the amount of analyte (SjAb) in the standard solution or sample. A direct electrochemical immunoassay based on silver-enhanced colloidal gold at electrode surfaces such as gold electrode, glassy-carbon electrode and carbon-paste electrode was also attempted. However, it was observed that the background arising from silver enhancement at the electrode surfaces was relatively high. Therefore, the polystyrene-based microwell was chosen as the support for the immunoassay. This support was found to give a very low background for silver enhancement. To our knowledge, the polystyrene matrix has not been exploited in silver-enhanced determinations except for stripping voltammetric analysis of the gold tagging system ASV determination of silver(i) ion Anodic stripping voltammetry (ASV) has been found to be a very sensitive method for trace ASV detection SjAg Ag + Colloidal gold-labeled goat anti-rabbit IgG antibody HNO 3 Rabbit SjAb Ag + Hydroquinone Silver enhanced gold Fig. 1. Schematic representation of the analytical procedure of silver-enhanced colloidal gold metalloimmunoassay for Schistosoma japonicum antibody detection. determination of metal ions. 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 detection of Ag I by ASV was investigated first. The study was carried out in a.6 M potassium nitrate solution containing.1 M HNO 3 (.6 M KNO 3 /.1 M HNO 3 ), since HNO 3 is required for the efficient dissolution of silver in the final step of the electrochemical immunoassay. The ASV scans of Ag I ions of different concentrations were recorded at a glassy-carbon electrode after electrodeposition at.5 V vs. SCE for 1 min (Fig. 2). A well-defined anodic peak located at about.4 V is clearly visible and it corresponds to the oxidation of electrodeposited silver. Experimental parameters, including the deposition potential and the deposition time, were selected according to the literature (Chu et al., 25). The anodic stripping peak current increased with the increase in the concentration of the Ag I ions. The inset of Fig. 2 shows the calibration plot of the integration of the anodic stripping peak current ( Q p ) against the concentration of the Ag I ions. A good linear dependence of the integration of the anodic stripping peak current with the Ag I concentration over the 51 9 to 11 5 M range was obtained, and the linear correlation

6 82 X. Chu et al. / Journal of Immunological Methods 31 (25) M M 25 2 i / µa M M M M M M Qp / µc [Ag I ] / µm E / V vs. SCE Fig. 2. ASV scans of Ag I ions of different concentrations at a glassy-carbon electrode after electrodeposition at.5v vs. SCE for 1 min. Inset calibration plot of the integration of the anodic stripping peak current ( Q p ) against the concentrations of the Ag I ions. coefficient was The same glassy-carbon electrode was used to obtain all of the data plotted in Fig. 2; the electrode was preconditioned at +1. V vs. SCE for 1 min between each measurement. The standard deviation (SD) of five measurements (n =5) of the background noise was 4.5 AC and the detection limit calculated from three times the standard deviation was M. These results show that ASV is a very sensitive and effective method for trace determination of metal ions and hence can be applied to the detection of Ag I ions produced by the electrochemical immunoassay based on a silver-enhanced gold nanoparticle label Optimization of immunoassay conditions The electrochemical metalloimmunoassay of S. japonicum antibody was performed as depicted in Fig. 1 using colloidal gold-labeled goat anti-rabbit IgG secondary antibody in conjunction with the silver enhancement process; the detailed optimization of the immunoassay procedure is reported below. The effect of the antigen antibody immunoreaction time upon the integration of anodic stripping peak current is shown in Fig. 3. The response increased rapidly with the reaction time between 1 and 4 min, then more slowly, and reached a constant value above 6 min. This indicates that the interaction of antigen with antibody has reached equilibrium after 6 min, and hence subsequent work employed a reaction time of 6 min. The influence of the reaction time of rabbit primary antibody (SjAb) with colloidal gold-labeled goat antirabbit IgG secondary antibody upon integration of the anodic stripping peak current is shown in Fig. 4. Similar to Fig. 3, the response increased rapidly with the reaction time between 2 and 5 min, then more slowly, and reached a constant value above 7 min. A reaction time of 7 min was consequently selected for further studies. The equilibrium time of SjAb with colloidal gold-labeled secondary antibody was longer than that of SjAb with SjAg, which may be related to the relatively large molecular volume of the colloidal gold-labeled secondary antibody. The goat anti-rabbit IgG secondary antibody was attached to a 2-nm gold nanoparticle via electrostatic interactions (Katz and Willner, 24). The quality of the colloidal gold-labeled goat anti-rabbit IgG secondary antibody strongly affected the response of the anodic stripping voltammetry, and hence its preparation should be performed strictly according to the method described in Section 2. In

7 X. Chu et al. / Journal of Immunological Methods 31 (25) Q p / µc Time / min Fig. 3. Effect of the immunoreaction time of immobilized SjAg with SjAb upon integration of the stripping peak current ( Q p ). Concentration of SjAb, 2 Ag/ml; reaction time of SjAb with colloidal gold-labeled secondary antibody, 7 min; dilution of colloidal gold-labeled secondary antibody, 1/4; silver enhancement time, 3 min. Other conditions as in Fig. 2. order to avoid aggregation between the gold nanoparticles, the colloidal gold solution should be used to synthesize the colloidal gold-labeled secondary antibody as soon as possible after its preparation. In addition, the modified nanoparticles were stabilized by addition of BSA at the end of the preparation. The addition of an excessive amount of BSA in the colloidal gold-labeled secondary antibody solution is beneficial to block the colloidal gold surface, thus lowering the non-specific adsorption of colloidal gold-labeled secondary antibody. Under these conditions, the colloidal gold-labeled secondary antibody can be stored at 4 8C for more than 1 month without any apparent decrease in activity. An enzyme immunoassay procedure was utilized to roughly estimate the number of active antibodies on the surface of a gold nanoparticle in the colloidal gold-labeled goat anti-rabbit IgG secondary antibody (Hirsch et al., 23). This procedure included an immunoreaction with excessive horseradish peroxidase (HRP)-labeled rabbit IgG followed by determination of the amount of HRP enzyme using color-developing reagent solution. The binding ratio of the second antibody to the gold nanoparticle was Q p / µc Time / min Fig. 4. Effect of reaction time of rabbit primary antibody (SjAb) with colloidal gold-labeled goat anti-rabbit IgG secondary antibody upon integration of the stripping peak current ( Q p ). Antigen antibody immunoreaction time, 6 min. Other conditions as in Figs. 2 and 3.

8 84 X. Chu et al. / Journal of Immunological Methods 31 (25) in the colloidal gold-labeled goat anti-rabbit IgG secondary antibody. The influence of the concentration of the colloidal gold-labeled secondary antibody upon the response of anodic stripping voltammetry is shown Fig. 5. The integration of the anodic stripping peak current ( Q p ) increased in a nearly linear fashion by decreasing the dilution factor (i.e., increasing the concentration) of the colloidal gold-labeled secondary antibody between 1/64 and 1/4, and then leveled off at more concentrated solutions. A dilution of 1/4 was consequently selected for further studies. Under these optimum conditions described above, the red color of the gold nanoparticle can be clearly observed at the walls of a polystyrene microwell after the incubation and washing steps, confirming that the colloidal goldlabeled goat anti-rabbit IgG secondary antibody has indeed been adsorbed on the walls of the polystyrene microwell by the indirect immunoassay format illustrated in Fig. 1. A comparison of gold nanoparticles of different sizes was made with regard to the signal amplification by silver staining. The 12 F 2-nm gold nanoparticles were prepared according to the literature (Jana et al., 21), and the electrochemical metalloimmunoassays using two gold nanoparticles of 12F2 and 2F4 nm were performed. The integration of the anodic stripping peak current was 325F21 AC and 338 F 25 AC, respectively, for gold nanoparticles of 12 F 2 nm and 2 F 4 nm in five parallel assays, indicating that the size of the gold nanoparticles might have little effect on the performance of the metalloimmunoassay procedure Effect of silver enhancement time The formulation of the silver-enhancer solution, including ph, the amount of silver salt and hydroquinone, was optimized to give adequate enhancement and minimize non-specific adsorption in the immunoassay. To mitigate the electrostatic adsorption of Ag + on the protein, an acidic solution of ph 3.5 was employed as the silver enhancement medium, in which the protein was also positively charged and the amino terminals exhibited very weak coordination to Ag +. In DNA analysis, the electrostatic adsorption of Ag + on the negatively charged DNA strands usually induces a high blank signal. When the formulation 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 enhancement time. Indeed, it was observed that the integration of the anodic stripping peak current resulting from the oxidation of deposited silver increased rapidly with the silver enhancement time from 1 to 3 min, then more slowly, and leveled off above 4 min (curve (a) in Fig. 6). However, increased silver enhancement time, while offering very favorable signal enhancement, leads to an increase in the background response Q p / µc /1 1/2 1/4 1/8 1/16 1/32 1/64 Dilution Fig. 5. Effect of dilution of colloidal gold-labeled secondary antibody upon integration of the stripping peak current ( Q p ). Antigen antibody immunoreaction time, 6 min. Other conditions as in Figs. 2 and 3.

9 X. Chu et al. / Journal of Immunological Methods 31 (25) a Q p / µc 3 2 b Time / min Fig. 6. Effect of silver enhancement time upon the integration of stripping peak current ( Q p ). (a) 2 Ag/ml SjAb; (b) rabbit negative control serum. Antigen antibody immunoreaction time, 6 min. Other conditions as in Figs. 2 and 3. resulting from the non-specific binding of silver ions onto the walls of the polystyrene microwell or the immobilized proteins, which also increase with the silver enhancement time (curve (b) in Fig. 6). This background contribution would limit detectability. With these two factors (high signal response and low detection limit) taken into account, silver staining time of 3 min was selected for further studies. The electrochemical deposition method (Lee et al., 25) was not utilized in the present study to reduce the background noise. This is due to the fact that electrocatalytic deposition requires direct electrical contact between the gold particles and the electrode surface. In immunoassay, the relatively big antibody molecules immobilized on the electrode surface and coated on the colloidal gold form two non-conductive electrical layers, inducing the blockage of direct electrical contact between the gold particles and the electrode surface. Therefore, it might be much more difficult to implement electrocatalytic silver deposition for signal amplification in immunoassay than in DNA detection. It was observed in experiments that the electrocatalytic deposition of silver could not be detected significantly in a sandwich immunoassay protocol based on a carbon-paste electrode with 12- and 2-nm gold nanoparticle labels Q p / µc lg[sjab] / (µg/ml) Fig. 7. Standard calibration plot of the integration of the anodic stripping peak current vs. SjAb logarithmic concentration. Error bars represent the standard deviation of four measurements. Antigen antibody immunoreaction time, 6 min. Other conditions as in Figs. 2 and 3.

10 86 X. Chu et al. / Journal of Immunological Methods 31 (25) Table 1 Reproducibility of the silver-enhanced colloidal gold metalloimmunoassay SjAb concentration Q p /AC a MeanFSD/AC RSD (%) b 2 Ag/ml 333.8, 322.1, 321.3, 31.1, F ng/ml 138.9, 155.7, 149.7, 151.9, F ng/ml 41.75, 4.79, 37.1, 37.28, F a Integration of stripping peak current of five parallel measurements. b Relative standard deviation Analytical performance The sensitivity of the silver-enhanced colloidal gold metalloimmunoassay was investigated by altering the concentration of SjAb over the 1.28 ng/ml to 5 Ag/ml range. The corresponding standard calibration plot is shown in Fig. 7. The integration of the anodic stripping peak current exhibited a highly linear response on SjAb logarithmic concentration between 6.4 ng/ml and Ag/ml and the linear correlation coefficient was The signal saturated above Ag/ml SjAb, due to the limited amount of SjAg available on the surface of the microwells. The detection limit was estimated to be 3. ng/ml (according to 3SD, where SD is the standard deviation of five measurements of a blank solution). The sensitivity of the method is better than the piezoelectric body acoustic wave sensor for the determination of SjAb (detection limit of 7.2 Ag/ml) (Wu et al., 1999) and the renewable amperometric immunosensor for SjAb assay (detection limit of.36 Ag/ml) (Liu et al., 21). Five repetitive measurements were performed using 2 Ag/ml, 16 ng/ml and 1.28 ng/ml SjAb standard solutions to estimate the precision of the analysis (Table 1). The mean integration of the stripping peak current was 318.3, 147. and AC and the relative standard deviation was 3.6%, 5.3% and 7.9%, respectively. Such signal variations reflect the good reproducibility of the protocol of the immunoassay and electrochemical detection Analytical application To demonstrate the use of the proposed electrochemical metalloimmunoassay for the determination of the SjAb in rabbit serum, two rabbit serum samples with different degrees of infection and a negative control rabbit serum, i.e., the serum of a rabbit not infected by adult worms, were assayed. The results are shown in Fig. 8. The integration of the stripping peak 4 3 a b Q p / µc 2 c 1/1 6 1/1 5 1/1 4 1/1 3 1/1 2 Dilution Fig. 8. Integration of the anodic stripping peak current of rabbit serum samples infected by adult worms for 42 days (a), 32 days (b) and negative control rabbit serum (c).

11 X. Chu et al. / Journal of Immunological Methods 31 (25) current increases with an increase in the degree of infection. The negative control serum gives only insignificant signals. This implies that the present method has desirable specificity, and could be used for the direct determination of SjAb concentration in serum as well as for evaluating the degree of infection. 4. Conclusions The silver-enhanced colloidal gold metalloimmunoassay for S. japonicum antibody detection was shown to be feasible. In the case of SjAb, the dynamic range and the detection limit of the proposed method were superior to the piezoelectric body acoustic wave sensor (Wu et al., 1999) and the renewable amperometric immunosensor (Liu et al., 21). The metalloimmunoassay showed good accuracy, acceptable precision and applicability for clinical analysis. The new method could be extended readily to the detection of other clinically important antigens. Moreover, the colloidal gold label is more stable than radioistopic or enzyme labels, and the colloidal gold labeling procedure is very simple and does not generally affect the biochemical activity of the labeled compound. Further studies to explore the feasibility of implementing silver enhancement in immunoassays may lead to a new approach with broad applicability in ultrasensitive immunoassays. Acknowledgments Financial support from the National Natural Science Foundation of China (Grant Nos. 2157) is gratefully acknowledged. References Authier, L., Grossiord, C., Brossier, P., Limoges, B., 21. Gold nanoparticle-based quantitative electrochemical detection of amplified human cytomegalovirus DNA using disposable microband electrodes. Anal. Chem. 73, 445. 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, Bordes, A.L., Limoges, B., Brossier, P., Degrand, C., Simultaneous homogeneous immunoassay of phenytoin and phenobarbital using a Nafion-loaded carbon paste electrode and two redox cationic labels. Anal. Chim. Acta 356, 195. Cai, H., Wang, Y.Q., He, P.G., Fang, Y.Z., 22. Electrochemical detection of DNA hybridization based on silver-enhanced gold nanoparticle label. Anal. Chim. Acta 469, 165. Cao, Y.W.C., Jin, R.C., Mirkin, C.A., 22. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297, Chu, X., Fu, X., Chen, K., Shen, G.L., Yu, R.Q., 25. An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label. Biosens. Bioelectron. 2, 185. Dequaire, M., Degrand, C., Limoges, B., 2. An electrochemical metalloimmunoassay based on a colloidal gold label. Anal. Chem. 72, Doyle, M.J., Halsall, H.B., Heineman, W.R., Heterogeneous immunoassay for serum proteins by differential pulse anodic stripping voltammetry. Anal. Chem. 54, Grabar, K.C., Freeman, R.G., Hommer, M.B., Natan, M.J., Preparation and characterization of Au colloid monolayers. Anal. Chem. 67, 735. Hayes, F.J., Halsall, H.B., Heineman, W.R., Simultaneous immunoassay using electrochemical detection of metal ion labels. Anal. Chem. 66, 186. Hirsch, L.R., Jackson, J.B., Lee, A., Halas, N.J., West, J.L., 23. A whole blood immunoassay using gold nanoshells. Anal. Chem. 75, Jana, N.R., Gearheart, L., Murphy, C.J., 21. Evidence for seedmediated nucleation in the chemical reduction of gold salts to gold nanoparticles. Chem. Mater. 13, Katz, E., Willner, I., 24. Integrated nanoparticle-biomolecule hybrid systems: synthesis, properties, and applications. Angew. Chem., Int. Ed. 43, 642. Kimura, H., Matsuzawa, S., Tu, C.Y., Kitamori, T., Sawada, T., Ultrasensitive heterogeneous immunoassay using photothermal deflection spectroscopy. 2: quantitation of ultratrace carcinoembryonic antigen in human sera. Anal. Chem. 68, 363. Lee, T.M.H., Li, L.L., Hsing, I.M., 23. Enhanced electrochemical detection of DNA hybridization based on electrode-surface modification. Langmuir 19, Lee, T.M.H., Cai, H., Hsing, I.M., 24. Gold nanoparticlecatalyzed silver electrodeposition on an indium tin oxide electrode and its application in DNA hybridization transduction. Electroanalysis 16, Lee, T.M.H., Cai, H., Hsing, I.M., 25. Effects of gold nanoparticle and electrode surface properties on electrocatalytic silver deposition for electrochemical DNA hybridization detection. Analyst 13 (3), 364. Limoges, B., Degrand, C., Brossier, P., Blankespoor, R.L., Homogeneous electrochemical immunoassay using a perfluorosulfonated ionomer-modified electrode as detector for a cationic-labeled hapten. Anal. Chem. 65, 154. Liu, Y.H., Liu, X.X., Song, C.C., Yu, X.H. (Eds.), Immunology and Immunodiagnosis Parasitic Diseases. Jiangsu Science-Technology Press, Nanjing.

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