Biosensor mabs imuno with silver nanoparticles metoda Chu - 2005 !

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Biosensors and Bioelectronics 20 (2005) 1805–1812

An electrochemical stripping metalloimmunoassay basedon silver-enhanced gold nanoparticle label

Xia Chua,b, Xin Fua, Ke Chena, Guo-Li Shenb,∗, Ru-Qin Yub

a Chemistry and Chemical Engineering College, Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research

(Ministry of Education), Hunan Normal University, Changsha 410081, PR Chinab State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China

Received 4 July 2004; accepted 9 July 2004

Available online 25 August 2004

Abstract

A novel,sensitive electrochemical immunoassay hasbeen developed based on the precipitation of silver on colloidal gold labelswhich, 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 27.25g ml−1 in a logarithmic plot. A detection limit as low as

1ngml−1 (i.e., 6× 10−12 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 5 × 10−9 M) and to the catalytic precipitation of a large number of

silver on the colloidal gold-labeled antibody.

© 2004 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 an-

tibodies 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 environmentalsamples. Accordingto the natureof a label, immunoassay can

be classified as label-free immunoassay, radio-immunoassay,

enzyme immunoassay, fluorescent immunoassay, chemilu-

minescent immunoassay, bioluminescent immunoassay and

so on. Since metalloimmunoassay, i.e., immunoassay involv-

ing metal-based labels, was developed, great progresses have

∗ Corresponding author. Tel.: +86 731 8821355; fax: +86 731 8821818.

E-mail address: [email protected] (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 an-alytical methods for example, atomic or colorimetric ab-

sorption 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 metalloim-

munoassays 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;

0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bios.2004.07.012



1806 X. Chu et al. / Biosensors and Bioelectronics 20 (2005) 1805–1812

Rapicault et al., 1996; Bordes et al., 1997) or metal ions

(Doyle et al., 1982; Hayes et al., 1994; Wang et al., 199 8)

remained insufficient compared with fluorescent europium

chelate labels for which picomolar levels could be deter-

mined. Recently, a new electrochemical metalloimmunoas-

say based on a colloidalgold label was reported which pushed

the sensitivity of the metalloimmunoassay to the picomolardomain (Dequaire et al., 2000).

In this work, a new silver-enhanced colloidal gold elec-

trochemical stripping detection strategy is presented, which

should further improve the sensitivity of the metalloim-

munoassay through the catalytic precipitation of silver on

the gold nanoparticles. Silver deposition on gold nanopar-

ticles is commonly used in histochemical microscopy to

visualize the distribution of an antigen over a cell sur-

face (Xu, 1997). Mirkin and co-workers have developed

a scanometric DNA array (Taton et al., 2000), a electri-

cal detection-based DNA array (Park et al., 2002) and the

Raman spectroscopic fingerprints for DNA and RNA de-

tection (Cao et al., 2002) based on silver deposition ongold nanoparticles. Silver enhancement was also used for

detecting the DNA hybridization event by the scanning elec-

trochemical microscopy (Wang et al., 2002) and the elec-

trochemical 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 elec-

trochemical metalloimmunoassay based on the use of gold

nanoparticle labels and silver enhancement has not been re-

ported.

The present protocol uses the electrochemical stripping

technique for detecting the silver deposited on the goldnanoparticles. Stripping analysis is a powerful electroanalyt-

ical 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 metal-

loimmunoassay. 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 metalloimmunoas-

say 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 devel-

oped metalloimmunoassay are reported in the following

sections.

2. Experimental

2.1. Materials

Human IgG, goat anti-human IgG, horseradish per-

oxidase (HRP)-labeled goat anti-human IgG and bovine

serum albumin (BSA) were purchased from ShanghaiHuamei Biochemical Reagents (Shanghai,China). Chloroau-

ric acid (HAuCl4), trisodium citrate, hydroquinone, silver

nitrate (AgNO3) and tetramethylbenzidine (TMB) were ob-

tained from Shanghai Chemical Reagents (Shanghai, China).

All of the solutions were prepared with doubly distilled

water.

2.2. Buffers and solutions

The following buffers were used in this study: (a) car-

bonate buffer (15 mM Na2CO3 and 35 mM NaHCO3, pH

9.6); (b) phosphate-buffered saline (PBS; 137 mM NaCl,

1.7 mM KH2PO4, 8.3mM Na2HPO4 and 3.0mM 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 C6H8O7·H2O and 0.163M Na3C6H5O7·2H2O,

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 (300g ml−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 mgAgNO3, 50 ml citrate buffer and 50 ml doubly distilled water

(Xu, 1997), which was prepared fresh as needed.

2.3. 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 HNO3), rinsed in doubly distilled wa-

ter, and oven-dried prior to use. In a 500-ml round-bottom

flask, 250 ml of 0.01% HAuCl4

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 antibody-

colloidal gold conjugate as soon as possible. The resulting

solution of colloidal particles was characterized by an absorp-

tion maximum at 520 nm. Transmission electron microscopy

(TEM) indicated a particle size of 20± 4.7 nm (100 particles

sampled).



X. Chu et al. / Biosensors and Bioelectronics 20 (2005) 1805–1812 1807

2.4. Preparation of the antibody-colloidal gold

conjugate

2.4.1. 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 1mgml−1 stock solution aliquots (0–50g) of goat

anti-human IgG and were added in 5-g increments to cu-

vettes 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 1.150 ml with doubly distilled wa-

ter and 100l 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 de-

crease in absorbances starts to be insignificant. For 20-nm

gold colloid, the optimum amount of goat anti-human IgGfor coating the gold nanoparticles is 30g per 1 ml colloidal

gold solution and is effective to prevent aggregation.

2.4.2. 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, dur-

ing which the goat anti-human IgG antibodies adsorbed onto

the gold nanoparticles through a combination of ionic and

hydrophobic interactions. The conjugate was then dividedinto 1-ml fractions in 1.5-ml microcentrifuge tubes and cen-

trifuged at 17 390 × g for 10 min. Two phases can be ob-

tained: a clear to pink supernatant of unbound antibody and

a dark red, loosely packed sediment of the antibody-labeled

immunogold. Thesupernatantwas discardedand thesoft sed-

iment of immunogold was rinsed by resuspending in 1ml

of TBS-BSA and collected after a second centrifugation at

17390×g for 10 min.Finally, the conjugate wasresuspended

in 250l 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.

2.5. Immunoassay procedure

Primary goat anti-human IgG antibody (200l,

300g ml−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 (200l) 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 l of

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, 200l 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, 300l of 1.5 M

HNO3 was added to the microwells and incubated at roomtemperature for 30 min to dissolve the metal silver deposited

on the walls of the microwells.

2.6. Electrochemical measurement

The glassy-carbon electrode (Jiangsu Electroanalytical

Instruments, Jiangsu, China) and platinum wire electrode

(Shanghai Exact Scientific InstrumentLtd.,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 elec-

trolyte solution by a double electrolytic salt bridge filled withsaturated KNO3 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 (300l)

were transferred from the microwells into a 10-ml beaker

containing 3 ml of 0.6 M KNO3 and 0.1 M HNO3 as elec-

trolyte solution, and the released silver (I) ions were then

quantified by ASV under the following instrumental con-

ditions: 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 preconditioningat +1.0 V versus SCE for 1 min between each measurement.

2.7. Enzyme linked immuno-sorbent assay (ELISA)

protocol

Primary goat anti-human IgG antibody (100l,

300g ml−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 100l per well of the

diluted human serum samples at 37 ◦C for 1 h. After another

washing step, 100l of the HRP-labeled goat anti-human

IgG antibody was added to the wells and incubated at 37◦

Cfor 1 h. After a final washing step, 100l per well of TMB

solution (400l of 0.6% TMB-DMSO and 100 l of 1%

H2O2 diluted with 25 ml of citrate-acetate buffer, pH 5.5)

was added. The reaction was stopped after an appropriate

time by adding 50l of 2M H2SO4, and absorbance was

read at 450 nm.

3. Results and discussion

The principle of the heterogeneous electrochemical im-

munoassay based on silver-enhanced colloidal gold is




Fig. 1. Schematic representation of the analytical procedure of the het-

erogeneous electrochemical immunoassay based on silver-enhanced gold

nanoparticle label.

depicted in Fig. 1, and it was applied to human IgG ana-

lyte. Primary antibodies specific for human IgG are adsorbed

passively on the walls of a polystyrene microwell. The hu-

man 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 thewalls of the polystyrene microwell through the catalytic re-

duction of the silver ions on the antibody-colloidal gold con-

jugate. The silver metal thus deposited is then dissolved in an

acidic solution and the silver ions (AgI) 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.

3.1. 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 AgI by ASV

was firstly investigated. The study was carried out in a 0.6 M

potassium nitrate solution containing 0.1 M HNO3 (0.6 M

KNO3 /0.1 MHNO3), since HNO3 is required for the efficient

dissolution of silver in the final step of the electrochemical

Fig. 2. CV curve (v = 100mV s−1) recorded at a glassy-carbon electrode

immersed in 3 ml of 0.6M KNO3 /0.1M HNO3 containing 10M AgI 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

1 × 10−3 AgI in 0.6M KNO3 /0.1M HNO3 without preliminary electrode-

position.

immunoassay. The CV curve (Fig. 2) recorded at a glassy-

carbon electrode after cathodic polarization at −0.5 V versus

SCE for 10 min in a magnetically stirred solution containing

10M AgI shows a well-defined anodic peak at 0.4 V (peak

potential E p,a) which is characteristic of the oxidation of elec-

trodeposited 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 AgI ions without

electrodeposition (Fig. 2, inset), it seems this small cathodic

peak corresponds to the reduction of AgI ions anodically

released and still present in the diffusion layer. The shift of reduction peak of AgI 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 (ip,a) and the integration

of the stripping peak current are directly proportional to the

concentration of AgI ions over a certain range.

Several parameters were investigated in order to establish

optimal conditions for the detection of AgI. The influence

of the electrodeposition potential ( E d) upon the stripping re-

sponse was tested (Fig. 3). As E d decreased from −0.1 to

−0.9 V, the anodic peak current (ip,

a) resulting from the ox-

idation 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 se-

lected for the further studies. Theeffect of the deposition time

upon the stripping response was also examined in Fig. 4. The

anodic peak current (ip,a) increased in a nearly linear fashion

up to 10 minand 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 lin-

ear dependence of the anodic stripping peak current (ip,a)

with the silver (I) concentration over the 5 × 10−9 M to 5




Fig. 3. Effect of the deposition potential upon the silver stripping peak cur-

rent. Deposition time, 10 min; electrolyte, 0.6M KNO3 /0.1M HNO3; con-

centration of AgI, 1 × 10−5 M.

× 10−5 M range was obtained, and the linear correlation co-

efficient was 0.9947 (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 precondition-

ing the electrode at+1.0V versusSCEfor 1 min between each

measurement. The standard deviation (S.D.) of five measure-

ments (n = 5) of the background noise was 2.5 A and the

detection limit calculated from three times of the standard

deviation was 5× 10−9 M. These results further showed that

ASV is a very sensitive and effective method for trace de-

termination 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 la-bel.

3.2. Optimization of immunoassay conditions

Theelectrochemical immunoassay of human IgG was per-

formed as depicted in Fig. 1 using colloidal gold-labeled goat

Fig. 4. Effect of the deposition time upon the silver stripping peak cur-

rent. Deposition potential, −0.5 V vs. SCE; electrolyte, 0.6M KNO3 /0.1 M

HNO3; concentration of AgI , 1 × 10−5 M.

Fig. 5. Calibration plots of AgI recorded by ASV at a glassy-carbon elec-

trode. Electrodeposition at −0.5 V vs. SCE for 10 min under magnetic stir-

ring in 3 ml of 0.6 M KNO3 /0.1M HNO3. Error bars represent S.D., n = 4.

anti-human IgG antibody in connection with the silver en-hancement, and the detailed optimization of each of these

steps was reported below.

The effect of the antigen–antibody reaction time upon the

anodic strippingpeakcurrent 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 beperformed 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. 6. Effect of the antigen–antibody immunoreaction time upon the an-

odic stripping peak current. Concentration of human IgG, 27.25 g ml−1;

dilution ratio of the antibody-colloidal gold conjugate, 1:4; silver staining

time, 30 min. Other conditions, as in Fig. 5.




Fig. 7. Effect of the dilution ratio of antibody-colloidal gold conjugate

upon the anodic stripping peak current. Concentration of human IgG,

27.25g ml−1; antigen–antibodyimmunoreaction time,60 min;silverstain-

ing time, 30 min. Other conditions, as in Fig. 5.

BSA was added to the TBS buffer solution used to resus-

pend the sediment of the antibody-colloidal gold conjugate,

since it was beneficial to retain the stability of the colloidalgold-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 voltam-

metry was also investigated in Fig. 7. The anodic stripping

peak current increased in a nearly linear fashion by decreas-

ing 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 so-

lutions. 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 af-ter 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 immunoas-

say format illustrated in Fig. 1.

Further amplification of the sensitivity of the electrochem-

ical immunoassay based on colloidal gold-labeled antibody

canbe achieved by catalytic precipitation of silver on the gold

nanoparticle tags, which can produce relatively large parti-

cles. 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 offer-

ing very favorable signal enhancement, leads to an increase

in the background response. In contrast to the analytical sig-

nal 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.

3.3. 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

(Qp) was chosen because it has a little more sensitive than

the ip,a response. The signal increased rapidly with the hu-

man IgG concentration at first (up to 1.7g ml−1), then

more slowly, and started to level off above 27.25 g ml−1

(Fig. 8A). Such curvature can be addressed by using a log-

arithmic scale, which resulted in a highly linear response

up to 27.25g ml−1 and the linear correlation coefficient

was 0.9989 (Fig. 8B). The dynamic range for the assay ex-

tended between 1.66 ng ml−1 and 27.25g ml−1. The sig-

nal saturated above 27.25g ml−1 human IgG, owing to




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 1.0ng ml−1, i.e., 6 × 10−12 M hu-

man IgG (according to 3S.D., where S.D. is the standarddeviation of five measurements of a blank solution, S.D. =

1.4C, n = 5). The sensitivity of the method is compet-

itive with another gold nanoparticle-based electrochemical

immunoassay recently reported for goat IgG (detection limit

of 0.5 ngml−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

27.25g ml−1 human IgG solution was used to estimate the

precision. This series yielded a mean integration of the strip-

ping peak current of 163C and a relative standard deviation

of 7.2%. Such signal variations reflect the good reproducibil-

ity of the protocol of the immunoassay and electrochemicaldetection.

Theoretically, the silver-enhanced colloidal gold electro-

chemical immunoassay developed in the present work will

have a lower detection limit than the electrochemical im-

munoassay 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 nanoparticletags in a beaker containing 3 ml electrolyte solution for ASV

analysis, while the reported colloidal gold-based immunoas-

say 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 deter-

mination. Then, it might be reckoned that the silver enhance-

ment 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 electro-

chemical microcell andavoiding thedilution of the silver ions

solution.

3.4. Analytical application

To demonstrate the applicability of proposed electro-

chemical immunoassay to clinical diagnostics, four human

serum samples provided by Xiangya Medical College, Cen-

tral 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 elec-

trochemical immunoassay to detecthumanIgG in serum sam-

ples.

Table 1

IgG concentrationin human serumsamplestested by proposedelectrochem-

ical immunoassay format and ELISA methoda

Serum sample IgG concentration (ngml−1)

Electrochemical immunoassay ELISA

1 48.5 ± 3.2 46.7

2 357.8 ± 25.7 369.73 874.6 ± 65.8 912.6

4 1278.4 ± 87.4 1287.5

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 bet-ter 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 sen-

sitivity 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 col-

loidal gold label is more stable than the radioistopic or en-zyme 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 simultane-

ous detection of several analytes by using different colloidal

metal labels with distinct anodic stripping potentials.

Acknowledgement

Financialsupport from the National Natural Science Foun-

dation of China (Grant No. 20105007) is gratefully acknowl-

edged.

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Biosensor mabs imuno with silver nanoparticles metoda Chu - 2005 !

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