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Tuesday Aug 31 12:47 PM StyleTag -- Journal: ELECOM (Electrochemistry Communications) Article: 81

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Electrochemistry Communications 1 (1999) 425–428

An electrochemical impedance immunoanalytical method for detectingimmunological interaction of human mammary tumor associated

glycoprotein and its monoclonal antibody

Ma Jie a,*, Chu Yi Ming a, Di Jing a, Liu Shun Cheng a, Li Huai na b, Feng Jun b, Ci Yun Xiang b

a Department of Chemistry, Capital Normal University, Beijing 100037, PR Chinab Department of Chemistry, Peking University, Beijing 100871, PR China

Received 13 April 1999; received in revised form 13 July 1999; accepted 15 July 1999

Abstract

An electrochemical impedance immunosensor has been developed for the specific detection of immunological interaction between humanmammary tumor associated glycoprotein and its monoclonal antibody (GP1D8). Antibody proteins were immobilized by spontaneousadsorption of antibody on gold. Consequently, electrochemical impedance spectroscopy (EIS) measurements of a gold electrode coated withthe antibody showed changes in a.c. current response after the addition of the specific antigen. The successful immunological reaction betweenthe immobilized antibody–antigen at the electrode surface could be monitored. q 1999 Elsevier Science S.A. All rights reserved.

Keywords: Electrochemical impedance spectroscopy; Immunological reactions; Immunosensors; Antibody immobilization

1. Introduction

Electrochemical detection of antibody–antigen interac-tions has been the subject of several research efforts. Moni-toring of capacity changes at the electrodeNelectrolyteinterface as a result of the antigen–antibody complex for-mation was applied to develop a series of capacitive affinitysensors [1–4]. Amperometric detection of antigen–antibodyinteraction was accomplished by the application of redox-modified antigens or antibodies and their competitive asso-ciation with an electrode interface in the presence of theanalyte substance (i.e., antigen or antibody, respectively)[5,6]. Redox enzymes were applied to probe the antigen–antibody association at monolayer interfaces fordevelopmentof amperometric immunosensor electrodes [7]. Electro-chemical impedance spectroscopy (EIS) has provided a non-damaging means of characterizing the electrical properties ofmany biological systems [8]. The electrochemical impedi-metric immunosensor has been rarely reported [9]. The newtechniques based on impedance measurements of the electri-cal equivalent circuit of the oscillator [10] and based on shearacoustic impedance measurements [11] have been reported.These methods can obtain an immunoaffinity signal.

* Corresponding author. Fax: q86-10-6890-2938; e-mail: jiema@public.bta.net.cn

In this paper, an electrochemical impedance immunosen-sor for detecting immunological interaction of human mam-mary tumor associated glycoprotein and its monoclonalantibody is described. The successful immunologicalreactionbetween the immobilized antibody–antigen could bemonitored.

2. Experimental

2.1. Reagents

Monoclonal antibody (McAb GP1D8, IgG1, MW 176.2KD) mouse against human mammary tumor associated gly-coproteins [12] in ascitic fluid was successively purified byprecipitation with caprylic acid and ammonium sulfate, andused within one month. The purity and mass of antibody weredetermined by analytical polyacrylamide gel electrophoresis(PAGE). Purification and storage temperature was 48C. Theantigen (Ag), mammary tumor-associated glycoprotein(MTGP) [12,13] (MW 66 KD), was extracted from frozenmetastatic ductal carcinomas of the breast, then purified usingmolecular exclusion chromatography on a Sephadex G-200column. The molecular weight was determined by a sodiumdodecylsulfate (SDS)–polyacrylamide gel electrophoresis

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Fig. 1. CV of self-assembled McAb monolayer on gold. Solution: 1 mmolly1 K3[Fe(CN)6]/K4[Fe(CN)6] in 0.1 mol ly1 phosphate buffer solution,pHs7.4. (1) Bare gold electrode; (2) gold electrode immersed in McAbsolution for 20 min; (3) gold electrode immersed in McAb solution for40 min.

(PAGE). The specific association between McAb and anti-gen was investigated by Western blot [13].

Water for all experiments was purified on a Milli-Q/orga-nex-Q system (Millipore). Other chemicals were reagentgrade.

Phosphate buffer saline (136.7 mmol ly1 NaCl, 2.7 mmolly1 KCl, 9.7 mmol ly1 Na2HPO4Ø12H2O, 1.5 mmol ly1

KH2PO4), with the pH adjusted to 7.4, was used as the elec-trolyte in the measuring system and for the preparation of1 mmol ly1 of both K3[Fe(CN)6] and K4[Fe(CN)6]. Inorder to clean the gold surfaces, ‘piranha’ solution was pre-pared (30% H2O2/conc. H2SO4, 1:3 v/v).

2.2. Instrumentation

Cyclic voltammetry (CV) was performed using EG&GPAR 273 potentiostats. Electrochemical impedance spectros-copy (EIS) was performed using an EG&G PAR impedancesystem (273/5208).

Impedance data were analyzed using a complex EG&GPARC M388 software system V2.90 computer program.

2.3. Antibody immobilization procedure

The area of the gold electrode was 0.2 cm2; it was rinsedwith 0.1 mol ly1 HCl, 0.1 mol ly1 NaOH, and then in absoluteethanol by ultrasonic rinsing for 10 min. The gold electrodewas immersed in ‘piranha’ solution for 5 min after rinsingwith distilled water. The electrode was then immediatelyimmersed a 0.01 mol ly1 phosphate buffer saline (pH 7.4)solution containing 3.016 mg mly1 monoclonal antibody(GP1D8). The McAb was immobilized onto the gold elec-trode at 48C for 24 h. A monolayer of antibody formed onthe gold electrode. Furthermore, this layer of the antibody onthe gold electrode could be reproducibly obtained by theabove procedure. The immunological sensitivity of the McAbcoating was checked by the immunofluorescence test usingan antibody labeled by fluorescein isothiocyanate (FITC).The antibody was polyclonal rabbit anti mouse immunoglob-ulin G.

2.4. Electrochemical cell and procedures

The impedance measurements were performed in a three-electrode system, which contained a working electrode (anti-body-coated gold electrode), a counter electrode (Ptelectrode), and reference electrode (saturated calomel elec-trode, SCE).

3. Results and discussion

3.1. Monitoring of the antibody adsorption

At first, the absorption of McAb onto the gold electrodewas monitored by CV using 1 mmol ly1 K3[Fe(CN)6]/

K4[Fe(CN)6] as a redox probe for the density of the self-assembled monolayers (Fig. 1). The adsorption of McAbcaused a decrease in the peak current of the K3[Fe(CN)6]/K4[Fe(CN)6]. After the addition (20 min) of McAb, theheight of the voltammetric peaks drastically decreased andthe separation of the remaining poorly defined voltammetricwaves increased. Only the capacitance current was left in thevoltammogram, which was taken after an adsorption time of40 min. The peak current decreased further with increasingadsorption time of the McAb. This indicates the formation ofa high-density layer of immobilized Ab protein. In order toobtain a stable self-assembled monolayer of McAb, anadsorption time of 24 h was used at 48C in the experiment.

3.2. Monitoring of protein activity in McAb self-assembledmonolayer

The immunofluorescence test with an antibody labeled byfluorescein isothiocyanate (FITC) was used to check theimmunological sensitivity of the McAb coating. Theantibodyused was polyclonal rabbit anti mouse immunoglobulin G.The self-assembled McAb/Au was incubated in a 1:2000solution of polyclonal rabbit anti mouse immunoglobulin G-FITC for 40 min. Then the McAb/Au electrode was rinsedwith 0.1 mol ly1 phosphate buffer saline. The fluorescencephotograph of the immune complex is shown in Fig. 2. TheMcAb self-assembled monolayer on gold was also shown tobe immunologically sensitive. This experiment shows thepresence of active antibody on the surface of the goldelectrode.

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Fig. 2. Fluorescence photograph of the immune complex of antibody and anti-antibody.

Fig. 3. EIS: (s) antibody/gold electrode; (d) non-specific antigen/gold electrode (from glycoprotein from human mammary tumor) to prove EIS changedue to the binding of specific antigen; (*) Ag–McAb/gold; (=) Ag–McAb-coated electrode treated with 6 mol ly1 urea. Solution: 0.1 mol ly1 phosphatebuffer saline pHs7.4; electrode area: 0.2 cm2; applied potentialssurface Eo9; amplitude: 10 mV; frequency range: 100 000–0.1 Hz.

3.3. EIS

Impedance spectra were taken at the following steps of theexperiment:1. The McAb-coated electrodes were immersed in 0.1 mol

ly1 phosphate buffer saline.2. The McAb-coated electrodes were incubated for 40 min

in a non-specific antigen (glycoprotein from human mam-mary tumor, 200 mg mly1 0.1 mol ly1 PBS, 200 ml).

3. The McAb-coated electrodes were incubated for 40 minin a solution of the specific antigen (MTGP, 200 mg mly1

0.1 mol ly1 PBS, 200 ml).4. The Ag–McAb-coated electrodes were treated with 6 mol

ly1 urea in order to split the antigen–antibody complex.The resulting set of impedance spectra is shown as a Nyqu-

ist plot (Fig. 3). A semicircle emerged in the Nyquist plot,which represents the charge-transfer resistance (RCT). Thischarge-transfer resistance is caused by charged species (pro-

tein) in a medium that has a constant dielectric [14]. RCT

shows no obvious change during the binding of the non-specific antigen (glycoprotein from human mammary tumor)to the McAb-coated gold surface. The change in the electro-chemical properties of the electrodes from their initial char-acteristics takes place during the binding of the specificantigen. The RCT increase resulted in the formation of stableantigen–antibody complex. Then, the RCT drasticallydecreased owing to the immune complex being split by thepresence of urea and is smaller than that for McAb/Au. Thereis a possible reason why this is so. From a mechanistic stand-point, the influence of a high concentration of urea not onlycauses the desorption of the antigen but also partial denatur-ation of the antibody. The three-dimensional flexibility struc-ture of McAb was damaged because of denaturation. Theapproximate agreement observed between experiment andthe ideal equivalent circuit simulation is encouraging. Thecomputer simulation data are shown in Table 1.

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Table 1Theoretical simulation parameters for impedance measurements of four stepsin the experiment

Step no. Rs/V cm2 RCT/V cm2 Cdl/F cmy2

(1) 1.8418=104 7.5458=104 4.9322=10y6

(2) 1.9023=104 7.7112=104 5.0112=10y6

(3) 1.7979=104 1.3083=105 2.0502=10y6

(4) 2.4743=102 2.4760=104 3.8795=10y5

As discussed above, the immobilized antibody–specificantigen complex conformation can be characterized by usingEIS. Moreover, the RCT changes are proportional to the spe-cific antigen concentration changes. This sensor has beenapplied to monitor immunological reactions. Furthermore,EIS can be used for affinity measurements of antibody–spe-cific antigen.

4. Conclusions

Antibodies can be immobilized by their spontaneousadsorption on gold. A denser antibody coating of theelectrodeis obtained by this direct immobilization process. The rec-ognition reaction between the immobilized antibody and thespecific antigen causes changes in the impedance spectra ofthe gold electrode. This sensor has been applied to monitorimmunological reactions by measuring the impedancespectra.

Using the self-assembled antibody directly onto the goldelectrode to measure the recognition reaction between anti-body and antigen provides some advantages: (1) theantibodyor antigen does not need secondary labeling; (2) the assaytime could be further shortened; (3) in future, the impedanceimmunosensor could be used to explore binding kineticsbetween antibody and specific antigen.

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