Working electrode design

Electrodes were screen-printed onto a PET substrate (175 mm thickness) as illustrated in Fig. 1. Initially, a silver connective layer was applied, followed by a carbon sensor layer. A non-conducting dielectric layer was then placed over the surface to define a circular electrode surface of 3 mm diameter.

Fig. 1: Design of the screen printed electrode. A conductive silver strip is printed onto a PVC substrate. This is followed by addition of the carbon electrode layer. The electrode is covered by a polymer dielectric layer which masks the conductive strip and exposes a defined area of the carbon layer with a diameter of 3 mm.

Electrode operation

The screen-printed electrode (SPE) was connected via an edge connector to a BAS 100/W electrochemical analyser, along with a Ag/AgCl reference electrode and a Pt auxiliary electrode. The three electrodes were placed in a stirred vessel of 4 ml capacity (Fig. 2). The electrodes were operated either using cyclic voltammetry (CV) or time base (TB) modes.

Fig. 2: Operation of the screen printed electrode. Reactions were performed in a stirred reaction vessel with a Ag/AgCl reference electrode and a Pt auxiliary electrode, connected to a BAS 100/W electrochemical analyser and PC.

Polymerisation of aniline on the electrode surface and immobilisation of the anti-biotin antibody was performed according to Minnett et al. [3].

Immunoanalytical interactions

Biotin at various concentrations from 0 to 1 mg/ml was applied to the surface of the working electrode for 30 min at room temperature in a volume of 10 ml. This was followed by the addition of 10 ml biotin-HRP at 0.05 mg/ml for a further 30 min. To perform this, the electrode was disconnected from the electrochemical apparatus and allowed to lie in a horizontal position. The electrode was rinsed gently with PBS/0.5% (v/v) Tween 20. The electrode was reattached to the electrochemical analyser as shown in Fig. 2 and placed in 10 ml 10 mM phosphate buffer, pH 6.8. When the electrode had settled to baseline current levels, 1 ml of 1 M hydrogen peroxide was added and the increase in catalytic current observed.

Aniline has the characteristic of becoming potentiodynamically polymerised to polyaniline on the surface of a carbon electrode. The redox peaks of polyaniline can be seen during the cyclic voltammetry of aniline on the electrode surface (Fig. 3).

Fig. 3: Cyclic voltamogram of the polymerisation of aniline on the carbon electrode surface. The four redox peaks increased as a result of deposition of polyaniline on the electrode.

Polyaniline acts as an electron mediator between the electrode and the bulk electrolyte solution. It can also act as a point of attachment of antibodies and other molecules such as enzymes. In this case, 1 mg/ml anti-biotin antibody was used. If a conjugate of biotin and HRP is subsequently introduced, this will bind to the electrode surface. Introduction of the substrate, hydrogen peroxide will then result in its catalysis. This reaction consumes electrons. At a steady potential, this will result in the increase in the current from the electrode. This is illustrated in Fig. 4.

Fig. 4: Binding of antibody with biotin-HRP results in the catalysis of H₂O₂ and the flow of electrons from the sensor causing an increase in catalytic current. Binding of free biotin displaces the biotin-HRP, reducing the current.

Reduction of the concentration of the biotin-HRP conjugate led to a decline in the steady state current (Fig. 5). Competition between free biotin and biotin-HRP showed a decrease in catalytic current from the sensor as the concentration of free biotin was increased (Fig. 6). This is as would be expected from a competition immunoassay format such as that presented here. The titration curve was sigmoidal as is typical of antibody/antigen interactions, with a linear range lying between 0.03 and 1 mg/ml free biotin.

Fig. 5: Competition assay with free biotin. This demonstrated the reduction in catalytic current from the sensor as the concentration of biotin was increased.

Fig. 6: Log plot of free biotin concentration. Titration yielded a sigmoidal plot, typical of antibody/antigen interactions. A linear range could be established between 0.03 and 1 mg/ml.

A competition immunoassay for the analysis of biotin has been successfully performed on the surface of a screen-printed electrode utilising an immobilisation and mediation system based on the polymerisation of aniline. We are presently extending this system for the analysis of cocaine and we hope to extend the range and sensitivity of the assay format.

1. Tijssen, P. (1985). Practice and theory of enzyme immunoassay. Elsevier, Oxford.
2. Diamond, D. (1998). Principles of chemical and biological sensors. Wiley and Sons, New York.
3. Minett, A., Iwuoha, E., Bogan, D., Killard, A., Smyth, M. and Wallace, G.G. (1998). Development of an immunological sensor based on polyaniline. Eirelec, Howth, Ireland.