(459d) Theoretical and Experimental Studies on an Electrochemical Enzyme Immunosorbent Biosensor

Electrochemical immunosensors integrate biological recognition molecules (e.g., antibodies) with redox enzymes (e.g., horseradish peroxidase (HRP) to combine the advantages of immunoassays (extremely high sensitivity and selectivity) with those of electrochemical biosensors (reproducible, quantitative electrical output). Mechanistic mathematical models that describe the multiple mass-transfer and chemical reaction steps that give rise to the electrical output are needed to help design, optimize, and validate electrochemical immunosensors for medical applications. In this study, we developed such a model of an electrochemical immunosensor that used HRP as a reporter enzyme on the secondary and validated it experimentally.

The model consists of a system of non-linear mass-balance equations that describe simultaneous mass transfer and chemical reactions of the reactants (catechol and hydrogen peroxide) that HRP oxidizes, as well as the HRP product, O-quinone, which is reduced at the working electrode to give the amperometric output. A bi-substrate, ping-pong reaction mechanism was used to describe the enzymatic reaction, and the Butler-Volmer kinetic model was used to describe the reduction of o-quinone back to catechol at the working electrode’s surface. The system of equations was solved numerically using the BVP4C function in MATLAB.

Experimental data for model validation was obtained using electrochemical immunosensors for a surrogate protein antigen (mouse IgG). The immunogens interface was fabricated on the gold working electrode of DropSens screen printed electrode arrays. The steady-state amperometric output of the resulting immunosensors was measured over a range of four independent variables: the concentrations of the two reactants, the voltage of the working electrode, and pH. The experimental results were used to determine optimal operating conditions and to validate the model. The validated model was then used to calculate Damkohler numbers and flux-control coefficients to identify mass-transfer and reaction steps that most strongly affected the magnitude of the immunosensors’ output and the output’s sensitivity to analyte concentration.