Effects of Diffusion Limitations and Partitioning on Signal Amplification and Sensitivity in Bienzyme Electrochemical Biosensors Employing Cyclic Product Conversion

In this study, the nonlinear and non-monotonic behavior of amperometric bienzyme biosensors employing an enzymatic trigger reaction is investigated analytically and computationally using a two-compartment model comprising an enzymatic layer and an outer diffusion layer. The trigger enzymatic reaction is coupled with a cyclic electrochemical–enzymatic conversion (CEC) process. The model is formulated as a system of reaction–diffusion equations incorporating nonlinear Michaelis–Menten kinetics and interlayer partitioning effects. Exact steady-state analytical solutions for substrate and product concentrations, as well as for the output current, are obtained for specific cases of first- and zero-order reaction kinetics. At the transition conditions, biosensor performance is further analyzed numerically using the finite difference method. The CEC biosensor exhibits the highest signal gain when the first enzyme has low activity and the second enzyme has high activity; however, under these conditions, the response time is the longest. When the first enzyme possesses a higher substrate affinity (lower Michaelis constant) than the second, the biosensor demonstrates severalfold higher current and gain compared to the reverse configuration under identical diffusion limitations. Furthermore, increasing external mass transport resistance or interfacial partitioning can enhance the apparent signal gain.