Simultaneous measurement of nonlinearity and electrochemical impedance for protein sensing using two-tone excitation

Impedance biosensors detect the binding of a target to an immobilized probe by quantifying changes in the impedance of the electrode-electrolyte interface. The interface´s I–V relationship is inherently nonlinear, varying with DC bias, and target binding can alter the degree of nonlinearity. We propose and demonstrate a method to simultaneously measure the nonlinearity and conventional small-signal impedance using intermodulation products from a two-tone input. Intermodulation amplitudes accurately reflect the impedance´s manually-measured voltage dependence. We demonstrate that changes in nonlinearity can discriminate protein binding. Our measurements suggest that target binding can alter nonlinearity via the voltage dependence of the ionic double layer.