The present work compares the responses of a model bioaffinity sensor based on a dielectric functionalization layer, in terms of specific and nonspecific binding, when interrogated simultaneously by Surface Plasmon Resonance (SPR), non-Faradaic Electrochemical Impedance Spectroscopy (EIS), and Plasmonic based-EIS (P-EIS). While biorecognition events triggered a sensitive SPR signal, the related EIS response was rather negligible. Contrarily, even a limited nonspecific adsorption onto the surface of the metallic electrode, allowed by the intrinsic imperfect compactness of the functionalization layers, was signaled by EIS and not by SPR. The source of this finding has been addressed from both theoretical and experimental perspectives, demonstrating that EIS signals are mainly sensitive to adsorptions that alter the current pathway through defects of the functionalization layer exposing the electrode. These observations are of importance for those developing biosensors analyzed by SPR, EIS, or the novel combination of the two methods (P-EIS). A possible application of the observed complementarity of the two methods, namely assessment of sample purity in respect to a target analyte is highlighted. Moreover, the possibility of false-positive EIS responses (determined by nonspecific binding) when assessing samples containing complex matrices or consisting of small molecular weight analytes is emphasized.

Electro-optical measurements, i.e., optical waveguides and plasmonic based electrochemical impedance spectroscopy (P-EIS), are based on the sensitive dependence of refractive index of electro-optical sensors on surface charge density, modulated by an AC electrical field applied to the sensor surface. Recently, P-EIS has emerged as a new analytical tool that can resolve local impedance with high, optical spatial resolution, without using microelectrodes. This study describes a high speed image acquisition and processing system for electro-optical measurements, based on a high speed complementary metal-oxide semiconductor (CMOS) sensor and a field-programmable gate array (FPGA) board. The FPGA is used to configure CMOS parameters, as well as to receive and locally process the acquired images by performing Fourier analysis for each pixel, deriving the real and imaginary parts of the Fourier coefficients for the AC field frequencies. An AC field generator, for single or multi-sine signals, is synchronized with the high speed acquisition system for phase measurements. The system was successfully used for real-time angle-resolved electro-plasmonic measurements from 30 Hz up to 10 kHz, providing results consistent to ones obtained by a conventional electrical impedance approach. The system was able to detect amplitude variations with a relative variation of ±1%, even for rather low sampling rates per period (i.e., 8 samples per period). The PC (personal computer) acquisition and control software allows synchronized acquisition for multiple FPGA boards, making it also suitable for simultaneous angle-resolved P-EIS imaging.