Optical biosensing in microfluidics using nanoporous microbeads and amorphous silicon thin-film photodiodes: quantitative analysis of molecular recognition and signal transduction

Abstract

Microfluidic devices with integrated optical-to-electrical signal transduction using miniaturized photodetectors can address the need for compact and portable biosensing platforms for medical, veterinary, environmental and food safety applications. However, for different applications the features of the microfluidic point-of-need device will differ in order to be fit-for-purpose, namely the minimum detectable limits, the target sensitivity, and the detection time. Therefore, in order to perform an adequate optimization of the assay parameters for a given detection challenge, the rapid estimation of (1) equilibrium constants, (2) binding kinetics, (3) rate at which photons are generated/absorbed per captured molecule, and (4) molecular capture efficiencies is highly advantageous.This work presents a general method for the quantitative analysis of molecular capture performed on microbeads trapped inside microchannels based on a simple mass balance. Focusing on the interaction of protein A with human IgG as a model system, three different signal transduction methods, based on fluorescence, chemiluminescence and colorimetry, were compared. Acquiring the optical signal with thin-film photodiodes, the minimum detectable IgG per volume of bead solution (resin) was determined as for fluorescence, 4.4 ± 1.4 for chemiluminescence, and 34 ± 3 for colorimetry. A difference in binding efficiency for IgG-Alexa 430 and IgG-HRP was also observed by estimating the respective dissociation constants (KD) as 180 ± 40 nM and 1.6 ± 0.6 µM. Furthermore, the molecular binding (kon, koff) and enzyme kinetic parameters (kcat) were also estimated and discussed, along with considerations regarding capture yields and total assay time. Overall, this methodology proved to be simple and general to study the performance of biosensing platforms, showing potential for improving assay engineering.