Deciphering mass transfer limitations of porous silicon based aptasensors for protein detection

Porous silicon (PSi) is an attractive nanomaterial for the design of optical biosensors, due to the combination of its large internal surface, versatile surface chemistry, and tunable optical properties. Specifically, interferometric-based PSi biosensors, in which detection of the target is carried out by monitoring changes in the PSi reflectivity spectrum, enable a direct and label-free target detection, with a simple experimental setup. Such biosensors have been utilized for detection of various targets, such as small molecules, DNA, proteins and whole cells. Despite the significant advantages that PSi-based biosensors offer, their application for clinical diagnostics has not been realized due to their insufficient sensitivity, usually in the micromolar range, in contrast to theoretical calculations. In fact, we show that for different PSi-based systems, in which aptamers are used as capture probes to target various proteins, a similar biosensing performance is observed (in terms of sensitivity, dynamic detection range and apparent dissociation constant) regardless of the aptamer-protein dissociation constant values.

To investigate these phenomena and the limiting factors of PSi biosensors, we develop a mathematical model which describes the target mass transport to and within the porous nanostructure, as well as the simultaneous reaction between the target and immobilized capture probes. This is supplemented by numerical simulations and experimental analysis of the real time diffusional transport of a fluorescently-labeled model protein in a PSi-based aptasensor, by confocal scanning laser microscopy. Furthermore, by experimental simulation of a well-mixed biosensing system, in which the diffusional transport to and within the PSi is significantly diminished, we show that diffusion is the main limiting factor of these biosensors.