Femtomole Detection of Proteins Using a Label-Free Nanostructured Porous Silicon Interferometer for Perspective Ultrasensitive Biosensing.

Abstract

Nanostructured porous silicon (PS) is a promising material for label-free optical detection of biomolecules, though it currently suffers of limited clinical diagnostic applications due to insufficient sensitivity. In this regard, here we introduce an ultrasensitive and robust signal processing strategy for PS biosensors that relies on the calculation of the average value over wavelength of spectral interferograms, namely IAW, obtained on PS interferometer by subtraction (wavelength by wavelength) of reflection spectra acquired after adsorption of biomolecules inside the nanopores from a reference reflection spectrum recorded in acetate buffer. As a case study, we choose to monitor bovine serum albumin (BSA) unspecific adsorption, which has been often employed in the literature as a model for proof-of-concept studies of perspective biosensing applications. The proposed IAW signal processing strategy enables reliable detection of BSA at concentrations in the range from 150 pM to 15 μM (down to 3 orders of magnitude lower than those targeted in the current literature) using a PS interferometer operating in label-free mode without any amplification strategies, with good sample-to-sample reproducibility over the whole range of tested concentrations (%CV = 16% over 5 replicates) and good signal-to-noise ratio also at the lowest tested concentration (S/N ≈ 4.6 at 150 pM). A detection limit (DL) of 20 pM (20 femtomoles, 1 mL) is estimated from the sigmoidal function best fitting (R(2) = 0.989) IAW experimental data over the whole range of tested concentrations. This is the lowest DL that has been reported in the literature since the seminal paper of Sailor and co-workers (1997) on the use of PS interferometer for biosensing, and lowers of 4 orders of magnitude DL attained with label-free PS interferometers using conventional effective optical thickness (EOT) calculation through reflective interferometric Fourier transform spectroscopy. Accordingly, the IAW signal processing strategy envisage bringing PS optical transduction at the forefront of ultrasensitive label-free biosensing techniques, especially for point-of-care clinical analysis where low analyte concentrations have to be detected in a small amount of biological samples.