Spatially Addressable Multiplex Biodetection by Calibrated Micro/Nanostructured Surfaces.

A challenge of any biosensing technology is the detection of very low concentrations of analytes. The fluorescence interference contrast (FLIC) technique improves the fluorescence-based sensitivity by selectively amplifying, or suppressing, the emission of a fluorophore-labeled biomolecule immobilized on a transparent layer placed on top of a mirror basal surface. The standing wave of the reflected emission light means that the height of the transparent layer operates as a surface-embedded optical filter for the fluorescence signal. FLIC extreme sensitivity to wavelength is also its main problem: small, e.g., 10 nm range, variations of the vertical position of the fluorophore can translate in unwanted suppression of the detection signal. Herein, we introduce the concept of quasi-circular lenticular microstructured domes operating as continuous-mode optical filters, generating fluorescent concentric rings, with diameters determined by the wavelengths of the fluorescence light, in turn modulated by FLIC. The critical component of the lenticular structures was the shallow sloping side wall, which allowed the simultaneous separation of fluorescent patterns for virtually any fluorophore wavelength. Purposefully designed microstructures with either stepwise or continuous-slope dome geometries were fabricated to modulate the intensity and the lateral position of a fluorescence signal. The simulation of FLIC effects induced by the lenticular microstructures was confirmed by the measurement of the fluorescence profile for three fluorescent dyes, as well as high-resolution fluorescence scanning using stimulated emission depletion (STED) microscopy. The high sensitivity of the spatially addressable FLIC technology was further validated on a diagnostically important target, i.e., the receptor-binding domain (RBD) of the SARS-Cov2 via the detection of RBD:anti-S1-antibody.

Fluorescence biosensing micropatterned surfaces based on immobilized human acetylcholinesterase.

Human acetylcholinesterase (AChE) is a widely studied target enzyme in drug discovery for Alzheimer's disease (AD). In this paper we report evaluation of the optimum structure and chemistry of the supporting material for a new AChE-based fluorescence sensing surface. To achieve this objective, multilayered silicon wafers with spatially controlled geometry and chemical diversity were fabricated. Specifically, silicon wafers with silicon oxide patterns (SiO(2)/Si wafers), platinum-coated silicon wafers with SiO(2) patterns (SiO(2)/Pt/Ti/Si wafers), and Pt-coated wafers coated with different thicknesses of TiO(2) and SiO(2) (SiO(2)/TiO(2)/Pt/Ti/Si wafers) were labelled with the fluorescent conjugation agent HiLyte Fluor 555. Selection of a suitable material and the optimum pattern thickness required to maximize the fluorescence signal and maintain chemical stability was performed by confocal laser-scanning microscopy (CLSM). Results showed that the highest signal-to-background ratio was always obtained on wafers with 100 nm thick SiO(2) features. Hence, these wafers were selected for covalent binding of human AChE. Batch-wise kinetic studies revealed that enzyme activity was retained after immobilization. Combined use of atomic-force microscopy and CLSM revealed that AChE was homogeneously and selectively distributed on the SiO(2) microstructures at a suitable distance from the reflective surface. In the optimum design, efficient fluorescence emission was obtained from the AChE-based biosensing surface after labelling with propidium, a selective fluorescent probe of the peripheral binding site of AChE.