Spatially Addressable Multiplex Biodetection by Calibrated Micro/Nanostructured Surfaces

Area of Science:

• Biophotonics
• Nanotechnology
• Biosensing

Background:

• Biosensing technologies face challenges in detecting low analyte concentrations.
• Fluorescence interference contrast (FLIC) enhances sensitivity but is highly sensitive to wavelength and vertical position.
• Existing FLIC methods can suffer from signal suppression due to minor variations.

Purpose of the Study:

• To introduce quasi-circular lenticular microstructured domes as continuous-mode optical filters for FLIC.
• To overcome the wavelength sensitivity limitations of traditional FLIC.
• To enable simultaneous separation of fluorescent patterns for diverse fluorophore wavelengths.

Main Methods:

• Fabrication of microstructures with stepwise or continuous-slope dome geometries.
• Utilizing lenticular structures to modulate fluorescence intensity and lateral position.
• Simulation and experimental validation using fluorescent dyes and stimulated emission depletion (STED) microscopy.

Main Results:

• Lenticular microstructures generated concentric fluorescent rings with diameters determined by fluorescence wavelengths.
• Shallow sloping side walls of the domes enabled simultaneous separation of fluorescent patterns across various wavelengths.
• FLIC effects induced by microstructures were confirmed experimentally, demonstrating high sensitivity.

Conclusions:

• The developed microstructured domes offer a novel approach to spatially addressable FLIC.
• This technique significantly improves the specificity and sensitivity of fluorescence-based biosensing.
• Validated for detecting SARS-CoV-2 receptor-binding domain (RBD), showing diagnostic potential.