On-the-flow pathogen characterization based on native fluorescence detection

Abstract

Pathogen detection and characterization on-the-flow based on native fluorescence spectroscopy is a promising approach that does not require specific binding or tagging of the analyte. However, the variety of cells is large compared to the number of basic molecular building blocks. Therefore, the fluorescence spectra of different species are often very similar, and sophisticated detection methods are required to reveal differences. The specificity of this approach can be further improved by implementing high spectral resolution and using multiple excitation wavelengths. We have developed a compact platform that combines a microfluidic quartz channel with chip-size wavelength-selective detection which records the fluorescence of particles as they traverse the channel. The interaction between the excitation light and the analyte is enhanced by anti-resonantly guiding the excitation light within the analyte-containing fluid. We have recorded the intrinsic fluorescence of single cells (e.g., yeast, e-coli and BT) as they transit the detection area even at high speed. Simultaneously monitoring total intensity and spectrally-resolved emission yields accurate spectra for particle discrimination. Knowing the particle speed and the physical dimensions of the observation window, we are able to determine particle positions with microscopic (~10 microns) resolution. A novel modulation technique allows us to achieve a high signal-to-noise ratio even at high particle speeds.