This Phase I NIH SBIR project will lead to a breakthrough in the performance to cost ratio of fluorescence microplate readers. By collecting fluorescence decay curves at several emission wavelengths simultaneously, we will achieve a true multiplexing capability. One will be able to determine the independent responses of more than one fluorescent probe present in the same microwell. Our approach does not rely on non-overlapping fluorescence and/or excitation spectra of the various probe molecules. Nor does it involve tedious repeat scans of the plate, each time at a different pair of excitation/emission wavelengths. In phase I we shall demonstrate that the multiplexing format's measurement speed (less than 60 seconds for a 384 well plate) and sensitivity (detection of less than 10 femtomoles in 100 uL) compares favorably with currently available commercial microplate readers. Our approach involves replacing the quartz halogen lamp or xenon flashlamp excitation source with a pulsed microlaser. Novel digitizer technology affords a huge cost advantage over the alternative of a digital oscilloscope for collecting the fluorescence decay curves. Heretofore, laser-induced fluorescence (LIF) detection has appeared only in expensive high throughput screening and DNA sequencing instruments. Our work will bring high performance LIF detection to the mid-range microplate readers that are workhorse tools for genotyping, gene expression, and many other applications in biomedical research. The new type of microplate reader we envision will play a vital role as diagnostic applications of genotyping expand.
