SBIR-STTR Award

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

During this NIH SBIR Phase II project, Dakota Technologies, Inc. (DTI) will complete the development of a revolutionary laser-induced fluorescence instrument that provides the flexibility and convenience of research-grade microplate readers, but collects data at speeds typical of high throughput screening. The Phase I project investigated and established proof of concept for novel fluorescence lifetime measurements applied to microplate reading. DTI has the ability to measure very high quality fluorescence decay curves two orders of magnitude faster than the competition. The Phase I research also demonstrated the value of globally analyzing fluorescence polarization data for improved characterization of drug-protein binding and an extremely sensitive means to monitor DNA hybridization without the need for labeling either probes or targets. By the end of Phase II, DTI will be able to acquire a complete fluorescence decay curve (waveform) every time a high repetition rate (10,000 pulses per second) solid-state microlaser fires. The compact passively Q-switched microlaser provides high peak power, short pulse duration, and outstanding shot-to-shot stability. As a consequence, the dominant influence on data quality is photon statistics, not noise of the measurement system. Compared to TC-SPC, where data is acquired for one fluorescence photon per 100 laser shots or more, we record the response for tens to hundreds of fluorescence photons per laser shot. Important Phase II activities include transforming the Phase I proof of concept microplate reader into a robust engineering prototype and developing optimized assays that take advantage of the system's unique features.

Thesaurus Terms:
biomedical equipment development, computer data analysis, computer program /software, computer system design /evaluation, fluorescence polarization, laser, method development, technology /technique development cell surface receptor, chemical kinetics, computer system hardware, cyclic AMP, gene expression, high throughput technology, metalloendopeptidase, photolysis, protein kinase, single nucleotide polymorphism, three dimensional imaging /topography