Supported by NIH Grant EB-006639 Highly parallelized detection of single fluorescent molecules: simulation and experiment Brian K. Canfield, Jason K. King, William N. Robinson, William H. Hofmeister, and Lloyd M. Davis Center for Laser Applications, University of Tennessee Space Institute Tullahoma, TN 37388 Steven A. Soper Louisiana State University, Dept. of Chemistry, Baton Rouge, LA 70803-1804 Supported by NIH Grant EB-006639 and

High-throughput Microfluidics 192 microfluidic processing channels reagent dispenser 96-well titer plates sample transfer chip polymer substrates inexpensive low autofluorescence imprint lithography femtoliter probe volumes reduced sample needs single-molecule detection Estimated performance: ~ 108 assays per day Evotec Commercial System: ~ 140,000 assays per day

Prototype Microfluidic Fabrication Laser machining of SiO2 200 fs pulses @ 800 nm, 1.2 W average power user-specified pulse rate 3D submicron positioning resolution custom patterning operated with LabVIEW Chips etched in 80 °C KOH, bonded to PDMS- coated coverslips 100 mm

Wide-field Fluorescence Microscope Beam focus ~ 5 × 500 mm Field of view: > 500 mm CCD: 512 × 512 pixels Pixel size: 16 × 16 mm Magnification: 16x → 1 : 1 mapping

Data Collection Device situated with slit open laser focused in channels Slit closed to laser focal region ~ 5 pixel rows Vertical binning by camera 5 rows → 1 “superpixel” Frames spooled to disk maximum rate = 7.5 kHz Data file analyzed in Matlab fast, array-level operations 100 mm

Single-molecule Detection: Simulation Time → Signal to noise: ~ 103 Pixel →

Single-molecule Detection: Experiment Time → Signal to noise: ~ 3 Pixel →

Fluorescence Autocorrelation

Summary Demonstrated high-throughput microfluidic chips with single-molecule detection sensitivity and rapid readout ~ 150 channels in field of view, ~ 1 mm2 cross-sectional area 7.5 kHz maximum readout rate fluorescence autocorrelation Next Steps Optimize microchannels achieve 192 channels, increase channel uniformity and quality Improve signal-to-noise ratio increase detection efficiency, standardize flow rate Enhance microscope for two-color detection enzyme inhibitor measurements