1. 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
Supported by NIH Grant EB
and

2. 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

3. Prototype Microfluidic Fabrication
Laser machining of SiO2
200 fs 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

4. 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

5. 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

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

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

8. Fluorescence Autocorrelation

9. 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