1. Tracking and Probing Single, Diffusing Molecules in Droplets Mark Arsenault, Peker Milas, Ben Gamari, Richard Buckman, Lori Goldner Biophysics Group MiniSymposium 19 May 2010

2. Droplet-Based Assays ~1  m diameter aqueous droplet fluorophore Target molecule Oil Phase

3. Outline Motivation Microfluidics Experimental Setup Droplet Tracking Preliminary Results

4. Single-Molecule Assays (surface)

5. Single-Molecule 3-Bead Assay (surface) Quadrant Photodiode ~ 5 pN “pretension” on ~1  m diameter beads

6. Single-Molecule 3-Bead Assay (surface) Quadrant Photodiode

7. Single-Molecule 3-Bead Assay (surface) Quadrant Photodiode ~ 5 pN motor force

8. Droplet-Based Assays Tightly focused IR laser

9. Droplet-Based Assays Tightly focused IR laser Tightly focused visible laser

10. Outline Motivation Microfluidics Experimental Setup Droplet Tracking Preliminary Results

11. Benefit of Miniaturization Faster Cheaper Better 50 years! ENIAC on a 7.44 by 5.29 sq. mm chip Historic Computer Images, ftp.arl.army.mil/ftp/historic-computers ENIAC-on-a-Chip, www.ee.upenn.edu/~jan/eniacproj.html Miniaturization

12. A. W. Chow AIChE, 2002 1 inch

13. IR Force Oil Aqueous A Aqueous B IR Beam Detection Region Microfluidics

14. Flow-focusing device producing both a) 20 mm diameter and b) ~1 mm diameter aqueous droplets. a)b)

15. Outline Motivation Microfluidics Experimental Setup Droplet Tracking Preliminary Results

16. APD Flow cell Objective Condenser Dichroic Mirror Detectors Position Sensitive Detector Lens Pinhole IR (tracking) beam Excitation Beam Fluorescent Emission Laser confocal volume Back-Focal-Plane Tracking Fluorescent Excitation

17. Back-Focal-Plane Tracking APD Flow cell Objective Condenser Dichroic Mirror Detectors Position Sensitive Detector Lens Pinhole IR (tracking) beam Excitation Beam Fluorescent Emission Laser confocal volume Fluorescent Emission

18. Solution FRET Large Volume Ensemble Measurement

19. Solution FRET Large Volume Ensemble Measurement Small Volume Ensemble Measurement

20. Solution FRET Large Volume Ensemble Measurement Small Volume Ensemble Measurement Small Volume, Single- Molecule Measurement

21. Back-Focal-Plane Tracking APD Flow cell Objective Condenser Dichroic Mirror Detectors Position Sensitive Detector Lens Pinhole IR (tracking) beam Excitation Beam Fluorescent Emission Laser confocal volume IR Trapping

22. Back-Focal-Plane Tracking PID control of Mad City Labs piezoelectric nanostage. [We will move the stage (microfluidic device) so as to remain in the trap/confocal spot] APD Flow cell Objective Condenser Dichroic Mirror Detectors Position Sensitive Detector Lens Pinhole IR (tracking) beam Excitation Beam Fluorescent Emission Laser confocal volume IR Tracking

23. Outline Motivation Microfluidics Experimental Setup Droplet Tracking Preliminary Results

24. Back-Focal-Plane Tracking x y 1 um bead APD Flow cell Objective Condenser Dichroic Mirror Detectors Position Sensitive Detector Lens Pinhole IR (tracking) beam Excitation Beam Fluorescent Emission Laser confocal volume

25. Back-Focal-Plane Tracking y x VxVx APD Flow cell Objective Condenser Dichroic Mirror Detectors Position Sensitive Detector Lens Pinhole IR (tracking) beam Excitation Beam Fluorescent Emission Laser confocal volume

26. Back-Focal-Plane Tracking x y z APD Flow cell Objective Condenser Dichroic Mirror Detectors Position Sensitive Detector Lens Pinhole IR (tracking) beam Excitation Beam Fluorescent Emission Laser confocal volume

27. Outline Motivation Microfluidics Experimental Setup Droplet Tracking Preliminary Results

28. Surface-based FRET

29. Solution FRET

30. 16-mer RNA duplex with Cy3 and Cy5 labeling + Trolox + Puglisi oxygen scavenging enzyme 5` 3` Cy3 G - C C - G U - A C - G A - U C - G U - A G - C U - A C - G A - U C - G U - A C - G G - C Cy5 3` 5`

31. Conclusions Solution FRET We are obtaining nice burst data from single, diffusing fluorescent RNA molecules Let’s do droplets! Droplet Tracking We need to calibrate much more quickly (i.e. 100 ms). Calibrating in oil will be that much easier, however, so once we make that jump, the oil should give us an added jump in performance.

32. Device Fabrication Silicon Spin negative photoresist: SU-8 Transparency mask Expose to UV light 100 um

33. Device Fabrication UV light exposed Develop – finished master! Pour PDMS

34. Device Fabrication Remove device 150 um thick Attach to glass + PDMS 100 um Punch plumbing

35. Tipstreaming Mechanism Microscale tipstreaming in a microfluidic flow focusing device, PHYSICS OF FLUIDS 18, 121512 2006

36. Capillary Number Relates viscous forces to capillary pressure Ca=  C Ga/  where  = oil viscosity G = elongation rate a = drop radius  = surface tension Ca =  C Q C a/  h  Z (1/W or – 1/2W up ) h = channel depth V D ~ 1 cm/s V C ~ 5 cm/s Microscale tipstreaming in a microfluidic flow focusing device, PHYSICS OF FLUIDS 18, 121512 2006

37. Dimensionless Parameters Navier Stokes eqn. Reynold’s Number Weber Number Inertia is not important!

38. Droplet Regimes Geometry-controlled Thread-formation drippingjetting Microscale tipstreaming in a microfluidic flow focusing device, PHYSICS OF FLUIDS 18, 121512 2006