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

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

Outline Motivation Microfluidics Experimental Setup Droplet Tracking Preliminary Results

Single-Molecule Assays (surface)

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

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

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

Droplet-Based Assays Tightly focused IR laser

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

Outline Motivation Microfluidics Experimental Setup Droplet Tracking Preliminary Results

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, Miniaturization

A. W. Chow AIChE, inch

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

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

Outline Motivation Microfluidics Experimental Setup Droplet Tracking Preliminary Results

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

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

Solution FRET Large Volume Ensemble Measurement

Solution FRET Large Volume Ensemble Measurement Small Volume Ensemble Measurement

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

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

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

Outline Motivation Microfluidics Experimental Setup Droplet Tracking Preliminary Results

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

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

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

Outline Motivation Microfluidics Experimental Setup Droplet Tracking Preliminary Results

Surface-based FRET

Solution FRET

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`

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.

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

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

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

Tipstreaming Mechanism Microscale tipstreaming in a microfluidic flow focusing device, PHYSICS OF FLUIDS 18,

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,

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

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