Microfluidics

Dave EddingtonAssistant Professor

Department of Bioengineering

July 10, 2008

Outline• Background of BioMEMS

– Microfluidics– Micropatterning– microstructures

• Background of soft lithography• Example projects

– Sickle cell device– Hypoxia– Brain slice device– Cnidocysts

Microtechnology• Photolithography

– Microelectronics• Deposit metals• Diffuse dopants

• Micromachining– Microelectromechanical Systems

• Deposit structural material• Bulk Etch• DLP, Accellerometer

• Soft Lithography– BioMEMS

• Mold PDMS– Microfluidics– Micropatterning– Micromolding

BioMEMS

MEMS

microelectronics

BioMEMS: Micropatterning• Use PDMS network to define surface chemistries

– Advesive– Non-Adhesive

• Control over cells/surfaces• Control cell-matrix interactions• Control cell-cell interactions• Control cell shape

McBaeth et al, Developmental Cell, 05

BioMEMS: Microstructures

• Use PDMS to measure cellular forces• Use PDMS stamp to create wells in gels

Nelson et al, Science, 2006Tan et al, PNAS, 2003

BioMEMS: Microfluidics

• Integrate multiple tasks onto single device• Short diffusion lengths• Laminar flow• Large surface to volume ratio• Similar length-scale as cells• Very small volumes

Lee et al, Science, 05

Why we like microfluidics?

• Leveraging the Microscale– Rapid diffusion– Large surface to volume ratio– Process integration– Microscale systems for microscale needs

Lithography• From Greek, meaning “writing in stone”• A method of patterning a layer of photosensitive material

based on radiation-induced structural degradation• Photosensitive material

– Material that experiences a change in its physical properties when exposed to a radiation source

– Photoresist– Depending on chemical nature, produce either a

positive or a negative image

Lithography

Lithography

SiPR

SiPR

SiSi

“+” “—”

David T. Eddington1*, John M. Higgins2,3, Lakshminarayanan Mahadevan2,4

and Sangeeta N. Bhatia1,5

1Division of Health Sciences and Technology, Harvard-MIT, 2School of Engineering and Applied Sciences, Harvard University,

3Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, 4Department of Systems Biology, Harvard Medical School,

5Department of Electrical Engineering and Computer Science, MIT,*Current Address: Department of Bioengineering, University of Illinois at Chicago

Microfluidic Model of a Sickle Cell CrisisMicrofluidic Model of a Sickle Cell Crisis

What is sickle cell disease?

• The molecular defect was identified over 60 years ago• A single nucleotide change leads to an amino acid

substitution• Sickle hemoglobin molecules polymerize when

deoxygenated• The polymers form long fibers and distort the red blood cell

membrane and “sickle” the cell• Life expectancy = 45 years with one hospitalization/year

http://fig.cox.miami.edu/~cmallery/150/chemistry/hemoglobin.jpghttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SickleMutation.gif

Vaso-occlusion is a MultiscaleProcess

• Multiscale processes (length & time)– 0.1s,10 nm: polymerization of hemoglobin S– 0.1s, 10 μm: cell sickling– 1000s, 100-μm: vessel jamming

In Vitro Model

• Map out phase space – f(O2, Q, x)– Each occlusion = point

1

2

3

4

5

Experimental Setup

Oxygen Drop

0 20 40 60 80 100 120−2

0

2

4

6

8

10

12

Time (s)

[O2] (

%)

Movies

• Video on method• 7 μm channels• In a 250 μm channel

Occlusion and Relaxation

0 100 200 300 400 500 6000

0.2

0.4

0.6

0.8

1

1.2

Time (s)

Vel

ocity

(no

rmal

ized

to in

itial

)

Occlusionτ = 124 sRelaxationτ = 22 s

0

10

[O2] (

%)

Time (s)

Controls

0 100 200 300 400 500 6000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Time (s)

Vel

ocity

(no

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ized

to in

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)

Normal (0% HbS)Heterozygous (33% HbS)

Phase Space of Occlusion

Medical Intervention Validation

0 200 400 600 800 10000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

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Time (s)

Vel

ocity

(no

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to in

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)

Untreated (HbS = 78%)τ = −90 sTreated (HbS = 31%)τ = −404 s

0

10

[O2] (

%)

Time (s)

Preventing Occlusion

0 50 100 150 200 250 300 350 4000

0.2

0.4

0.6

0.8

1

1.2

1.4

Time (s)

Vel

ocity

(no

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0% O

2; 0% CO

τ = −35 s0% O

2; 0% CO

τ = −31 s0% O

2; 0% CO

τ = −35 s0% O

2; 0.01% CO

τ = −358 s0% O

2; 0.01% CO

τ = −95 s

0

0.01

[CO

] (%

)

0 0

10

[O2] (

%)

Time (s)

Microfluidic Sickle Cell Model

• It is possible to evoke, control and inhibit vaso-occlusion in a minimal microfluidic model

• Oxygen-dependant sickle hemoglobin polymerization and melting are enough to recreate vaso-occlusion

• Clinical interventions can be validated• Test bed for new therapeutics

– Generalizable to other hematological diseases

Characterizing MultiCharacterizing Multi--Modal Tissue Function in a Modal Tissue Function in a Microfluidic DeviceMicrofluidic Device

Javeed Shaikh Mohammed, Wang Yong, Tricia Harvat, Jose Oberholzer,

David Eddington

Islet Quantification: Pre Implantation

• Islet characterization: viability, purity, and sterility

• Transplant η ↔ Islet function • Our goal: high-throughput

platform for use in islet isolation arena

http://transplant.hospital.uic.edu/transplant/islets.html

Function = ?

• Specific aims: – Simple microfluidic device for perfusion and

imaging– Evaluate islet functionality

Cross-sectional view of device

Mouse Islets: Dynamic Perfusion

C57B6 mice islets (25 per perfusion chamber)

Balb/c mice (25 per perfusion chamber), Fura-2: 5 μM

Multimodal Function AnalysisMouse Islets: Ca2+ imaging & insulin

ELISA

Human islets (100 per perfusion chamber) [n=6]

Human Islets: Dynamic Perfusion

Human islets (100 per perfusion chamber), Fura-2: 5 μM

Multimodal Function AnalysisMouse Islets: Ca2+ imaging &

insulin ELISA

Human Islets Transplanted to Mice (Gold Standard to Quantify Islet

Function)

Non potent islets

Potent islets

1000 human islets transplanted into nude mice

Evaluating Human Islets: Highly Variable

Batch # KRB2 14 mM KRB2 KCl KRB2 KRB2 14 mM KRB2 KCl KRB2H249 0.13 1.80 0.42 1.56 0.14 2.26 82.40 5.28 140.60 29.03H251 0.21 0.65 0.12 1.43 0.08 0.05 40.56 2.54 154.50 2.31H256 0.00 0.17 0.03 0.51 0.05 0.71 89.40 9.41 210.00 0.10H259 0.01 0.35 0.11 0.82 0.27 0.88 375.50 58.06 263.20 3.33H261 0.01 0.16 0.05 0.20 0.03 1.43 125.00 2.18 94.36 7.61

AUC-FURA2 AUC-Insulin ELISA

Batch # KRB2 8 mM KRB2 KRB2 12 mM KRB2 KRB2 16.7 mM KRB2H249 0.79 104.10 15.52 38.43 197.20 17.95 1.45 165.20 21.19H251 0.21 188.40 17.07 0.71 201.40 4.71 0.24 193.90 12.65H252 0.13 293.40 1.61 11.42 480.40 35.88 26.70 802.10 24.41H253 0.00 105.00 4.11 1.87 94.17 4.88 0.39 171.60 3.10H256 2.48 101.20 8.83 1.41 360.00 8.07 5.33 420.80 38.14H259 0.17 105.90 7.80 4.70 196.20 14.32 0.32 160.10 23.50

AUC-8 mM AUC-12 mM AUC-16.7 mM

High Throughput High Throughput Oxygenation Oxygenation

Oxygen is a Key Metabolic Variable

• Current Tools: Hypoxic Chambers– Crude, inefficient, and

problematic• Cannot replicate

gradients of oxygen– found across all tissues in

every animal• Need a better too to study

– Development– Angiogenesis– Cancer– Hematopoiesis– Drug Toxicity

System Design: Add-on for Standard Lab Materials

• Modular Platform– Multiwell format – Diffusion through

PDMS

Oxygen Quantification

Microfluidic Brain Slice DeviceMicrofluidic Brain Slice Device

Hugo Caicedo, Javeed S. Mohammed, Chris P. Fall, and David Eddington

Current Approaches for Delivery

• Bathe entire slice – Imprecise– Simple

• Micropipette picospritzer– Precise– Bulky– Separate controller for each pipette

Simple

• Moudular add on for a standard perfusion chamber

Precise

• Microfluidic Stimulation– Simultaneous stimulation of multiple regions

– High spatial and temporal precision

Hans-Ulrich Dodt, Nature Methods 2007

Device Fabrication

http://www.jove.com/index/Details.stp?ID=302

Fluid Delivery: Passive Pumping

• Steady flow without external pumps

Microfluidic Brain Slice Device Design

• Adapt to commonly used materials

Fluorescence Quantification

• “Paint” neuromodulators

Fluorescence Quantification

• Deliver a bolus

Compatible with electrophysiology

3D Modeling

Nematocysts as Part of Drug Nematocysts as Part of Drug Delivery PlatformDelivery Platform

Shawn C. Oppegard, Peter Anderson*, and David Eddington

*University of Florida, Whitney lab for Marine Biology

Overview• Cnidarian biology• Aim of project

– Bioleverage nematocysts• Preliminary results

– Material puncture tests– Lectin binding– Optical Tweezing

• Future directions– Nematocyst patterning

and immobilization

Nematocysts biology• Nematocysts are the venom

delivery system in cnidariananimals

– Phylum cnidaria includes the jellyfish

– Nematocysts are specialized organelles contained within cnidocytes

• Prey contact induces discharge of functional stinger

• Discharge is one of the fastest movements in animal kingdom

– Penetrates hard fish scales– Occurs in less than a microsecond– 5x106 g acceleration– 7 GPa pressure at thread tip

@

http://oceanexplorer.noaa.gov

http://www.reefland.com

http://www.beachhunter.net

Nematocyst discharge in an ex vivotentacle

Nematocysts as Part of a Drug-Delivery Platform

• Ultimate Goal:– Bioleverage nematocysts– Microfabricate containment

wells for nematocysts

• Nematocysts attractive as miniature hypodermic needles– Efficient– Very stable– Can be triggered chemically or

electrically– Very small thread diameter

• Aim is to genetically re-engineer– Dr. Peter Anderson at the

Whitney Marine Biology Lab-University of Florida

DischargeStimulation

Nematocyst

Drug-Delivery“Patch”

Incorporation

Isolated nematocyst discharge studies

• Dry in 25 mM EDTA and then rehydrate in water– EDTA chelates calcium– Discharge is a calcium-dependent process

• Problem: Reorientation of cysts after firing when not anchored– Do not puncture most test materials

Rehydration

Tentacle-contained nematocyst discharge studies

• Best case scenario– Immobilized– Physiological discharge due

to mechanical stimulation of cnidocil

• Tentacles from 3 different animals tested– Chrysaora

• Initial trials– Cladonema

• Stenotele nematocysts– Physalia

• Very long threads (~1 mm vs. ~15 µm capsule diameter)

www.palaeos.org

www.paleobio.org

www.ocean-life.info

Chrysaora

Cladonema

Physalia

5 cm

10cm

Szczepanek, J. Cell Science, 2001www.palaeos.org

Stenotele

Puncture Mechanics Assessment

• Need to assess the puncture mechanics of the thread– Trigger discharge of nematocysts into adjacent material

• Tested materials with gamut of elastic moduli (Eskin=75 kPa)

– Gelatin ~ 20 kPa– Polyacrylamide ~ 60 kPa– Teflon ~ 0.1 MPa– Latex ~ 0.8 MPa– Polydimethyl siloxane (aka silicone, PDMS) ~ 1 MPa Starting Point– Nitrile ~ 2.6 MPa– PVC (Saran wrap) ~ 250 MPa– Polycarbonate ~ 2 GPa– Aluminum foil ~ 70 GPa– Glass ~ 90 GPa

• Elastic modulus as the material characteristic– Not measuring actual puncture stress– E is order of magnitude approximation

Soft

Hard

Physalia: Puncture tests• Went to Florida

– Physalia cultured at Whitney marine biology lab

• Method– Excised tentacles– Nematocysts discharge in

response to:• EGTA solution• Mechanical stimulation

– Tweezer prodding

• Note: A similar protocol was followed for Chrysaora at UIC

Physalia and Chrysaora: Puncture tests (cont.)

• Started with PDMS microchannels because easiest– Clear visualization of cross-sectional penetration– Tentacle pulled inside– Stimulated discharge with EGTA– Observe penetration with dissecting and compound scope

• Used films for all other materials– Place test material films on top of Physalia tentacle– Stimulate discharge with tweezers– Observe penetration with dissecting scope

600 microns

200 microns

Physalia nematocysts elastic modulus puncture threshold is ~1

MPa– Gelatin ~ 20 kPa– Polyacrylamide ~ 60 kPa– Teflon ~ 0.1 MPa– Latex ~ 0.8 MPa– PDMS ~ 1 MPa

----------THRESHOLD-----------– Nitrile ~ 2.6 MPa– PVC ~ 250 MPa– Polycarbonate ~ 2 GPa– Aluminum foil ~ 70 GPa– Glass ~ 90 GPa

Penetration

NoPenetration

Soft

Hard

•Chrysaora could not puncture PDMS

•Should use Physalia for patch

Future Work: Lectin binding as means of nematocyst

immobilization• Isolated nematocysts to be used, not

tentacles– Need to immobilize and possibly orient

• Lectins– Sugar-binding proteins– Sugar moeities present on surface of

nematocysts• Fluorophore-conjugated lectin binding to

nematocyst:– Lectins bind to apical surface of

nematocysts in Cladonema and Physalia• Could bind nematocysts to PDMS

membrane

• Explore other, basal-localized receptors to Physalia

Summary• BioMEMS is an enabling technology• Simple device design streamlines dissemination

– Device complexity limits practicality• We have lots of exciting projects

– Enabling projects• Microfluidic sickle cell model • Islet quantification• Brain slices• High throughput hypoxia

– Bioleveraged• Cnidocyst drug delivery

Acknowledgements• BML lab

– Javeed Mohammed– Hugo Caicedo– Kihwan Nam– Shawn Oppegard

• Collaborators– Jose Oberholzer (UIC)- islets– Yong Wang (UIC) - islets– Chris Fall (UIC) – brain slice– Peter Anderson (UF) – jellyfish

• HST (sickle cell)– Sangeeta Bhatia– John Higgins– Lakshminarayanan Mahadevan

• Funding– NIH - NRSA– DARPA – Alfred P. Sloan Foundation