Development of a Cellular Isolation System for Real-Time Single Cell Oxygen

Consumption Monitoring

Joe DragavonMarch 21, 2007

•Measure multiple parameters in individual living cells in real-time to correlate cellular events with genomic information.

•Develop modular, affordable microsystems for analyzing complex cellular processes.

•Application: To monitor and increase the understanding of programmed cell death, apoptosis (ap-a-tow’-sis)

• This is done by determining the cellular oxygen consumption rate before and after a stress is applied (LPS)

• Too much apoptosis = cell-loss disorders• Too little apoptosis = uncontrolled cell growth (cancer)

Highly interdisciplinary undertaking!!

Microscale Life Sciences Center (MLSC)

Comprehensive Understanding ofComplex Cellular Processes

www.nst.co.il/frontiers/apoptosis.htm

MajorContributors

My Objective

• To develop a Cell Isolation System (CIS) with which we can monitor oxygen consumption rates at the single cell level– Must be user friendly– Must be adaptable for performing stimulus

response experiments– Cells must be kept viable and in a “low-stress”

environment– Hopefully inexpensive (relatively)

Why Oxygen

• History of O2 sensors at the UW– Professor Callis: pressure sensitive paints

• Direct indicator of cell viability and metabolic activity

• Difficult analyte to exclusively monitor– If we can do this, we should be able to do other

analytes in a similar manner

Why Single Cell

• Heterogeneous subpopulations

1. Cell death due to inflammatory pathology (pyroptosis)

2. Malignancy due to unregulated cell growth

3. Susceptibility and resistance to cell death leading to clones with aberrant survival

electronic ground state

T1

T2

S0

S1

S2

Sn

IC

ISC ICA F

P

Ene

rgy

electronic ground state

T1

T2

S0

S1

S2

Sn

IC

ISC ICA F

P

Ene

rgy

electronic ground state

T1

T2

S0

S1

S2

Sn

IC

ISC ICA F

P

Ene

rgy 0% 20%

Dy, E. S.; Kasai, H. e-Journal of Surface Science and Nanotechnology 2005, 3, 473-475.

McGraw, C. M., Callis, J. B. Experiments in Fluids 2006, 40, 2, 203-211.

Porphyrin O2 Sensor

Clark Electrode Response

y = -2E-05x + 0.0006R2 = 0.9997

0.0000

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Bead response

y = -2E-05x + 0.0006R2 = 0.955

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Clark Electrode Response

y = -2E-05x + 0.0006R2 = 0.9997

0.0000

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Clark Electrode Response

y = -2E-05x + 0.0006R2 = 0.9997

0.0000

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Bead response

y = -2E-05x + 0.0006R2 = 0.955

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Bead response

y = -2E-05x + 0.0006R2 = 0.955

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

microspheres Clark Electrode

Strovas, T.; Dragavon, J.; et al. Appl. Environ. Microbiol. 2006, 72 (2), 1692-1695.

Sensor Deposition2-Photon photo polymerization

Laser

Substrate

63x 1.4 NAobjective

Nano-cube

Dichroic mirror

Shutter

Camera

YoungDragavon

• Good for individual sensor deposition• Low throughput• Porphyrin embedded into polymer

100µm

Young, A. C., Dragavon, J. et al. Two-Photon Lithography of Platinum-Porphyrin Oxygen Sensors. IEEE Sensors. Accepted

Big Idea: Integrated ParallelCell Fluorescence System

NutrientSupply

CCDOnly those pixels above the tube are excited. Collects fluorescence data and allows feedback via computer to LED layer.

Tubular Cage Layer3D microfabricated polymer pressed into the layer below to trap individual cells. The tubes are perforated to allow nutrients to pass through. The inner surface of the tubes is functionalized to act as an external sensor.

Specimen LayerFluorescent bacteria cells on a flat transparent slide. Cells are sustained at temperature by a continuous flow of nutirents.

LED Excitation LayerMulticolored LED with microfabricated half-spherical lens array shown here to be uniformly illuminating blue. The intensity and color can either be static or dynamic to meet experiment needs.

NutrientSupply

CCDOnly those pixels above the tube are excited. Collects fluorescence data and allows feedback via computer to LED layer.

Tubular Cage Layer3D microfabricated polymer pressed into the layer below to trap individual cells. The tubes are perforated to allow nutrients to pass through. The inner surface of the tubes is functionalized to act as an external sensor.

Specimen LayerFluorescent bacteria cells on a flat transparent slide. Cells are sustained at temperature by a continuous flow of nutirents.

LED Excitation LayerMulticolored LED with microfabricated half-spherical lens array shown here to be uniformly illuminating blue. The intensity and color can either be static or dynamic to meet experiment needs.

NutrientSupply

CCDOnly those pixels above the tube are excited. Collects fluorescence data and allows feedback via computer to LED layer.

Tubular Cage Layer3D microfabricated polymer pressed into the layer below to trap individual cells. The tubes are perforated to allow nutrients to pass through. The inner surface of the tubes is functionalized to act as an external sensor.

Specimen LayerFluorescent bacteria cells on a flat transparent slide. Cells are sustained at temperature by a continuous flow of nutirents.

LED Excitation LayerMulticolored LED with microfabricated half-spherical lens array shown here to be uniformly illuminating blue. The intensity and color can either be static or dynamic to meet experiment needs.

JenYoung

Microwell Array

• High throughput• Approaching the requirements for

commercial implementation• Decent uniformity

1 2 3

4 5 6

7 8 9

• All microwells seeded simultaneously (random seeding)

• Can sequentially perform oxygen consumption measurements on multiple locations

Sensors

1 2 3

4 5 6

7 8 9

20 40 60 80 100

10

20

30

40

50

60

70

80

90

100

1000

2000

3000

4000

5000

6000

7000

8000

9000

Phase Modulation Data Processing

• System calibrated using a PMT, MLE, and the OOFD

• Improved fit by going to higher order polynomial

• Multiple phosphorescent lifetimes being detected (non-linear Stern-Volmer)

DragavonMolter

]O[1 2SV0 K

Ψ

Ψ

mKΨ

Ψ]O[1 2

0

])O[1/(])O[1/(

1

2SV2022SV101

0

KfKfΨ

Ψ

1)

2)

3)

Model

HollDragavonMolter

ActuatorSystem

• System acts as a manual linear actuator

• Load cell allows for accurate monitoring of the exerted force

• Rigid structure allows for a significant amount of force to be applied

• V-groove system ensures proper piston alignment

Living Cell Array

• Increased support– Aluminum plate

– 1/8” quartz window

• One macrowell format– ~5mL volume

• Reusable seal!– VERY IMPORTANT!!

• The glass lid is also made of borosilicate, just like the chips

• The thickness of the PDMS layer is ≈ the same as the lid

• Adhesive not optimized

Plunger(stainless steel)

PDMS

Glass

LaserScanningModule

Lid Actuator

Heat Source

405nmExcitation

TemperatureControl

PressureControl

QA/QC

bad seal

unsealed sealed (visual confirmation!) cleaned

PDMS delaminationdense array sealand other flaws

Cell Test 6/26/06

-0.05

0

0.05

0.1

0.15

0.2

0.25

0 1 2 3

# of cells

delta

O2

(ppm

/min

)de

lta O

2 (p

pm/m

in)

O

2 (

pp

m/m

in)

• 2 Locations (3X3)• 1 chip (81 wells)• 4 sequential

repetitions• Mouse macrophage

– very fragile!

• 10x objective

Sequential Drawdowns

Cell Drawdown Histogram

0

2

4

6

8

10

12

14

0 0.02 0.04 0.06 0.07 0.09 0.11 0.13 0.15

Bin

Freq

uenc

y

Δ O2 (ppm/min)

Fre

qu

en

cy

HistogramSingle Cell Drawdown

# of cells

Cell Test 6/26/06 1

-0.05

0

0.05

0.1

0.15

0.2

1 2 3 4 5 6 7 8 9

w ell #

de

lta

O2

(p

pm

/min

)

1

2

3

4

Average

2

1

0

2

1

0 00 0

del

ta O

2(p

pm

/min

)

Location 1Cell Test 6/26/06 1

-0.05

0

0.05

0.1

0.15

0.2

1 2 3 4 5 6 7 8 9

w ell #

de

lta

O2

(p

pm

/min

)

1

2

3

4

Average

2

1

0

2

1

0 00 0

del

ta O

2(p

pm

/min

)

Location 1 Cell Test 6/26/06 2

-0.05

0

0.05

0.1

0.15

0.2

0.25

1 2 3 4 5 6 7 8 9

w ell #

de

lta

O2

(p

pm

/min

)

1

2

3

4

Average

2

1

0

1 1 1

0 0 0

Location 2

del

ta O

2(p

pm

/min

)

Cell Test 6/26/06 2

-0.05

0

0.05

0.1

0.15

0.2

0.25

1 2 3 4 5 6 7 8 9

w ell #

de

lta

O2

(p

pm

/min

)

1

2

3

4

Average

2

1

0

1 1 1

0 0 0

Location 2

del

ta O

2(p

pm

/min

)

• General linearity• Not centered around 0• Superimposed biological

and system variance

paper in progress

0

100000

200000

300000

400000

500000

600000

700000

800000

0 5 10 15 20 25 30 35

step

int 20%

0%inte

nsi

ty

Power Spectrum

0

5E+11

1E+12

1.5E+12

2E+12

2.5E+12

3E+12

0 5 10 15 20

frequency

Fo

uri

er c

oef

fici

entMethod

# of data points

# of laser shots

time (sec)

Original 42 210 42

Current 21 105 21

In Progress 31 31 6.2*

TBD (Nyquist) 6 6 1.2*

For a Single Oxygen Determination

* Camera Limited

Final Work:New Data Collection

DragavonYoungBurgess

Φ = tan-1(I/R)

My Objective

• To develop a Cell Isolation System (CIS) with which we can monitor oxygen consumption rates at the single cell level– Must be user friendly– Must be adaptable for performing stimulus

response experiments– Cells must be kept viable and in a “low-stress”

environment– Hopefully inexpensive (relatively)

(working on this still)

HIBISCUS

• Hybrid Integraged BIophotonic Sensors Created

by Ultrafast laser Systems

• 7 universities and companies

• 6 different countries

• Aiming to develop a single production machine

for biophotonic chips

• Use a high-power femtosecond laser to write

waveguides and etch the channels

Proposed Synthesis

• Gramicidin S, a cyclic pentapeptide antibiotic

ONH

NH

O

O

(9H -f luoren-9-yl)methyl 3-(benzylamino)-3-oxopropylcarbamate

Chemical Formula: C25H24N2O3Exact Mass: 400.1787

Molecular Weight: 400.4697m/z: 400.18 (100.0%), 401.18 (28.2%), 402.19 (3.6%)

Elemental Analysis: C, 74.98; H, 6.04; N, 7.00; O, 11.99

ONH

O

O

O

F

F

F

F

F

perf luorophenyl 3-(((9H -f luoren-9-yl)methoxy)carbonylamino)propanoate

Chemical Formula: C24H16F5NO4Exact Mass: 477.0999

Molecular Weight: 477.3802m/z: 477.10 (100.0%), 478.10 (26.5%), 479.11 (3.3%)

Elemental Analysis: C, 60.38; H, 3.38; F, 19.90; N, 2.93; O, 13.41

H2N

phenylmethanamine

Chemical Formula: C7H9NExact Mass: 107.0735

Molecular Weight: 107.1531m/z: 107.07 (100.0%), 108.08 (7.7%)

Elemental Analysis: C, 78.46; H, 8.47; N, 13.07

• Representative reaction to be used to test various analytical methods

• Experimental parameters already determined by current Watts group members

+

Optical TechniquesMethod Information How

SPR RI, Binding Spreeta™ by TI

ATR RI, Binding Waveguides in glass

µMS RI, Diffusion PSD

Raman Compound Identification Microscope

Fluorescence Functional Groups, Protected vs. Unprotected Spectral Deconvolution

HPLC Product Separation, Standard Technique HPLC

Costin, c. D., Synovec, R. E. Anal. Chem. 2002, 74, 4558 – 4565

Multi-Channel Chip

• Promotes phase separation

• Diffusion properties can be used to improve analysisBen Wahab,

Watts group

Phase Separation

• Multiple channels will promote the separation/isolation of desired compounds

• Increases the effectiveness of the optical analysis

• Good for:– µMS– SPR– Fluorescence

Acknowledgements

University of Washington

MLSC-NIH

CPAC

Cody Young, Tim Molter,

Sarah McQuaide

Drs. Jen, Lidstrom, Meldrum, Holl, Chao, Lutz, Marquardt, Strovas

Lloyd Burgess, Gordon Mitchell

University of Hull

HIBISCUS

Paul Watts

and Watts group members

Phase Modulated Determination(OOFD)

Delay of the intensifier is established using the camera software

Laser period = Intensifier periodThe period is variable

ExternalTrigger

Laser

Camera

Intensifier

-180°

-90°

0°

90°

180°

1

2

3

4

5

StepDegree Shift

100μsec

Shonat, R., Kight, A. Annals of Biomedical Engineering, Vol. 31, pp. 1084–1096, 2003

Thermoelectric Cooler Effect

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9

well #

ave

-20

-10

0

Offav

era

ge

pp

mAccumulation Comparison

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9

well #

aver

age

ppm

1

2

3

4

5av

era

ge

pp

m

Gain Comparison

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9

well #

aver

age

ppm 50

100

150

av

era

ge

pp

m

Focus Comparison

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9

well #

aver

age

ppm

-75um

-25um

0um

25um

75umav

era

ge

pp

m

N = 30

OXYGEN SENSOR CALIBRATION AND REPRODUCIBILITYThermoelectric Cooler Effect

Gain Comparison Focus Comparison

Accumulation Comparison

well #

well #

well #

well #

Bin Comparison

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9

well #

aver

age

ppm

4x4

6x6

10x10

12x12

20x20av

era

ge

pp

mShot Comparison

0

50000

100000

150000

200000

250000

1 2 3 4 5 6 7 8 9

well #

coun

tsa

ve

rag

e i

nte

gra

ted

in

ten

sit

y

Microwell Location Comparison

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9

well #

Av single

wholeav

era

ge

pp

m

1 hour Stability

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9

well #

ava

ve

rag

e p

pm

Long-term Stability

17,000 x 50sec exposures( >150 O2 determinations)

OXYGEN SENSOR CALIBRATION AND REPRODUCIBILITYShot Comparison

1800 x 50µsec exposures

Microwell Location Comparison

Bin Comparison

N = 30well #

well #

well #

well #

-0.1

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0.08

0.1

1 2 3 4 5 6 7 8 9

well #

delta

O2 1

2

3

delta

O2

(ppm

/min

)

10 Acquisitions

-0.1

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0.08

0.1

1 2 3 4 5 6 7 8 9

well #

delta

O2 1

2

3

delta

O2

(ppm

/min

)

10 Acquisitions

-0.1

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0.08

0.1

1 2 3 4 5 6 7 8 9

well #

delta

O2 1

2

3

delta

O2

(ppm

/min

)

42 Acquisitions

-0.1

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0.08

0.1

1 2 3 4 5 6 7 8 9

well #

delta

O2 1

2

3

delta

O2

(ppm

/min

)

42 AcquisitionsΔ O2 (15 min, 42 Kinetic Series) Δ O2 (11 min, 10 Kinetic Series)

well # well #

Table 1. Integrated Intensities of One Microwell Over Multiple Kinetic Series

Kinetic Series Step #

Series # 1 2 … 20 21 Average StDev CV

1 252649 252012 … 254735 254643 252552.4 1397.15 0.553

10 250113 251660 … 248569 247600 249158 1707.178 0.685

20 246669 243976 … 248299 248096 246771.5 1650.655 0.669

30 244905 247621 … 246993 248892 247372 1530.93 0.619

Average 248584 248817.3 … 249649 249807.8

STDEV 3467.195 3792.816 … 3459.799 3267.134

CV 1.394778 1.524338 … 1.385865 1.307859

Integrated intensity at a fixed phase shift

Coefficient of Variance = CV = (StDev/Average)*100

* will return to this camera issue

Benzylamine

Pentafluorophenol

http://www.aist.go.jp/RIODB/SDBS/cgi-bin/direct_frame_top.cgi?lang=eng

Ongoing Work:External Stimulus

• External Stimulus Response– LPS Application

• Monitor O2 consumption prior to and after application

• Monitor O2 over time

• This would add the metabolic rate of the cell as another dimension to the data set

5 bead

1920 x 1200 pixels

Jesacher et. al., 23 August 2004 / Vol. 12, No. 17 / OPTICS EXPRESS 4129

Ongoing Work: SLM used to generate dynamic optical tweezers

Size exclusion optical trapping

BurgessYoung

High-throughput cell deposition

Ongoing Work:Sample Cassette

Holl

Technology from the current CIS is the foundation for the cassette

Ongoing Work: Multispectral Analysis

HollBurgessPowellKarlsgodt Cheaper than the current set-up!

Clark Electrode Response

y = -2E-05x + 0.0006R2 = 0.9997

0.0000

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Bead response

y = -2E-05x + 0.0006R2 = 0.955

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Clark Electrode Response

y = -2E-05x + 0.0006R2 = 0.9997

0.0000

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Clark Electrode Response

y = -2E-05x + 0.0006R2 = 0.9997

0.0000

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Bead response

y = -2E-05x + 0.0006R2 = 0.955

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Bead response

y = -2E-05x + 0.0006R2 = 0.955

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

4 6 8 10 12 14 16

Time (min)

Mol

O/L

Bulk O2 Consumption and Biological Compatibility

y = 5160.2x + 1.0671R2 = 0.9978

0

0.5

1

1.5

2

2.5

3

3.5

4

0 0.0001 0.0002 0.0003 0.0004 0.0005

Mol O/L

To/

T

0

10

20

30

40

50

60

70

Life

time

s (

sec)

DragavonStrovas

Strovas, T.; Dragavon, J.; et al. “Measurement of Respiration Rates of Methylobacterium extorquens AM1 Cultures by Use of a Phosphorescence-Based Sensor”, Appl. Environ. Microbiol. 2006, 72 (2), 1692-1695.

microspheres Clark Electrode