Alfredo M. Alfredo M. MoralesMoralesaa, William D. , William D. MunslowMunslowbb, , Scott M. Scott M. MaurerMaurerbb, Blake A. , Blake A. SimmonsSimmonsaa

a a Sandia National Laboratories, Livermore, CA 94550Sandia National Laboratories, Livermore, CA 94550b b Lockheed Martin MS2, Manassas, VA 20110Lockheed Martin MS2, Manassas, VA 20110

Authorized for Unlimited Public Release by SNL SAND2009Authorized for Unlimited Public Release by SNL SAND2009--3326C3326CAnd by LMC BAL200902002And by LMC BAL200902002--02 02

NBC 2009 SymposiumNBC 2009 SymposiumJyvJyvääskylskylää, Finland, Finland8 8 –– 11 June 200911 June 2009

Insulator Based Insulator Based DielectrophoresisDielectrophoresis ((iDEPiDEP):):A Tool to Detect, Separate and Concentrate A Tool to Detect, Separate and Concentrate

Aerosolized Biological ParticlesAerosolized Biological Particles

2

Biological Sensor RequirementsBiological Sensor Requirements

•• Requirements and Performance Measures:Requirements and Performance Measures:Affordability Affordability -- Early detection Early detection -- Low false alarm rateLow false alarm rate

•• Current Biological Detection System Deficiencies:Current Biological Detection System Deficiencies:Expensive Expensive -- Hours/days for analysis Hours/days for analysis -- Sensitivity Sensitivity vsvs false alarm ratefalse alarm rate

•• Desirable Characteristics of Typical DetectDesirable Characteristics of Typical Detect--toto--Warn Sensor:Warn Sensor:Spider Charts: metrics shown by length of legsSpider Charts: metrics shown by length of legs

Response Time (min)

FAR (per year)

Maintenance Interval (wks)

Size (m3)

Operating Cost ($/year)

Unit Cost ($M)

Detection Confidence

100

1

2

1000 100

1000500

98

110

.5

4

.1

0.5

1.5

1.0

4

95

90

10.000

1001,000

Sensitivity (ACPLA)

52

26

Response Time (min)

FAR (per year)

Maintenance Interval (wks)

Size (m3)

Operating Cost ($/year)

Unit Cost ($M)

Detection Confidence

100

1

2

1000 100

1000500

98

110

.5

4

.1

0.5

1.5

1.0

4

95

90

10.000

1001,000

Sensitivity (ACPLA)

52

26

Response Time (min)

FAR (per year)

Maintenance Interval (wks)

Size (m3)

Operating Cost ($/year)

Unit Cost ($M)

Detection Confidence

100

1

2

1000 100

1000500

98

110

.5

4

.1

0.5

1.5

1.0

4

95

90

10.000

1001,000

Sensitivity (ACPLA)

52

26

Ideal Biological Detection System:Ideal Biological Detection System:•• Low cost, low consumables and simple Low cost, low consumables and simple

operationoperation•• Operational flexibility: Configurable as either Operational flexibility: Configurable as either

detectdetect--toto--warn or as a detectwarn or as a detect--toto--treat system treat system •• Expensive bioassays may be used if Expensive bioassays may be used if

inexpensive orthogonal triggers limit the inexpensive orthogonal triggers limit the number of times bioassays are runnumber of times bioassays are run

3

Why an Why an iDEPiDEP Approach?Approach?

Flow

500 VDC 750 VDC 1000 VDC+ -

Flow

Separation by size: 2 μm (green) and 1 μm (red) Time

Time

Bacillus subtilisspores (red) trapped. 1 μm polystyrene beads (green) pass.

iDEP Selectively Traps Particles and May be Used as a TriggeriDEP Selectively Traps Particles and May be Used as a Trigger

Lapizco-Encinas, B. H.; Simmons, B.A., et al. , Anal. Chem. 2004, 76, 1571-1579

+-

4

iDEPiDEP Can Concentrate Bacteria SelectivelyCan Concentrate Bacteria Selectively

•• iDEPiDEP Separation of Live B. Separation of Live B. subtilissubtilis and B. cereus (similar bacteria)and B. cereus (similar bacteria)•• Mechanism: Different Size and ShapeMechanism: Different Size and Shape•• Also Separated E. coli, B. cereus, B. Also Separated E. coli, B. cereus, B. subtilissubtilis, B. , B. megateriummegaterium, B. , B. subtilissubtilis

spores, TMV virus, and polystyrene particles of various sizesspores, TMV virus, and polystyrene particles of various sizes

0

10

20

30

40

50

60

70

80

E. coli B. megaterium B. subtilis B.cereus

Min

imum

E re

quire

d fo

r DEP

trap

ping

(V/m

m)

medium conductivity = 2.2 microS/mm

medium conductivity = 10.4 microS/mm

Solution Conductivity=2.2microS/cmSolution Conductivity=2.2microS/cm

Solution Conductivity=10microS/cmSolution Conductivity=10microS/cm

Selectively trapping B. cereus (green)while passing B. subtilis (red)

E. coli < B. megaterium < B. subtilis < B. cereus

Lapizco-Encinas, B. H.; Simmons, B.A., et al. , Electrophoresis 2004, 25,1695-1704.

- +

5

iDEPiDEP is a Physically Selective Phenomenonis a Physically Selective Phenomenon

•• DEP: Motion of Conductive Object Toward DEP: Motion of Conductive Object Toward (away from) High Electric Field(away from) High Electric Field

–– Along electric field gradientsAlong electric field gradients–– Nonlinear in the applied electric fieldNonlinear in the applied electric field

•• DEP Force Proportional to:DEP Force Proportional to:–– Particle volumeParticle volume–– Conductivity + Conductivity + polarizabilitypolarizability difference difference

between particle and liquidbetween particle and liquid

---

+++

+

Net movement

Lower charge density

Electrode

Elec

trode

Nonuniform electric field

---

+++

+

-Net movement

Higher charge density

Lower charge density

Electrode

Elec

trode

Nonuniform electric field

-

•• DEP Force depend on:DEP Force depend on:–– Conductivity of the particle, Conductivity of the particle, pp–– Conductivity of the medium, Conductivity of the medium, mm–– Size of the particle, Size of the particle, rr

23 ~,~Re2 EfrF mpmoDEP

mpmpmpf 2),( Vdc,For

iDEPiDEP Uses Insulating Post Uses Insulating Post Features to Shape EFeatures to Shape E--fieldfield

Sabounchi, P.; Morales, A.M. et al. Biomed Microdevices, 2008, 10, 661–670.

6

iDEPiDEP with Pressure Driven Flowwith Pressure Driven Flow

•• Large Sample Volumes Must be Processed for Many Clinical ApplicaLarge Sample Volumes Must be Processed for Many Clinical Applicationstions•• Only Pressure Driven Flow can Process Such VolumesOnly Pressure Driven Flow can Process Such Volumes

Electrokinetic Flow Profile

Pressure Driven Flow Profile

iDEP Force

Force due to flow

•• Potential Attributes of Biological Potential Attributes of Biological Sensor Incorporating Sensor Incorporating iDEPiDEP::

–– Affordability: Affordability: iDEPiDEP trigger for bioassaytrigger for bioassay–– Early Detection: Response within Early Detection: Response within

seconds of turning onseconds of turning on–– Low False Alarm Rate: Orthogonal Low False Alarm Rate: Orthogonal

iDEPiDEP triggertrigger

FUparticleparticle )(u-dt

dum

)( EEEUu DEPEKparticle

Fluid FlowFluid Flow ElectrokineticElectrokineticVelocityVelocity

DielectrophoreticDielectrophoreticVelocityVelocity

A Significant Amount of Work A Significant Amount of Work Done in the Lab.Done in the Lab.

What Will it Take to Field What Will it Take to Field iDEPiDEP??

7

MicroMicro--molded molded iDEPiDEP ZeonorZeonor ChipsChips

•• Polymer Polymer iDEPiDEP Chips:Chips:–– Low Cost & Ease of ManufactureLow Cost & Ease of Manufacture–– Amenable to Large Scale ProductionAmenable to Large Scale ProductionR1 R2 R3 R4

x y

8

Chip Surface and Buffer Chemistry Is CriticalChip Surface and Buffer Chemistry Is Critical

•• Surface & buffer chemistry enable repeatable trapping with pressSurface & buffer chemistry enable repeatable trapping with pressure driven flowure driven flow

•• MOPA Treatment Lowers Contact angle, MOPA Treatment Lowers Contact angle, 90 90 22°° w/o MOPA w/o MOPA vsvs 65 65 44°° w/ MOPAw/ MOPA

•• Background Buffer pH Adjusted to 8 Background Buffer pH Adjusted to 8 While Keeping Conductivity LowWhile Keeping Conductivity Low

•• Compatible with TWEEN used in Compatible with TWEEN used in Aerosol Sample CollectorAerosol Sample Collector

O

O

8

OOH

OO

OR

OH

+ + NaIO4

MOPA (Methoxy polyethylene glycol acrylates)

“Chip polymer” Functionalized “chip polymer”

WithMOPA

Without MOPA

9

Waste ManifoldWaste Manifold

System Layout and ArchitectureSystem Layout and Architecture

Aerosol CollectorAerosol Collector Test SampleTest Sample

iDEPiDEP ChipChipBackground BufferBackground Buffer

10

Robust Automated Robust Automated iDEPiDEP SystemSystem

•• Designed a Robust System Capable of Operating in the FieldDesigned a Robust System Capable of Operating in the Field•• Both Software & Hardware were Simplified and Optimized with Both Software & Hardware were Simplified and Optimized with

Reliability and Safety in MindReliability and Safety in Mind•• Prototype Field Test Unit Fabricated, Integrated and TestedPrototype Field Test Unit Fabricated, Integrated and Tested

Modular iDEP chip holder

11

Commercial microfluidic connectors

Robust Automated Robust Automated iDEPiDEP SystemSystem

•• Designed a Robust System Capable of Operating in the FieldDesigned a Robust System Capable of Operating in the Field•• Both Software & Hardware were Simplified and Optimized with Both Software & Hardware were Simplified and Optimized with

Reliability and Safety in MindReliability and Safety in Mind•• Prototype Field Test Unit Fabricated, Integrated and TestedPrototype Field Test Unit Fabricated, Integrated and Tested

12

Electronics, HVS, and aerosol collector integrated

Robust Automated Robust Automated iDEPiDEP SystemSystem

•• Designed a Robust System Capable of Operating in the FieldDesigned a Robust System Capable of Operating in the Field•• Both Software & Hardware were Simplified and Optimized with Both Software & Hardware were Simplified and Optimized with

Reliability and Safety in MindReliability and Safety in Mind•• Prototype Field Test Unit Fabricated, Integrated and TestedPrototype Field Test Unit Fabricated, Integrated and Tested

13

Rack mountable packaging

Robust Automated Robust Automated iDEPiDEP SystemSystem

•• Designed a Robust System Capable of Operating in the FieldDesigned a Robust System Capable of Operating in the Field•• Both Software & Hardware were Simplified and Optimized with Both Software & Hardware were Simplified and Optimized with

Reliability and Safety in MindReliability and Safety in Mind•• Prototype Field Test Unit Fabricated, Integrated and TestedPrototype Field Test Unit Fabricated, Integrated and Tested

14

Dedicated control and data logging software

Robust Automated Robust Automated iDEPiDEP SystemSystem

•• Designed a Robust System Capable of Operating in the FieldDesigned a Robust System Capable of Operating in the Field•• Both Software & Hardware were Simplified and Optimized with Both Software & Hardware were Simplified and Optimized with

Reliability and Safety in MindReliability and Safety in Mind•• Prototype Field Test Unit Fabricated, Integrated and TestedPrototype Field Test Unit Fabricated, Integrated and Tested

15

Sample Automated Sample Automated iDEPiDEP RunRun

Free Flow (0 V)Free Flow (0 V) Trapping (1600 V)Trapping (1600 V) Release (0 V)Release (0 V)Time

Fluorescently labeled2 μm polymer beads

(106 particles/ml)

-

+

16

Fluorescence Detector is Immune to Fluorescence Detector is Immune to iDEPiDEP ArtifactsArtifacts

•• OffOff--chip, Capillarychip, Capillary--based based Conductivity and Absorbance Conductivity and Absorbance Detectors are Affected by Detectors are Affected by iDEPiDEP--trapping Related Artifactstrapping Related Artifacts

•• Fluorescence Detection not Fluorescence Detection not Affected by Artifacts. OnAffected by Artifacts. On--chip chip Fluorescence Detection Senses:Fluorescence Detection Senses:

•• Trapping & concentration between Trapping & concentration between postsposts

•• Trapped particles shortly after Trapped particles shortly after trapping voltage is turned ontrapping voltage is turned on

•• System response in secondsSystem response in seconds•• Biological particles by detecting Biological particles by detecting

native fluorescencenative fluorescence

•• Chip Holder Design Becomes Chip Holder Design Becomes ComplexComplex

Fluorescently Labeled Polymer Beads(106 particles/ml)

Fluorescence Analysis Areas

Fluorescence Imaging

20 40 60 80 100Time(sec)

Trap

ping

Vol

tage

(v

Fluo

resc

ence

(au

Trapping VoltageFluorescence

17

OffOff--Chip Capillary Based Fluorescence DetectorChip Capillary Based Fluorescence Detector

•• These LIF Detectors Have Been Used for Many These LIF Detectors Have Been Used for Many Years Both in the Lab and in the Field at Years Both in the Lab and in the Field at Various LocationsVarious Locations

•• The Detector Units were Developed to The Detector Units were Developed to bebe Miniature, Rugged, &Miniature, Rugged, & Consume Little PowerConsume Little Power

•• They Measure Approximately 2.5 X 3 X 2 in, They Measure Approximately 2.5 X 3 X 2 in, requirerequire 5.3 volts, < 100 milliamps, Excluding 5.3 volts, < 100 milliamps, Excluding the Laser Sourcethe Laser Source

•• Detector has been used with 405, 488, 532, 635, Detector has been used with 405, 488, 532, 635, and 650 nm Excitation Sources and 650 nm Excitation Sources

Off-Chip Miniaturized Laser Induced Fluorescence Detector Unit

Renzi, R. et al, Anal. Chem., 2005, 77(2), pp 435–441

Embedded Capillary Fluorescence Provides Real-Time DetectionEmbedded Capillary Fluorescence Provides Real-Time Detection

18

0 50 100 150 200 250 300 350 400Time (sec)

Trap

ping

Vol

tage

(v)

Ave

rage

Cou

nts

Per

Sec

ond

Trapping VoltageAvg CPS

0 50 100 150 200 250 300 350 400Time (sec)

Trap

ping

Vol

tage

(v)

Ave

rage

Cou

nts

Per

Sec

ond

Trapping Voltage

Avg CPS

Fluorescence Indicates Trapping and ReleaseFluorescence Indicates Trapping and Release

•• No Redesign of Chip Holder Needed No Redesign of Chip Holder Needed •• Fluorescence CPS Changes as Soon as Trapping StartsFluorescence CPS Changes as Soon as Trapping Starts•• Trapping is Seen a Minute after Voltage is Applied Trapping is Seen a Minute after Voltage is Applied vsvs Concentration!Concentration!•• A Slight Delay is Introduced by Locating Sensor OffA Slight Delay is Introduced by Locating Sensor Off--chip chip

Fluorescently Labeled Polymer Beads(106 particles/ml)

Fluorescently Labeled Bacillus atrophaeus(106 particles/ml)

Trapping Trapping

19

SummarySummary

•• iDEPiDEP Has Several Features and Can:Has Several Features and Can:–– Selectively trap and concentrate particlesSelectively trap and concentrate particles–– Be used as a trigger in a cost effective, fast Be used as a trigger in a cost effective, fast biodetectorbiodetector

•• Automated, Prototype Automated, Prototype iDEPiDEP Field Unit has been Built, Field Unit has been Built, Integrated and TestedIntegrated and Tested

•• Embedded Fluorescence Detection has been shown to Embedded Fluorescence Detection has been shown to Sense Trapping and Concentration in < 1 minute After Sense Trapping and Concentration in < 1 minute After Applying Applying iDEPiDEP VoltageVoltage

20

Questions?Questions?

AcknowledgmentsAcknowledgments

Sandia National Labs:Sandia National Labs:•• Tim Tim ShepoddShepodd•• Josh WhaleyJosh Whaley•• Mark D. ZimmermanMark D. Zimmerman•• Benjamin Van Benjamin Van BlariganBlarigan•• Karen L. Karen L. KrafcikKrafcik•• April April NissenNissen•• Joanne Joanne VolponiVolponi•• Ron Ron RenziRenzi•• Nicholas Nicholas MascarenhasMascarenhas

Previous SNL and LMC fundingPrevious SNL and LMC funding

Lockheed Martin:Lockheed Martin:•• TyesiaTyesia PompeyPompey•• Stephanie GrovesStephanie Groves•• Frank Frank RotondoRotondo•• Robert Robert DD’’ItaliaItalia