CPAC Summer Institute - University of...

CPAC Summer InstituteCPAC Summer InstituteJune 15-17, 2008

Real Time Biosensor Systems & Protein ProductionReal Time Biosensor Systems & Protein Production

••Clement E. Furlong, Scott D. Soelberg, Richard Stevens and Peter Kaufmann Clement E. Furlong, Scott D. Soelberg, Richard Stevens and Peter Kaufmann Departments of Medicine Departments of Medicine

(Div. Medical Genetics) & Genome Sciences(Div. Medical Genetics) & Genome Sciences

0.8

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Fundamentals of Fundamentals of Fundamentals of Fundamentals of

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Ref

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ivity

Surface Plasmon ResonanceSurface Plasmon ResonanceSurface Plasmon ResonanceSurface Plasmon Resonance

0

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0 20 40 60 80

Ө DegreesӨ Degrees

1.33761.3377

System software

Sensorgram

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ive

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Sensorgram

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Ti MiR

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Our goal is to reduce the size and cost of SPR Our goal is to reduce the size and cost of SPR technology from a ~$300 000 desk top device to a technology from a ~$300 000 desk top device to a technology from a ~$300,000 desk top device to a technology from a ~$300,000 desk top device to a portable system that costs under $30,000portable system that costs under $30,000

This system is designed around gthe miniature TI-SPR chip, designed by José Melendez and teamand team.

Spreeta sensing componentsSpreeta sensing componentsSpreeta sensing componentsSpreeta sensing componentsS SPRS SPR•• Spreeta SPR components Spreeta SPR components developed in collaboration developed in collaboration with UW with TIwith UW with TI

Each Spreeta chip contains Each Spreeta chip contains all of the optical all of the optical components needed for components needed for

•• Miniaturized, robust, high Miniaturized, robust, high performance devices. performance devices.

I i i l titI i i l tit components needed for components needed for sensitive SPR measurement sensitive SPR measurement of biomolecular interactionsof biomolecular interactions

•• Inexpensive in large quantityInexpensive in large quantity

•• Excellent manufacturing Excellent manufacturing capabilities and qualitycapabilities and qualitycapabilities and quality capabilities and quality controlcontrol

The SPIRIT systemThe SPIRIT systemThe SPIRIT systemThe SPIRIT systemyy(Surface Plasmon Instrumentation for the Rapid Identification of Toxins)(Surface Plasmon Instrumentation for the Rapid Identification of Toxins)

yy(Surface Plasmon Instrumentation for the Rapid Identification of Toxins)(Surface Plasmon Instrumentation for the Rapid Identification of Toxins)

•• Compact, Compact, lightweight (lunchbox lightweight (lunchbox size, 6 lb.)size, 6 lb.)

Current laboratoryprototype

•• High performanceHigh performance•• 24 simultaneous 24 simultaneous

measurementsmeasurementsmeasurements measurements •• Low power (5W) Low power (5W)

allows portable allows portable titioperationoperation

•• SemiSemi--AutomatedAutomated

Touchscreen data displayTouchscreen data displayTouchscreen data displayTouchscreen data display

Selected SPR curveSelected SPR curve Detected levelsDetected levels(bargraph)(bargraph)

Sensor channelSensor channel

Detected levelsDetected levels(numeric)(numeric)

Sensor surface chemistrySensor surface chemistrySensor surface chemistrySensor surface chemistryEach Spreeta chip has 3 Each Spreeta chip has 3 useable channelsuseable channels

Soluble protective coatingSoluble protective coating(dextran/trehalose)allows long-term dry storage at

useable channelsuseable channels

room temperatureControl receptorsControl receptors(usually antibodies)Designed NOT to respond to that agent

Y Y Y Y Y Ψ Ψ Ψ Ψ ΨGold layer (50 nM)Gold layer (50 nM)

Target receptors:Target receptors:(usually antibodies)Designed to capture a specific agent or

l t Y Y Y Y Y Ψ Ψ Ψ Ψ Ψ

Glass substrateGlass substrate

analyte e.g.:

•Toxins•Viruses•Spores•Bacteria Spreeta Spreeta

sensor chip

Storage of Gold SlidesStorage of Gold Slidesgg120140

wet Dextran 1

6080

100of

1 d

ay w

02040

erce

nt o

Control

Dextran 2

00 50 100 150 200 250 300 350

Time (days)

Pe

Anti-Alkaline-phosphatase(AP)-antibody-coated slides were dried with a thin layer of 10 mM Tris, pH 8.0, 2.5% trehalose

d 2 5% d t Aft t d d t lid tt dand 2.5% dextran. After extended storage, slides were wetted, exposed to AP and analyzed for AP activity.

Silicone moldingSilicone moldinggg

•• Versatile technique for production of Versatile technique for production of q pq pprecision flowcells & other fluidic precision flowcells & other fluidic componentscomponents

Antibody 1

Flow channelsformed by fishing line

Antibody 2

Antibody 3

Flowcell cast from mold

Fixture for derivatizing individual channelsFixture for derivatizing individual channelsFixture for derivatizing individual channelsFixture for derivatizing individual channels

SPIRIT performs 24 simultaneous SPIRIT performs 24 simultaneous ppmeasurements of antibody bindingmeasurements of antibody binding

Eight sensor chipsDetection event

Eight sensor chips

Toxin

Silicon flow cell Silicon flow cell i f i hi f i hinterfaces with interfaces with TE controller TE controller ((±±0.010.01oo C)C)

Three active spots per sensor

Flowcell

Snap-in block of 8 sensor chips

Examples of Assays Possible with SPRExamples of Assays Possible with SPRExamples of Assays Possible with SPRExamples of Assays Possible with SPR

•• Whole microbial cells Whole microbial cells --((F.tularensis, E. coli, Y. pestisF.tularensis, E. coli, Y. pestis))

•• Spores Spores (e g anth a )(e g anth a )--(e.g., anthrax)(e.g., anthrax)

•• Viruses with or without amplification Viruses with or without amplification --(e.g. Norwalk, flu)(e.g. Norwalk, flu)

P t i b di t d t ti ith ith tP t i b di t d t ti ith ith t•• Proteins by direct detection with or without Proteins by direct detection with or without amplification/verificationamplification/verification--(protein toxins, industrial proteins, therapeutics)(protein toxins, industrial proteins, therapeutics)

•• Small molecular weight analytes using displacement orSmall molecular weight analytes using displacement orSmall molecular weight analytes using displacement or Small molecular weight analytes using displacement or competition assayscompetition assays--(e.g., domoic acid, cortisol, insecticides, toxic chemicals, TNT & other small (e.g., domoic acid, cortisol, insecticides, toxic chemicals, TNT & other small organics)organics)

Detection of Larger AnalytesDetection of Larger AnalytesDetection of Larger AnalytesDetection of Larger AnalytesDetection of Larger AnalytesDetection of Larger AnalytesDetection of Larger AnalytesDetection of Larger Analytes

• Microbes• Microbes• Microbes

• Spores

• Microbes

• Spores

• Viruses

•Proteins/Toxic Proteins

• Viruses

•Proteins/Toxic Proteins•Proteins/Toxic Proteins

•Small molecule toxins

•Proteins/Toxic Proteins

•Small molecule toxins

Analyte Detection and Signal AmplificationAnalyte Detection and Signal Amplification

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Ti i

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Signal Detection

Analyte Detection and Signal AmplificationAnalyte Detection and Signal AmplificationAnalyte Detection and Signal AmplificationAnalyte Detection and Signal Amplificationy py p

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Time, min

Signal Detection

Detection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawater

1.33973

1.33974

n

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dex,

n

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ract

iv

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1.33967

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Detection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urine

Amplification

500 pM SEBWash(urine)

From: Naimushin et al., Biosensors and Bioelectronics 17:573

Detection of MicrobesDetection of MicrobesC t D t ti Li it 10C t D t ti Li it 1033 f / lf / l

Detection and Verification of F. Tularensis (105 cfu/ml)

Current Detection Limit: 10Current Detection Limit: 103 3 cfu/mlcfu/ml

1.3396

dex

Detection

Amplification/verification

1 3392

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ive

Ind

anti-F.T #1anti-F.T #2anti-F.T. #3anti Bot A NT #1Active channels

e ec o

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r anti-Bot A NT #1anti-Bot A NT #2anti-Bot A NT #3

Active channels

1.3388

1.3390

Rel

a Reference channels

0 2 4 6 8 11 13 15 17 19 21 23Time (min)

Virus DetectionVirus DetectionNorwalk VLP Detection

Reference Subtracted, 0.75 mL Sample, p

180

10^6 PFU/ml Norwalk VLPs Anti-Norwalk Amp

120140160180

Norwalk

Amplification

406080

100

RIU

Amplification

-40-20

020

0 2.5 5 7.5 10 12.5 15 17.5 20 22.5Time (min)

-6040 Time (min)

Quantitative Detection of Quantitative Detection of QQStaphylococcal Enterotoxin BStaphylococcal Enterotoxin B

9 0E-05

1.33515

1.33520

1.33525

, RIU 6.0E-05

7.0E-05

8.0E-05

9.0E-05

RIU/

min

1.33505

1.33510

enso

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3.0E-05

4.0E-05

5.0E-05

B bi

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6.0E-06

8.0E-06

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Se

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SEB t ti MSE

B

0.0E+00

2.0E-06

0 1 2 3 4

Time, min SEB concentration, nM

Detection of 5 ng/mL (5 ppb; 33pM) Detection of 5 ng/mL (5 ppb; 33pM) g pp pg pp pBotNT BotNT (denatured botulinum toxin)(denatured botulinum toxin)

1.332261.33228

1.3323Anti-Bot-toxin

Reference

1.33221.332221.33224

RI AmplifyDetect

1.332141.332161.33218

1.33211.33212

0 10 20 30 40Time (min)

Direct Detection of Ricin A Direct Detection of Ricin A Direct Detection of Ricin A Direct Detection of Ricin A Direct Detection of Ricin A Direct Detection of Ricin A Chain (64 ppbChain (64 ppb--320 ppb)320 ppb)

Direct Detection of Ricin A Direct Detection of Ricin A Chain (64 ppbChain (64 ppb--320 ppb)320 ppb)( pp( pp pp )pp )( pp( pp pp )pp )

14)

8

10

12

14

x10-

6RIU

/min

)

0.00005

0.00007

0.00009

ubtr

acte

d R

IU 100 nM Ricin A Chain50 nM Ricin A Chain20 nM Ricin A ChainNo Ricin A Chain

0

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6

Bin

ding

Rat

e (x

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0.00003

Bac

kgro

und-

s

00 100 200 300 400

Ricin A Chain Concentration (nM)

B-0.000010 200 400 600 800 1000

Time (seconds)

B

Detection of small molecule toxins by SPRDetection of small molecule toxins by SPRDetection of small molecule toxins by SPRDetection of small molecule toxins by SPRis a more difficult taskis a more difficult taskis a more difficult taskis a more difficult task

Displacement AssayDisplacement AssayR

espo

nse

Time

R

Displacement AssayDisplacement Assay

Antibody Loading

Sensor Ready Condition

Antibody Loading

Res

pons

e

Time

R


Introduction of Target

Sample Detection Mode

Introduction of Target

Res

pons

e

Time

R


Antibody Displacement

Displacement of Target Analog

Antibody Displacement

Res

pons

e

Rate proportional to analyte (target) concentration

Time

R

The main advantage of the displacement The main advantage of the displacement i th t th i t i th t th i t assay is that the expensive components assay is that the expensive components

of the assay (antibodies or receptors) of the assay (antibodies or receptors) b t i d d b d b t i d d b d can be retained under a membrane and can be retained under a membrane and

reused many times.reused many times.

Competition AssayCompetition Assay

ANALYTE ATTACHED TO THE SURFACEANALYTE ATTACHED TO THE SURFACE

••Small Analytes:Small Analytes:••Estriol, Cortisol, Domoate…Estriol, Cortisol, Domoate…••Same analyte on the surfaceSame analyte on the surfaceSame analyte on the surfaceSame analyte on the surface

Competition AssayCompetition AssayNO ANALYTE PRESENTR

espo

nse

Time

R

Competition AssayCompetition AssayANALYTE PRESENT

Res

pons

e

Time

R

Competition AssayCompetition Assayp yp y

No target presentNo target present

nse

Ti

Res

po

Samples 1, 2, and 3

Time

Detection of Cortisol by Competition AssayDetection of Cortisol by Competition Assay

Cortisol Competition 2-24-04c 10 nM

1.3388

1.3389 BSABSA Cortisol

1 nM 750 pM

1.3387 RIU

BSA-CortisolHSA-GD

2 nM 1000 nM Estriol

13385

1.3386 5 nM Lower arrows indicate returnto no analyte

1.3384

1.3385

0 1000 2000 3000 4000 50000 1000 2000 3000 4000 5000Time (seconds)

External Compound Flow CellExternal Compound Flow CellExternal Compound Flow CellExternal Compound Flow Cell

Vacuum

Detection of cortisol in saliva Detection of cortisol in saliva Detection of cortisol in saliva Detection of cortisol in saliva using the compound flow cellusing the compound flow cell

0 0073

0.0074

RIU

Saliva plus 28 nM Cortisol

0 0073

0.0074

RIU

Saliva plus 28 nM Cortisol

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0.0073

ubtr

acte

d

Saliva plus 14 nM 0.0072

0.0073

ubtr

acte

d

Saliva plus 14 nM

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eren

ce-s

u

Saliva only

pCortisol0.0071

eren

ce-s

u

Saliva only

pCortisol

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0.007

Ref

e

0.0069

0.007

Ref

e

17 27 37 47 57 67 77Time (minutes)

17 27 37 47 57 67 77Time (minutes)

Quantification of CortisolQuantification of CortisolQuantification of CortisolQuantification of Cortisol100

ol s

ampl

e)

100

of n

o co

rtiso

10

ion

slop

e (%

1

1 10 100

Det

ect

0.1

Cortisol concentration (ng/ml)

1 10 100

Detection of Domoic Acid by Detection of Domoic Acid by yyCompetition AssayCompetition Assay

Collaboration with Dr. Vera Trainer’s team at NOAATrainer s team at NOAA

Standard Domoic Acid ConcentrationStandard Domoic Acid ConcentrationC i Cl E t tC i Cl E t tCurve in Clam ExtractsCurve in Clam Extracts

Stevens et al. Harmful Algae

Comparison of HPLC/SPR AssaysComparison of HPLC/SPR AssaysComparison of HPLC/SPR AssaysComparison of HPLC/SPR Assays

(GOLD STANDARD)(GOLD STANDARD)

Rapid Sample Clean UpRapid Sample Clean Up--p p pp p pConcentrationConcentration

Analyte

Analyte-immuno b dmagnetic bead

complex

Rapid Immuno Magnetic Bead Separation (IMS)Rapid Immuno Magnetic Bead Separation (IMS)Rapid Immuno Magnetic Bead Separation (IMS)Rapid Immuno Magnetic Bead Separation (IMS)

Rapid Immuno Magnetic Bead Separation (IMS)Rapid Immuno Magnetic Bead Separation (IMS)p g p ( )p g p ( )

Design for Ciguatoxin Detection ProtocolDesign for Ciguatoxin Detection Protocol

50 nm bead

50 nm bead

50 nm bead

Concentration, Verification, Amplification ProtocolConcentration, Verification, Amplification Protocol

1400 StreptavidinStreptavidin Microbeads (1075 RIU) 3

50 nM900

RIU 10 ng/ml

SEB

anti-SEB-biotin

(24 RIU)

50 nMParamagnetic

Bead

400

SEB (13 RIU)

(24 RIU)

1 21Sensor 2 3-100

0 20 40 60Time (min)

11 Ready 2 3

Concentration, Amplification and Verification Concentration, Amplification and Verification

of SEB from a Stool Sampleof SEB from a Stool Sample500

of SEB from a Stool Sampleof SEB from a Stool Sample

300

400 1 ng/ml SEB from a spiked stool sample

200

300

RIU

100

-100

0

0 2 4 6 8 100 2 4 6 8 10Time (min)

1/03 1/03 –– 6/03: Airborne SPR Sensing 6/03: Airborne SPR Sensing ggTest FlightsTest Flights

Variviggen instrumented with SPR sensors

Protein Nucleic Acids as Recognition Protein Nucleic Acids as Recognition El t f DNA/RNAEl t f DNA/RNA

Protein Nucleic Acids as Recognition Protein Nucleic Acids as Recognition El t f DNA/RNAEl t f DNA/RNAElements for DNA/RNAElements for DNA/RNAElements for DNA/RNAElements for DNA/RNA

Very stable receptor on chip

(Protein Nucleic Acid)

Allows detection of target

Binding of a 79 bp DNA Probe to a Complementary PNA Binding of a 79 bp DNA Probe to a Complementary PNA 16 mer on the Sensor Surface16 mer on the Sensor Surface

60

70

1000 ng/ml probe

40

50

U

1000 ng/ml probe

100 ng/ml probeApprox 60 RIU bound

10

20

30RIU 10 ng/ml probe

Reference (Avidinw ithout PNA)

Approx 30 RIU bound

-10

0

10

0 2 4 610

Time (min)Approx 2 RIU bound

Sequential Detection of 8 AnalytesSequential Detection of 8 Analytes

200

100

150Ovalbumin 10 ng/ml

SEB 5 ng/ml

F. tularensis 5 x 103 CFU/ml

Norwalk VLPs 5 x 109 particles/ml BG Spores

0

50RIU

Y. pestis

106 CFU/ml B. anthracis5 x 106 CFU/ml

5 x 109 particles/ml

Ricin A chain 20 ng/ml

9 x 104CFU/ml

-50

0

-1000 20 40 60 80 100 120 140 160 180

Time (min)

Inexpensive Production of AntibodiesInexpensive Production of AntibodiesInexpensive Production of AntibodiesInexpensive Production of Antibodies

Advantages of Shark IgNARsAdvantages of Shark IgNARsAdvantages of Shark IgNARsAdvantages of Shark IgNARs

Single poly peptide chain

12-15kD

Single poly peptide chain

Stanfield et al Science 305-1770Stanfield et al. Science 305 1770

Large scale production in E. coli – based bioreactors

Staby Expression SystemStaby Expression SystemStaby Expression SystemStaby Expression System

Poison = CcdB

Antidote = CcdA

Target = DNA

From Eurogentec Staby manual

Expression of IgNARExpression of IgNAR--cytb cytb fusionfusionfusionfusion

IgNAR-cytb

uced

uced

uced

pSCodon-IgNAR-cytB

BlaT7 pro

lac op

ccdA Not

indu

cIn

duce

d

Not i

nduc

Indu

ced

Not i

nduc

Indu

ced

6465 bp

pBR Rep

lac I

66

45

66

45

66

45

ileX glyT

proLleuW

argU

45

36

29

24

20

45

36

29

24

20

45

36

29

24

2020

14

6

20

14

6

20

14

6

The heme-binding domain of mouse cytochrome b5 has a red color allowing has a red color allowing visualization of expression.

Generation of Leaker StrainsGeneration of Leaker StrainsGeneration of Leaker StrainsGeneration of Leaker Strains

Phosphate binding protein productionPhosphate binding protein productionPhosphate binding protein productionPhosphate binding protein production

Non-leaker LeakerNon-leaker LeakerPB

P

PBP

Shoc

k

Shoc

k

bioreactor output(increasing time)

bioreactor output(increasing time)

Furlong and Sundstrom, J. Indus. Microbio. 30: 141-148

Protein purification systemProtein purification system

Pump 1

n 1

n 2

Fil 1

Pump 2 Col

umn

Col

umn

r 1

Filter 1

Fermentor

Sens

or

enso

r 2

Pump 3Fermentor S

Pump 4

Filter 2

Product OutWastePump 4 Product OutWaste

ConclusionConclusionThe portable SPR sensing system can The portable SPR sensing system can provide near realprovide near real--time monitoring for time monitoring for many different applications in many different applications in environmental monitoring, the food environmental monitoring, the food industry, pharmaceutical industry, medical industry, pharmaceutical industry, medical diagnostics and general research needs. diagnostics and general research needs.

SPR Team & SponsorsSPR Team & Sponsors•• Medical Genetics/Genome SciencesMedical Genetics/Genome Sciences

Dr. Clement FurlongDr. Clement FurlongScott Soelberg Scott Soelberg Dr. Gary GeissDr. Gary GeissDr Rick StevensDr Rick Stevens

•• Electrical Engineering Group:Electrical Engineering Group:Dr. Sinclair YeeDr. Sinclair YeeTim ChinowskyTim ChinowskyPeter KauffmanPeter KauffmanJared TritzJared TritzMichael GrowMichael GrowDr. Rick Stevens Dr. Rick Stevens

Steve NearSteve NearMatthew Probert Matthew Probert Joshua ProbertJoshua ProbertPeter KaufmannPeter Kaufmann

Michael GrowMichael GrowTony MactutisTony Mactutis

•• Texas Instruments: Texas Instruments: Jose MelendezJose MelendezJerry Elkind Jerry Elkind

•• NOAA TeamNOAA TeamDr. Vera TrainerDr. Vera TrainerDr. Jack Weckel Dr. Jack Weckel BB--TL EberhartTL EberhartS SpencerS Spencer

yyDwight Bartholomew Dwight Bartholomew John QuinnJohn Quinn

•• Seattle Sensor SystemsSeattle Sensor SystemsNathaneal SwansonNathaneal SwansonDr. Paul BakerDr. Paul BakerS SpencerS Spencer Dr. Paul BakerDr. Paul Baker

Financial Interests:Financial Interests:Sponsors:Sponsors: CEF SDS PK & PB holdSponsors:Sponsors: CEF, SDS, PK & PB hold

DOD stock in Seattle Sensor SystemsTexas Instruments Center for Process Analytical Chemistry (CPAC), UW, SeattleCenter for Process Analytical Chemistry (CPAC), UW, SeattleWashington State Sea Grant NSF/NIEHS NW Center for Human Health and Ocean Studies

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