Cavitands, Liquid Crystals and Antibodies: Materials for Integrated Chem/Bio. Sensing

Devanand K. Shenoy (PI), Elias Feresenbet, George Anderson and @Enrico Dalcanale

Center for Bio/Molecular Science and EngineeringNaval Research Laboratory

Washington, DC 20375

@ Department of Organic and Industrial ChemistryUniversity of Parma, Italy

11/17/04

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4. TITLE AND SUBTITLE Cavitands, Liquid Crystals and Antibodies: Materials for IntegratedChem/Bio. Sensing

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Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18

Outline

Technology Gaps

Goal, approach

Cavitands for chemical vapor sensing

Liquid Crystals for sensitivity amplification

Label-free antibodies for SPR biosensing

Conclusions

Technology Gaps

Chemical detectors - limited sensitivity - still use canines for bomb detection- high false alarm rates – limited specificity- slow response time – due to sensing material used

Biological Detectors- need reagents, labels, not real time

Separate Detectors- for chem. and bio. threats increase logistical burden/cost

Goal

Develop an integrated SPR chem/bio detector with

False Alarm Rate < 10-3 for chem. and < 10-8 for bio.Sensitivity – below permissible exposure level (PEL) for chem.

- LOD of 100 organisms/liter of air for bacteria/viruses- LOD of 10 nanograms/liter of air for toxins

Response time - < 1 minute for chem. and < 10 min for bio.

Military and civilian need

Chem/Bio detectors with high sensitivity, specificity, stability, and speed in portable format for airborne threats

Technique - SPR spectroscopy

Inputfiber

Outputfiber

Collimatinglens

Polarizer

Prism

Focusinglens

LC layerGold layerSubstrate

FlowcellSensitive optical transduction

technique – part in 10^7 refractive index changes can be measured

AuCrCover glassSubstrate for SPR

oo o

o o o oo

S S S S

oo o

o o o oo

S S S S

oo o

o o o oo

S S S S

oo o

o o o oo

S S S S

Cavitand sensing layer

Sensing with - Cavitands for chem. threats

- Liquid Crystals for chem. threats

- Antibodies for bio. threats

Approach

oo o

o o o oo

S S S S

Integration of chem/bio.

Common optical and data processing platformDifferent front ends for chem. and bio. samplesIsolated channels for chem. and bio. sensing layers

Cavitand 1 layer

Liquid crystal layer

Antibody

Cavitand 2 layer

Top View of SPR substrate

Chem. analyte

Bio. analyte Sensing layers separated

Different front ends

Sensor Components: TI Spreeta 2000 (3-channel)

Spreeta 2000 SPR components developed in collaboration with UWMiniaturized, robust, high performance devices Inexpensive: ~$4 in large quantityExcellent manufacturing capabilities and quality control.

Spreeta 2000 SPR sensing chip

CBW Agents Detectable with SPR

Antibodies tested atECBC

50 nM (15 ppb)750 pM (271 ppt)~1 uM (340 ppb)

VXSomanSarintabunDPMP(stimulantDomoateCortisolDNP

CW (organics)

YesNot yet doneYes

In progress~5x104 cfu/ml (prelim)In progress

Y. pestisF. tularensisE. Coli

Microbial cells

Yes (prelim)~109 pfu (prelim expts)

Small pox, Marburg, Ebola, Encephalitis, Hemorrhagic fever Flu (as a model system)

Viruses

Not yet done~105 cfu/ml (prelim)B. Anthracis, BG spores (simulant)

Spores

Yes (2.8ppt)Not yet doneYes

100 pM (2.8 ppb)20 nM (64 ppb; current level)

Cavitand Selectivity

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

MeCav PzCav QxCav PIB PECHSensing Layer

∆θ

p, D

egre

e

TOLUENE

Cavitand with deepest cavity shows largest response

Selectivity for aromatic vapors confirmed

PECH and PIB polymers for comparison

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

MeCav PzCav QxCav PIB PECH

Sensing layers

∆θ

P, D

egre

e

BENZENE

Feresenbet, E.; Dalcanale, E.; Dulcey, C.; Shenoy, D.K.,Sensors and Actuators B, 97, 211 (2003)

100 ppm

100 ppm

A B

C

polymers

A B C

A B

Cpolymers

R RRR

NN

OOO OOO O O

N NN NN N

O

R RRR

OO OOO O O

R RRR

NN

OOO OOO O O

N NN NN N

8.3 Å

4.6 Å

3.3 Å

MeCav PzCav QxCav

0

0.1

0.2

0.3

0.4

0.5

0 200 400 600Benzene, ppm

∆θp

, Deg

rees

Concentration Dependence of Signal

Saturation due to analyte “filling” of cavitands

QxCav completely encloses benzene molecule within cavity

QxCav-Benzene complex

0

0.005

0.01

0.015

0.02

0.025

0.03

Perch

loroe

thylen

e

Tolue

neNi

trome

thane

Aceto

nitrile

Benz

ene

n-Hex

ane

Analyte Vapors

∆θP

(arb

. uni

ts)

MeCavMeCav-thioether

MeCavMeCav-thioether

Devanand K. Shenoy, Elias B. Feresenbet, Roberta Pinalli, Enrico Dalcanale;Langmuir 2003, 19, 10454

Spin coated Self-assembled

Materials Processing:Spin Coated vs Self-Assembled Cavitands

Cavitand Morphology does not effect signal response

Signal response normalized for thickness

13 14 15 16 17

704.76

704.78

704.80

704.82

704.84

704.86

Res

onan

cew

avel

engt

h, n

m

Time. min.

COOH OUT

Cavitands for DMMP

OO

CH2Cl

CH2Cl

Cavitand shows high selectivity for DMMP

Cavitand shows good reversibility

O

O

O

O CF3

CF3

CF3

CF3

HO

CF3

CF3

CF3

CF3

HO

9 12 15 18 21

659.70

659.72

659.74

659.76

659.78

659.80

Res

onan

ce

Wav

elen

gth,

nm

Time, min.

FPOL

7 8 9 10

650.42

650.44

650.46

650.48

650.50

650.52

Res

onan

ce

Wav

elen

gth,

nm

Time, min.

PECH

1 2 3 4 5

713.24

713.26

713.28

713.30

713.32

713.34

Res

onan

ce

wav

elen

gth,

nm

Time, min.

MHDA

0

0.02

0.04

0.06

0.08

0.1

0.12

FPOL PECH MHDA CAVITAND

Sensing Layers

Resonance wavelength

shift, nm

2 ppb

Polymer Sensing layer

Liquid CrystalSensing layer

Liquid Crystal-based Sensing

Analyte

Local perturbation Global perturbation

Liquid Crystal (LC) perturbation causes signal amplification

Nematic order probed by optical method

Optical signal directly proportional to amount of vapor

One side Adhesive Mylar punch

Liquid crystalDrop (1ul)

LC film deposition

Cover Glass

Polyimidecoated on gold

5mm

100um

Liquid crystal molecule

LC alignment

Cover glass

PI2556

5CB liquid crystal

Gold (Polyimide)

A uniform planar orientation achieved

A B

LC exposure to benzene vapors

Wavelength shift due to vapor exposure and resulting decrease in LC order

Shift is reversible

E. Feresenbet, F. Taylor, T. M. Chinowsky. S. S. Yee, D.K Shenoy, Sensor Letters 2, 145-152, 2004

0 20 40 60

623.4

623.6

623.8

624.0

624.2

624.4

624.6

624.8

vapor on

vapor off / purge

Res

onan

ce w

avel

engt

h, n

m

Time, min.

0 5 10 15 20

623.58

623.60

623.62

623.64

vapor on

vapor off / purge

Res

onan

ce w

avel

engt

h, n

m

Time, min.

0 1500 3000 4500 6000 7500

623.7

624.0

624.3

624.6

624.9

Res

onan

ce

Wav

elen

gth,

nm

Concentration, ppm

Selectivity pattern of LC towards vapors

LC shows differential response to chemical vapors

Selectivity between isomers observed

-0.04

-0.02

0

0.02

0.04

0.06

0.08

Benzene Toluene m-Xylene p-Xylene Acetonitril Ethylacetate

Analyte Vapors

Res

onan

ce W

avel

engt

h Sh

ift (n

m)

Sensivity enhanced by two orders closer to phase transition

Shift is reversible

LC exposure closer to phase transition

8504

6803

3802

2701

ppb

60 80 100 120 140 160 180 200 220

683.6

683.8

684.0

684.2

684.4

684.6

684.8

4321

Vapor on

Vapor off / purge

Res

onan

ce

Wav

elen

gth,

nm

Time, min.

nematic isotropic35.1°c

m-xylene

623.59

623.595

623.6

623.605

623.61

623.615

623.62

623.625

0 0.1 0.2 0.3 0.4 0.5 0.6

Time, min

Res

onan

ce w

avel

engt

h, n

m

626.52

626.53

626.54

626.55

626.56

626.57

626.58

626.59

626.6

626.61

0 0.5 1 1.5 2 2.5 3

Time, min

Res

onan

ce w

avel

engt

h, n

m

Second order kinetics describes data for vapor diffusion into LC

Time response additional selectivity parameter

Kinetics of Response

m-Xylene0.0012 (p/ps)

Benzene0.0012 (p/ps)

dλ/dt =kλ2λ = resonance wavelength (nm)t = time (min)k = rate constant

E. B. Feresenbet, F. Taylor, T. M. Chinowsky, S. S. Yee, and D. K. Shenoy, Sensor Letters, 2, 145-152, 2004

-1

0

1

2

3

4

5

6

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

Time (min)

SPR

Shi

ft (n

m)

Biotin-Blocked NeutrAvidinNeutrAvidin

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0.0 1.0 2.0 3.0 3.9 4.9 5.9 6.9 7.9 8.9 9.8

Time (min)

SPR

Shi

ft (n

m)

Biotin-blocked NeutrAvidinNeutrAvidin

NeutrAvidin blocked by biotin bindspoorly to the thiol biotin surface

Subsequent binding of the biotinylated antibody is also poor

• Neutravidin-Biotin mediated antibody immobilizationEffect of blocking binding to

thiol-biotin coated gold surfaceEffect on binding of biotinylated

antibody

SPR Biosensing

0

1

2

3

4

5

6

7

8

0.01

0.91

1.81

2.71

3.61

4.51

5.41

6.31

7.21

8.11

9.01

9.91

10.8

11.7

12.6

13.5

14.4

15.3

16.2

Time (min)

SPR

Shi

ft (n

m)

Reused Thiol-Biotin Treated Chip

Reused gold Chip

Re-cycling the sensor surface

Neutravidin binds as well after cleaning of the gold surface and re-application of the thiol biotin layer

Neutravidin binding to different sensor surfaces

Bt-Rabbit anti-Goat IgG binding Goat-IgG

-0.5

0

0.5

1

1.5

2

2.5

0.0

1.1

2.2

3.4

4.5

5.6

6.7

7.8

8.9

10.1

11.2

Time (min)

SPR

Shi

ft (n

m)

0.01 0.1 1.0 10 ug/ml

Amplifier Donkey anti-Goat IgG binding

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0

0.5

0.9

1.4

1.9

2.3

2.8

3.3

3.7

4.2

4.7

Time (min)

SPR

Shi

ft (n

m)

0 0.01 0.1 1.0 10 ug/ml

SPR model immunoassay

SEB Direct Detection

-0.10

0.10.20.30.4

0.50.60.7

0.80.9

0.0 2.0 4.0 6.0 8.0 10.0 12.0Time (min)

SPR

Shi

ft (n

m)

2.5 ng/ml

25 ng/ml

100 ng/ml

1000 ng/ml

Amplifier Sheep anti-SEB Binding (10 ug/ml)

0

0.5

1

1.5

2

2.5

3

3.5

4

0.0

0.5

0.9

1.4

1.9

2.3

2.8

3.3

3.7

4.2

4.7

5.1

5.6

Time (min)SP

R S

hift

(nm

)

02.5251001000 ng/ml SEB

-0.05

0

0.05

0.1

0.15

0.2

0.0

0.5

0.9

1.4

1.9

2.3

2.8

3.3

3.7

4.2

4.7

5.1

5.6

Time

SPR

Shi

ft (n

m) 0

2.5 ng/ml25 ng/ml

Staphylococcal Enterotoxin B detection by SPR

Amplified Detection

Direct Detection of Ricin

-0.3

-0.1

0.1

0.3

0.5

0.7

0.9

0.0

0.8

1.7

2.5

3.3

4.2

5.0

5.8

6.7

7.5

8.3

9.2

10.0

10.8

11.7

Time (min)

SPR

shi

ft (n

m)

0 ng/ml10 ng/ml100 ng/ml1000 ng/ml

Ab-Amplified Detection of Ricin

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2

Time (min)SP

R s

hift

(nm

)

0 ng/ml10 ng/ml100 ng/ml1000 ng/ml

Ricin detection by SPR

Ab-Amplifed Detection of B. globigii

-0.5

0

0.5

1

1.5

2

2.5

3

0.0 0.5 0.9 1.3 1.7 2.1 2.6 3.0 3.4 3.8 4.2 4.6

Time (min)SP

R s

hift

(nm

)

0 spores/ml10^5 spores/ml10^6 spores/ml10^7 spores/ml

Direct detection of B. globigii

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

0.0

1.0

2.0

3.0

4.0

5.0

6.0

6.9

7.9

8.9

9.9

10.9

11.9

12.8

13.8

Time (min)

SPR

shi

ft (n

m)

0 spores/ml

10^5 spores/ml

10^6 spores/ml

10^7 spores/ml

Bacterial spore detection by SPR

Conclusions

Cavitands show high selectivity for chemical vapors Cavitand film morphology does not affect signal responseLiquid Crystal materials for sensitivity amplificationLabel-free, real-time SPR biosensingIntegration of chem. and bio. approaches appears feasible

Mr. Marco Busi, U. Parma, Italy

Prof. Enrico Dalcanale, U. Parma, Italy

Mr. Francis Taylor, U. Washington, Seattle

Prof. Tim Chinowsky, U. Washington, Seattle

Prof. Sinclair Yee, U. Washington, Seattle

Dr. T. Yu, UC Berkeley, CA

Dr. Francois Lagugne, UC Berkeley, CA

Prof. Y. Ron Shen, UC Berkeley, CA

Dr. Elias Feresenbet, formerly at NRL, Washington, DC

Dr. Charles Dulcey, NRL, Washington, DC

Dr. George Anderson, NRL, Washington, DC

Dr. William Barger, NRL, Washington, DC

Team Members

Funding: Naval Research Laboratory, Joint Services