cavitands, liquid crystals and antibodies: materials for integrated chem… · 2011. 5. 13. ·...
TRANSCRIPT
-
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
-
Report Documentation Page Form ApprovedOMB No. 0704-0188Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering andmaintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information,including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, ArlingtonVA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if itdoes not display a currently valid OMB control number.
1. REPORT DATE 17 NOV 2004
2. REPORT TYPE N/A
3. DATES COVERED -
4. TITLE AND SUBTITLE Cavitands, Liquid Crystals and Antibodies: Materials for IntegratedChem/Bio. Sensing
5a. CONTRACT NUMBER
5b. GRANT NUMBER
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Center for Bio/Molecular Science and Engineering Naval ResearchLaboratory Washington, DC 20375
8. PERFORMING ORGANIZATIONREPORT NUMBER
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
11. SPONSOR/MONITOR’S REPORT NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release, distribution unlimited
13. SUPPLEMENTARY NOTES See also ADM001849, 2004 Scientific Conference on Chemical and Biological Defense Research. Held inHunt Valley, Maryland on 15-17 November 2004 . , The original document contains color images.
14. ABSTRACT
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT
UU
18. NUMBEROF PAGES
27
19a. NAME OFRESPONSIBLE PERSON
a. REPORT unclassified
b. ABSTRACT unclassified
c. THIS PAGE unclassified
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