PittConn 2012 FI CS as Invited SERS Talks BA Assay

8/12/2019 PittConn 2012 FI CS as Invited SERS Talks BA Assay

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Providing Chemical Information When & Where You Need It

Pittcon 2012

RTA Booth

#2110

Frank E. Inscore, Atanu Sengupta, Chetan Shende

Mike Donahue, Hermes Huang,Stuart Farquharson

www.rta.biz

[email protected]

Detection of single-digitBacillus anthracis sporesin ~12-min by SERS

FundingNSF

DARPA

Materials and BSL-2/3 FacilitiesProf. Sperry (Chair Cell & Molecular Biology)

Center for Biotechnology and Life Sciences

University of Rhode Island


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The Overall Need/Problem

Detection of Bioagents Intentional Dispersal by Terrorist

Domestic/Military Targets e.g. weaponized aerosols, contamination of water & food supplies

B. anthracis, Y. pestis, F. tularensis, C. botulinum A,P. hanta

Detection of Waterborne Pathogens Unintentional Water Contamination

Domestic/Military

Cryptosporidium, Giardia, V. cholerae, Campylobacter jejuni

Detection of Foodborne Pathogens Unintentional Food Contamination

Domestic/Military

Listeria monocytogenes, E. coli (O157:H7), Salmonella enterica

Detection of Clinical Pathogens Unintentional Spread of Infections

Domestic/Military Hospitals

S. aureus (MERSA), Tuberculosis, AIDs

BA spores almost perfect bioagent.

Most likely to be employed by terrorist :

right size so can be inhaled most lethal route.

RTA is developing complete package toaddress each of these application areas.


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The Challenge & GoalDetect bioagents and pathogens

on surfaces, in aerosols, water, in biofluids, and food.(Category A agents 1st priority:Bacillus anthracis spores)

The device must provide the following:

Sensitivity: Detect 10,000 spores Anthrax LD50 ~10,000 spores (100 ng)

Speed: Within 15 minutes Specificity: Identify and discriminate pathogens

(No False Positives!)

Reproducibility: Accurate and Repeatable(No False Negatives!)

Current methods:

DNA or RNA enumeration: (Culture growth - 24 hours)

or Polymerase Chain Reactions (PCR, >4 hours)

Test kits (limited shelf life, very high false positive rate)


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The Solution: SERSInherent specificity: all chemicals (drugs/metabolites) produce a unique

Raman spectrum allowing unequivocal identification (no false-positives).

Improved sensitivity: Ag and Au nanoparticle substrates used to generateSERS amplify Raman signals (increase scattering efficiency) by 1 million times

or more with potential detection at sub-ppb, i.e. 10-8 M (no false-negatives).

Enhancement Factor ~ 105

SERS 1ppm DPA (aq)

RS 20,000 ppm DPA (aq)

RS 83,000 ppm DPA (KOH)

RS solid DPA

100 mW, 1-min

290 mW, 5-min

100 mW, 5-min

290 mW, 5-min

DPA (dipicolinic acid)

LMC = ~ 1 ppb

1ppm DPA

83,000 ppm DPA

SERS

RS

FT-R 785 nm


5/24

ALSO, chemical contribution

can provide additional 103

enhancement

Sub part-per-billion detection

becomes possible with SERS

SERS

provides

Single Molecule Detection

some argue this requires

enhancement factor (EF)

of 1012 -1014

Others say 105 -108

Surface-enhanced Raman Spectroscopy

Raman, although weak effect,

provides molecular specificity

BUT, when a molecule is within

a laser induced plasmon field,

the efficiency of Raman scattering

increases by 106 i.e. 1 million times!Sub part-per million detection possible

h

Plasmon Field

H

NH

H

H

H

N

NN

N

Ag

Surface-EnhancedRaman Photon


6/24

Commercial SERS Substrates:

Benzenethiol

10-3M

10-5M

10-8M(~10ppb)

benzenethiol

RTA: LMC ~10-11M (0.01 ng/mL or 10 ppt)

Conc. EF

102

104

107


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Approach: RTA SERS Patented Sampling Systemsprovide instant response in seconds as opposed to 30-min or more!

Metal Particle

Sol-Gel Matrix

AdsorbedMolecules

Moleculesin Solution

Laser

RamanScattering

2001: Simple SERS Sample Vials

1 10

2004: SER-Active Capillary

silver gold

2003: SERS Microplate

High Throughput Screening Extraction and Pre-Concentration

RTA Patents6,623,977; 6,943,031; 6,943,032; 7,312,088; 7,393,691; 7,393,692; 7,462,492; and 7,462,493

2005: SERS spin-coated Disk


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2006: Functionalized Sol-Gel SERS

(affords greater selectivity and sensitivity)

PC OTC

Std chromatographic media

Primary chemically selective SER-active sol-gel (SG) sub-sets developed at RTA.

Patents: 6623977, 6943031, 6943032, 7312088,7393691, 7393692, 7462492, 7462493, 7713914

Chem. Type Metal

cap/ 800 Analyte Selectivity M

SG1

SG2

polar - negative

non-polar - negative

Ag

silver

silver

SG3 more non-pol - neg. silver

SG6 very polar - neg. Ag

SG1-PDMS

SG2-PEG

SG2-PDMS

SG3-PDMS

less polar - neg.

less non-polar - neg.

more non-pol - neg.

very non-pol - neg.

Ag

silver

silver

silver

silver

SG4 very polar-positive Au


9/24

R&D: SERS Lab-On-ChipDifferent wafer, glass and plastic LOC designs


10/24

RTAs SERSID - Trace Chemical Analyzers for Field and Lab Use

2010

Providing Chemical Information When & Where You Need It

2011


11/24

Preliminary Analysis: Raman Spectroscopy

B. cereus

B. subtilis

B. anthracis

CaDPA

Core Wall(proteins-cysteine,

)Ca dipicolinateCortex

(peptidoglycan)

Exosporium

Spore Coat

DNA

RibosomesC C

O O

O ON

2-

Ca2+

J. Raman Spectrosc., 35, 82-86 (2004); Spectrosc., (2005)

Caveat: no consensus spectra in literature.Variability: growth/media conditions.

Limitations: sensitivity/sample issues.

Portable 1064 nm system: Chem ID, Hazmat & Hoax Material Analyzer

i i A i


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Preliminary Analysis: Surface-Enhanced Raman Spectroscopy

Appl Spec, 58, 351 (2004)

IJHSES, 20, 12-18 (2007)

US Patent: 7713914 B2

SPIE, 5585, 53-57 (2005)

BC in DDA (78C) ~2-min

BC in DDA (22C) ~60-min

BC in AA (22C) ~2-min

BC in 0.02M HNO3

(heat+sonicate) ~10-min

BC spores in nasal mucus

in AA

BC spores in saliva in AA

BC spores

0.03 mg/mL(aq)

80 mW 785nm 1-minBS 0.01 mg/mL (aq) 80 mW 785nm 1-min

BS 0.01 mg/mL (aq) 160 mW 785nm 1-min

* *


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Area is 0.2 mm x 0.2 mm

Depth is 0.1 mm

Volume = 0.004 mm3

= 4 nL

Microscope Cell Counting: Counting Grid

Average for 10 grids

= 87 spores

87 spores/4 nl

=21,725/microL

This Image = 60 spores

Microscope Image: Quantifying Spores in Counting Chamber

Diluted by 10

=2200 spores/microL

IJHSES, 20, 12-18 (2007)Bioterrorism, S. Morse, Ed., ISBN 978-953-307-636-2 (2012)

220 spores in actual volume measured


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SERS: sensitivity great! 220 Spores detected ~2-min!

100 pg/microL (ppb)

DPA in AA reference spectrum

220 BC spores with AA on SG1PDMS

1 spore = 10 pg, DPA =10% spore weight

Internal AA

Reference

IJHSES, 20, 12-18 (2007)

*

*


15/24

Problem: need both high specificity & sensitivity

10^9 spores/mL + AA

BC

BS

BAS

RS

DPA (s)

Na2DPA (s)

CaDPA (s)

SERS 1 mg/mL

DPA (aq)

Na2DPA (aq)

CaDPA (aq)

16


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The Proposal: SERS-Active Capture Assay

Incorporate Molecular Recognition Elements for Specificity

Target Specific

Molecular

Recognition

Elements

Ag Nanoparticles

Sol-Gel Layer

Glass Surface

Pathogens

16

Dipicolinic acid25 ppm B.

cereus

cysS-Ag

No DPAsignature!

250 ppm

B. subtilis

spectrum same as before

adding BS

Patent Application: (2011)

Proof of Concept:

NSF (July 2008)IJAC(March 2012)SPIE (March 2012)


17/24

Peptide-Functionalized Silver Sol-gel

Ag nanoparticles immobilized within porous sol-gel in a glass capillary were successfully functionalized

with a short peptide that specifically bindsBacillus anthracis spores.

Peptide functionalization and spore binding are verified by SERS. The functionalized spectrum dominated by sulfur of the pep-cys-Ag bond (660 cm-1 ).

SERS ofBA specific peptide

cys-Ag

Cysteine-Ag

Peptide

Ag

Sol-Gel

Glass Capillary Wall

wash

pictures are not to scale

pep-cys-Ag

tf= 0.5-72 hrs

Use Immediately or Seal (stable >6-months)functionalize


18/24

Assay: Spore Capture: Incubation and WashA 5-step SERS assay was successfully developed.

Peptide

Ag

Sol-Gel

Glass Capillary Wall

Spore

1) Draw 10 L sample into a peptide functionalized capillary, wait 5-15 minutes.

2) Perform washes to minimize non-specific interactions (40 sec)

3) Treat sol-gel capture matrix with new proprietary reagent wash (10-sec)

4) Perform additional treatment using AA wash (10-sec)

sol-gel & spore treatmentscleaning washes

washes applied after spore incubation:

minimize non-specific interactions within capture matrix

while

treatments amplify spore signal via DPA enhancement

incubation


19/24

5) Place capillary in Raman analyzer, measure spectrum (1 minute).

Spore Detection & Sensitivity: SERSA 5-step SERS assay for BA was successfully developed.

(also similar BC and BS assays)

Sensitivity:

10 spore spectrum!

NO False Negatives!

1/100th of 1000 spores/mL sample is in capillary

measure

t = 60 secondsTotal Assay: 7-17 min

depends on spore

incubation time

(5-15 min)


20/24

Spore Assay SpecificityA 5-step SERS assay for BA was successfully developed.

1/100th of 10000 BA spores/mL sample is in capillary

Selectivity:

BA 100 spore assay challenged:

10-100X [higher] BC, BM, & BS.

NO False Positives!

BA-S BCBMBS


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Reproducibility Assessed: ROC Curve Analysis


22/24

Reproducibility Assessed: ROC Curve Analysis

15-min incubation K=4.8

5-min incubation K=2.3

50/50 probability line

Concentration Number of

Capillaries

Mean 1007

Peak Height

Standard

Deviation

Mean Standard

Deviation ()

K Value

Blank (BC,BM,BS) 90.1

0.02 0.18

104 BAS (5-min) 12 0.52 0.4 2.3

104 BAS (15-min) 3 0.96 0.12 4.8

Goal is a 95% confidence level (bioagent concentration that can be detected 95% of time).

Determine K-value (indicates a statistically significant separation between a true and false response).

K > 3.29 meet Armys requirement (of 95% probability of detection & 5% probability of false alarm).

Since some non-specificity of other Bacillus spores with sol-gel,use of BC blank as opposed to water blank is more realistic!


23/24


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Executive Summary

Proposed device (SERS analyzer/BA assay) provides the following:

Sensitivity: Detect < 10,000 BA spores (as low as 10 BA-S spores)

Speed: Less than 15 minutes (within 12 minutes)

Specificity: Identify and discriminate BA (against BC, BS, BM)(No False Positives!)

Reproducibility: Accurate and Repeatable (95% confidence 100 spores)(No False Negatives!)

Possible assay time = 8.5-min + 40-sec + 20-sec + 30-sec = 10-min!

Complete publication for BA assay (ames and sterne) pending (2012).

Assay validation at US Army Edgewood facilities (June 2012)

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PittConn 2012 FI CS as Invited SERS Talks BA Assay

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