Providing Chemical Information When & Where You Need It

Pittcon 2012RTA Booth

#2110

Frank E. Inscore, Atanu Sengupta, Chetan Shende Mike Donahue, Hermes Huang,

Stuart Farquharson

www.rta.bizinscore@rta.biz

Detection of single-digit Bacillus anthracis spores in ~12-min by SERS

Funding NSF

DARPA

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

Center for Biotechnology and Life SciencesUniversity of Rhode Island

The Overall Need/ProblemDetection 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 to address each of these application areas.

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)

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 generate SERS 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

ALSO, chemical contribution can provide additional 103

enhancementSub 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 increase’s by 106 i.e. 1 million times!

Sub part-per million detection possible

h

Plasmon Field

ν

H

NH

H

H

H

N

NN

N

Ag

Surface-EnhancedRaman Photon

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

Approach: RTA SERS Patented Sampling Systems provide 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-ConcentrationRTA Patents

6,623,977; 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

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 µ Analyte Selectivity M

SG1 SG2

polar - negativenon-polar - negative

Agsilversilver

SG3 more non-pol - neg. silverSG6 very polar - neg. Ag

SG1-PDMSSG2-PEGSG2-PDMSSG3-PDMS

less polar - neg.less non-polar - neg.more non-pol - neg.very non-pol - neg.

Agsilversilversilversilver

SG4 very polar-positive Au

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

RTA’s SERSID - Trace Chemical Analyzer’s for Field and Lab Use2010

Providing Chemical Information When & Where You Need It

2011

Preliminary Analysis: Raman SpectroscopyB. cereus

B. subtilis

B. anthracis

CaDPA

Core Wall(proteins-cysteine,

)Ca dipicolinateCortex

(peptidoglycan)

Exosporium

Spore Coat

DNARibosomes

C CO O

O ON

2-

Ca 2+

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

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 mucusin AA

BC spores in saliva in AA

BC spores0.03 mg/mL(aq)

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

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

* *

Area is 0.2 mm x 0.2 mmDepth 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

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)

*

*

Problem: need both high specificity & sensitivity

10^9 spores/mL + AABC

BS

BAS

RSDPA (s)

Na2DPA (s)

CaDPA (s)

SERS 1 mg/mLDPA (aq)

Na2DPA (aq)

CaDPA (aq)

The Proposal: SERS-Active Capture AssayIncorporate Molecular Recognition Elements for Specificity

Target SpecificMolecular

Recognition Elements

Ag Nanoparticles

Sol-Gel Layer

Glass Surface

Pathogens

16

Dipicolinic acid25 ppm B.

cereus

cysS-Ag

No DPA signature!

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)

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 binds Bacillus 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

Cys

tein

e-A

g

Pept

ide

AgSol-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

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

Pept

ide

AgSol-Gel

Glass Capillary Wall

Spor

e

1) Draw 10 µL 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

whiletreatments amplify spore signal via DPA enhancement

incubation

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

measuret = 60 seconds

Total Assay: 7-17 mindepends on spore incubation time

(5-15 min)

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

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

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

Repeatability:100 BA spores - 12 for 12

Capillaries!No False Negatives!

Reproducibility Assessed: ROC Curve Analysis15-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 (σ)

K Value

Blank (BC,BM,BS) 9 0.1 0.02 0.18

104 BAS (5-min) 12 0.52 0.4 2.3104 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 Army’s 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!

BA Assay Goal: IMPROVE PERFORMANCE

ACCOMPLISHED!New Silver Sol-Gel Chemistry

New Additional Post-Functionalization Wash New Spore Incubation Time 10-min @95% Confidence

Concentration Number of Capillaries

Mean 1007 Peak Height

Standard Deviation

Mean Standard Deviation (σ)

K Value

Blank (105 BC) 7 0.02 0.0161 0.047104 BAS (10-min) 7 0.181 0.078 3.43

Concentration Number of Capillaries

Mean 1007 Peak Height

Standard Deviation

Mean Standard Deviation (σ)

K Value

Blank (Water) 5 0.025 0.0064 0.037104 BAS (10-min) 7 0.181 0.078 4.19

Specific goal of this ROC analysis: determine if modifications reduce spore incubation time from 15 to 10 min, without compromising sensitivity & specificity of the assay, i.e. detecting 10,000 BAS spores with 95% specificity (K value greater than 3.29).

~12-min, Assay Specificity: 98.5%

~12-min, Assay Specificity: 96%

This clearly indicates that the non-specific interaction of BC with the BA assay substrate is around 2.5%. (∆=4%-1.5%)

No False Positives/Negatives

t = 10-min + 40-sec + 20-sec + 60-sec = 12-min

K = 3.43

K = 4.19

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)