1. Detection of single-digit Bacillus anthracis spores
in ~12-min by SERS
Frank E. Inscore, Atanu Sengupta, Chetan Shende
Mike Donahue, Hermes Huang,
Stuart Farquharson
Funding
NSF
DARPA
Materials and BSL-2/3 Facilities
Prof. Sperry (Chair Cell & Molecular Biology)
Center for Biotechnology and Life Sciences
University of Rhode Island
Pittcon 2012
www.rta.biz RTA Booth
inscore@rta.biz Providing Chemical Information When & Where You Need It #2110

2. The Overall Need/Problem
Detection of Bioagents BA spores –almost perfect bioagent.
Most likely to be employed by terrorist :
• Intentional Dispersal by Terrorist right size so can be inhaled – most lethal route.
• 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
RTA is developing complete package to
address each of these application areas.

3. The Challenge & Goal
Detect bioagents and pathogens
on surfaces, in aerosols, water, in biofluids, and food.
(Category A agents 1 st 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)

4. The Solution: SERS
Inherent 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).
100 mW, 1-min SERS 1ppm DPA (aq)
SERS
1ppm DPA
290 mW, 5-min
RS 20,000 ppm DPA (aq)
DPA (dipicolinic acid)
100 mW, 5-min RS 83,000 ppm DPA (KOH) LMC = ~ 1 ppb
RS
290 mW, 5-min 83,000 ppm DPA
RS solid DPA
Enhancement Factor ~ 105
FT-R 785 nm

5. Surface-enhanced Raman Spectroscopy
Raman, although weak effect,
provides molecular specificity
hν SERS
BUT, when a molecule is within Ag
provides
a laser induced plasmon field, H
N
Single Molecule Detection
H N
N
the efficiency of Raman scattering
H
N N H
H
some argue this requires
increase’s by 10 6 i.e. 1 million times! Plasmon Field
Surface-Enhanced enhancement factor (EF)
Sub part-per million detection possible Raman Photon of 1012 -1014
ALSO, chemical contribution
3 Others say 105 -108
can provide additional 10
enhancement
Sub part-per-billion detection
becomes possible with SERS

6. Commercial SERS Substrates:
Benzenethiol
benzenethiol Conc. EF
10-3M 102
10-5M 104
10-8M 107
(~10ppb)
RTA: LMC ~10-11M (0.01 ng/mL or 10 ppt)

7. Approach: RTA SERS Patented Sampling Systems
provide instant response in seconds as opposed to 30-min or more!
2001: Simple SERS Sample Vials
Molecules Sol-Gel Matrix
Raman
in Solution
Scattering
Laser
Adsorbed
silver gold Molecules Metal Particle
2003: SERS Microplate 2004: SER-Active Capillary 2005: SERS spin-coated Disk
1 10
High Throughput Screening Extraction and Pre-Concentration
RTA 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

8. 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.
Chem. Type Metal
cap/ 800 µ Analyte Selectivity M
Ag
SG1 polar - negative silver
SG2 non-polar - negative silver
SG3 more non-pol - neg. silver
SG6 very polar - neg. Ag
Ag
SG1-PDMS less polar - neg. silver
SG2-PEG less non-polar - neg. silver
SG2-PDMS more non-pol - neg. silver
SG3-PDMS very non-pol - neg. silver
SG4 very polar-positive Au
Patents: 6623977, 6943031, 6943032, 7312088,
7393691, 7393692, 7462492, 7462493, 7713914

9. R&D: SERS Lab-On-Chip
Different wafer, glass and plastic LOC designs

10. RTA’s SERSID - Trace Chemical Analyzer’s for Field and Lab Use
2010
2011
Providing Chemical Information When & Where You Need It

11. Preliminary Analysis: Raman Spectroscopy
B. cereus
B. subtilis Core Wall
Cortex (proteins-cysteine,
B. anthracis (peptidoglycan) Ca dipicolinate)
DNA 2-
Ribosomes O O
Ca 2+
O C N C O
CaDPA Spore Coat
Exosporium
Portable 1064 nm system: Chem ID, Hazmat & Hoax Material Analyzer
Caveat: no consensus spectra in literature.
Variability: growth/media conditions.
Limitations: sensitivity/sample issues.
J. Raman Spectrosc., 35, 82-86 (2004); Spectrosc., (2005)

12. Preliminary Analysis: Surface-Enhanced Raman Spectroscopy
BC spores
0.03 mg/mL(aq) BS 0.01 mg/mL (aq) 80 mW 785nm 1-min
80 mW 785nm 1-min
BS 0.01 mg/mL (aq) 160 mW 785nm 1-min
Appl Spec, 58, 351 (2004)
BC in DDA (78C) ~2-min BC in AA (22C) ~2-min
* *
BC in DDA (22C) ~60-min
BC in 0.02M HNO3
(heat+sonicate) ~10-min
BC spores in nasal mucus IJHSES, 20, 12-18 (2007)
in AA
BC spores in saliva in AA US Patent: 7713914 B2
SPIE, 5585, 53-57 (2005)

13. Microscope Image: Quantifying Spores in Counting Chamber
Microscope Cell Counting: Counting Grid
This Image = 60 spores
Average for 10 grids
= 87 spores Diluted by 10
Area is 0.2 mm x 0.2 mm
Depth is 0.1 mm 87 spores/4 nl =2200 spores/microL
Volume = 0.004 mm3 =21,725/microL
= 4 nL 220 spores in actual volume measured
IJHSES, 20, 12-18 (2007)
Bioterrorism, S. Morse, Ed., ISBN 978-953-307-636-2 (2012)

14. SERS: sensitivity great! 220 Spores detected ~2-min!
1 spore = 10 pg, DPA =10% spore weight
220 BC spores with AA on SG1PDMS
100 pg/microL (ppb)
* DPA in AA reference spectrum
Internal AA
*
Reference
IJHSES, 20, 12-18 (2007)

15. Problem: need both high specificity & sensitivity
10^9 spores/mL + AA
BC
BS
BAS
RS SERS 1 mg/mL
DPA (s) DPA (aq)
Na2DPA (aq)
Na2DPA (s)
CaDPA (aq)
CaDPA (s)

16. 16
The Proposal: SERS-Active Capture Assay
Incorporate Molecular Recognition Elements for Specificity
Pathogens
Target Specific
Molecular
Recognition Proof of Concept:
Elements
Ag Nanoparticles NSF (July 2008)
Sol-Gel Layer IJAC (March 2012)
Glass Surface
SPIE (March 2012)
Dipicolinic acid
25 ppm B.
cereus
Patent Application: (2011)
cysS-Ag 250 ppm
B. subtilis
No DPA spectrum same as before
signature! adding BS

17. 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 ).
tf = 0.5-72 hrs
functionalize wash Use Immediately or Seal (stable >6-months)
SERS of
BA specific peptide
pep-cys-Ag
Peptide
cys-Ag
Cysteine-Ag
Ag
Sol-Gel
Glass Capillary Wall
pictures are not to scale

18. Assay: Spore Capture: Incubation and Wash
A 5-step SERS assay was successfully developed.
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)
incubation cleaning washes sol-gel & spore treatments
Spore
washes applied after spore incubation:
minimize non-specific interactions within capture matrix
while
treatments amplify spore signal via DPA enhancement
Peptide
Ag
Sol-Gel
Glass Capillary Wall

19. Spore Detection & Sensitivity: SERS
A 5-step SERS assay for BA was successfully developed.
(also similar BC and BS assays)
5) Place capillary in Raman analyzer, measure spectrum (1 minute).
Sensitivity:
10 spore spectrum!
NO False Negatives!
measure
t = 60 seconds
Total Assay: 7-17 min
depends on spore
incubation time
(5-15 min)
1/100th of 1000 spores/mL sample is in capillary

20. Spore Assay Specificity
A 5-step SERS assay for BA was successfully developed.
Selectivity:
BA 100 spore assay challenged:
10-100X [higher] BC, BM, & BS.
NO False Positives!
BA-S BC
BM
BS
1/100th of 10000 BA spores/mL sample is in capillary

21. Spore Assay Repeatability
A 5-step SERS assay for BA was successfully developed.
Repeatability:
100 BA spores - 12 for 12
Capillaries!
No False Negatives!
1/100th of 10000 spores/mL sample is in capillary

22. Reproducibility Assessed: ROC Curve Analysis
15-min incubation K=4.8
5-min incubation K=2.3
50/50 probability line
Concentration Number of Mean 1007 Standard Mean Standard K Value
Capillaries Peak Height Deviation Deviation (σ)
Blank (BC,BM,BS) 9 0.02 0.18
0.1
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 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!

23. BA Assay Goal: IMPROVE PERFORMANCE
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).
ACCOMPLISHED!
K = 3.43 New Silver Sol-Gel Chemistry
New Additional Post-Functionalization Wash
New Spore Incubation Time 10-min @95% Confidence
t = 10-min + 40-sec + 20-sec + 60-sec = 12-min
Concentration Number of Mean 1007 Standard Mean Standard K
Capillaries Peak Height Deviation Deviation (σ) Value
Blank (105 BC) 7 0.02 0.0161 0.047
104 BAS (10-min) 7 0.181 0.078 3.43
~12-min, Assay Specificity: 96%
No False Positives/Negatives
K = 4.19 Concentration Number of
Capillaries
Mean 1007
Peak Height
Standard
Deviation
Mean Standard
Deviation (σ)
K
Value
Blank (Water) 5 0.025 0.0064 0.037
104 BAS (10-min) 7 0.181 0.078 4.19
~12-min, Assay Specificity: 98.5%
This clearly indicates that the non-specific interaction of BC
with the BA assay substrate is around 2.5%. (∆=4%-1.5%)

24. 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)
Possible assay time = 8.5-min + 40-sec + 20-sec + 30-sec = 10-min!
• Specificity: Identify and discriminate BA (against BC, BS, BM)
(No False Positives!)
• Reproducibility: Accurate and Repeatable (95% confidence 100 spores)
(No False Negatives!)
Complete publication for BA assay (ames and sterne) pending (2012).
Assay validation at US Army Edgewood facilities (June 2012)