1. Food Borne Pathogen Analysis by
Surface-Enhanced Raman Spectroscopy
Atanu Sengupta, Chetan Shende, Hermes Huang,
Stuart Farquharson and Frank Inscore
www.rta.biz Visit our Booth
2. 2
Outline
The Need - Detection of Foodborne Pathogens
The Solution - Surface-Enhanced Raman Spectroscopy
• Basic Theory and Instrumentation
• Proposed Assay Concept
• Proposed Field Analysis
• Previous (Relevant) RTA Successes
The Results
3. 3
The Need/Problem
Detection of Foodborne Pathogens
• 76 million foodborne illnesses in the US/Year
• 325,000 hospitalization in the US/Year
• > 5000 deaths in the US/Year
• Cost US economy $4 Billion/Year
Examples
• 2010: Salmonella contaminated eggs
• 2009: Salmonella in peanut butter
• 2008: Salmonella in peppers
• 2007: E. coli in meat (Topps Meat Co. closes)
4. 4
The Goal
Detect Foodborne pathogens (Listeria and Salmonella)
on equipment surfaces and on/in food.
The device must provide the following:
• Sensitivity: Detect 1 cell (colony forming units)
per mg sample
• Speed: Within 2-3.5 hours
• Specificity: Identify and discriminate pathogens
(No False Positives!)
• Reproducibility: Accurate and Repeatable
(No False Negatives!)
5. 5
The Goal: Feasibility
SERS? Culture Growth
Culture Growth /PCR is measured
after the stationary phase is reached.
Goal, can SERS be used to detect
cells long before the stationary
Salmonella phase. . . within 2 to 8 hours if
possible.
Listeria
6. 6
How it works: Raman
Light Chemical
virt
H H Transmitted
hνo
H H hνvib
Absorbed (IR) hνo hνscat
H H
Raman
Raman
Scattered
hνscat vib1
Rayleigh hνvib
vib0
Laser light directed at a chemical generates Raman light.
7. 7
How it Works: SERS
30-80 nm diameter
When a molecule is within a plasmon field,
the efficiency of Raman scattering can increase by 1 million times!
Part-per billion detection becomes possible.
Single Molecule Detection:
requires 1012 - 1014
8. 8
How it Works: Instrument
Sample
Grating
Phenylalanine O
_
C O
Raman Intensity
+
Filter H3N CH
CH 2
Laser
CCD
500 750 1000 1250 1500 1750
Bin Columns Raman Shift (cm-1)
Grating acts like a prism separating light into component colors
CCD is just like a digital camera
9. 9
How it Works: RTA’s SERS-ID Analyzer
A Portable, Field Usable Analyzer
3.4x5x10”, 5 pounds
10. 10
The Solution: SERS
Specificity: Every chemical produces a unique Raman spectrum
allowing unequivocal identification.
Sensitivity: Silver and gold nanoparticles increase Raman signals
by 1 million times or more allowing < ppm detection.
SERS: Dipicolinic Acid
1 ppm Dipicolinic Acid
Raman:
Pure Farquharson,
Dipicolinic Acid Maksymiuk
& Inscore
Appl Spec, 58, 351 (2004)
12. 12
How it Works: RTA SERS Sampling Systems
2001: Simple SERS Sample Vials
Molecules Sol-Gel Matrix
Raman
in Solution
Scattering
2001
Laser
Adsorbed
Molecules Metal Particle
2007: SERS LOCs
2004: SERS-Active Capillary
1 10
RTA Patents: 6,623,977; 6,943,031&2, 7,312,088, 7,393,691&2, 7,462,492&3, 7,713,914
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The Proposal: The Analysis
The proposed SERS-FBPD will extract and identify
the presence of ~1-10 cells of Salmonella and Listeria on surfaces
in 2.5 and 3.5 hours from sample collection, respectively.
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The Proposal: Feasibility
Task 1 – Develop Pathogen Capture.
Attach molecular recognition elements (MREs) to gold and silver nanoparticles.
Task 2 – Demonstrate Pathogen Capture
Measure SERS of both Listeria monocytogenes and Salmonella typhimurium.
Task 3 – Determine Sensitivity & Selectivity
Measure 105 cfu/mL if possible.
Show selective and discriminative binding.
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The Proposal: SERS-Active Capture Assay
Pathogens
Target Specific
Molecular
Recognition
Elements
Ag Nanoparticles
Sol-Gel Layer
Glass Surface
16. 16
The Results: Task 1 – Develop Pathogen Capture.
1. Identify best SERS-active sol-gel for Pathogens.
Both silver-doped and gold-doped sol-gels produced surface-enhanced Raman spectra
for Listeria monocytogenes (G+) and Salmonella typhimurium (G-).
Gold
SERS of and 109 cfu/mL L. monocytogenes
using gold-doped and silver-doped sol-gels.
Spectral Conditions: 80 mW of 785 nm Silver
laser excitation, 1 minute acquisition.
L. monocytogenes
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The Results: Task 1 – Develop Pathogen Capture.
2. Functionalize best SERS-active sol-gels with Molecular Recognition Elements (MREs).
Two types of MREs were investigated for both pathogens. Initially, both MREs worked
better on gold. However, slight modifications improved the silver measurements.
3: Go/No Go: Do the MREs produce a signal?
Yes, weak, but unique spectral signatures
proved successful functionalization.
Listeria
SERS of MRE2 functionalized
Salmonella
gold for Listeria and Salmonella.
MRE2 on Gold
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The Results: Task 2 – Demonstrate Pathogen Capture.
4. Measure SERS of L. monocytogenes using MRE1 & 2 functionalized gold-doped sol-gels.
SERS were obtained for 107 and 109 cfu/mL L. monocytogenes using MRE1 & 2,
respectively.
5. Measure SERS of S. typhimurium using MRE1 & 2 functionalized gold-doped sol-gels.
SERS was obtained for 107 cfu/mL S. typhimurium using MRE1 only.
6: Go/No Go: Do L. monocytogenes and S. typhimurium produce SERS signals on their
respective assays at nominal concentrations? Yes, very good spectra were obtained for
both pathogens at 107 cfu/mL using MRE1.
Listeria
Listeria
SERS of 109 cfu/mL L. monocytogenes and
S. typhimurium using MRE1 functionalized Salmonella
Salmonella
gold.
MRE2 on Gold
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The Results: Task 3 – Determine Sensitivity & Selectivity.
7. Demonstrate selectivity by measuring both pathogens on each others assays.
Selective discrimination is at least 3-orders of magnitude. SERS was not obtained at 108
cfu/mL pathogen using the wrong assay, and only modest signals were obtained for 109
cfu/mL pathogen.
105 cfu/mL Listeria 105 cfu/mL Salmonella
109 cfu/mL Salmonella
109 cfu/mL Listeria
SERS of L. monocytogenes and SERS of S. typhimurium and
S. typhimurium measured on L. monocytogenes measured on
Listeria assay using MRE1 Salmonella assay using MRE1
functionalized silver. functionalized silver.
20. 20
The Results: Task 3 – Determine Sensitivity & Selectivity.
8. Measure SERS of L. monocytogenes and S. typhimurium to lowest concentration.
Exceptional surface-enhanced Raman spectra were obtained for both pathogens at
105 cfu/mL using MRE1 functionalized gold-doped and silver-doped sol-gels. This
concentration represents detection of ~ 103 cells in the measured 10 microL sample
volume (~300 cells within the focus of the laser). SERS using MRE2 were 2-3 orders of
magnitude less sensitive.
9: Go/No Go: Do L. monocytogenes and S. typhimurium produce SERS signals on their
respective assays at least as low as 105 cells/mL Yes, in fact both pathogens were
detected at 103 cells in the measured 10 microL sample volume.
Listeria
SERS of and 105 cfu/mL (300 cells) L.
monocytogenes and S. typhimurium using
MRE1 functionalized gold.
Salmonella
21. 21
The Results: Feasibility
Task 1 – Develop Pathogen Capture.
Two types of molecular recognition elements (MREs) for both the Listeria and
Salmonella genus were successfully attached to gold and silver nanoparticles.
Task 2 – Demonstrate Pathogen Capture
Surface-enhanced Raman spectra were obtained for both Listeria monocytogenes and
Salmonella typhimurium. The 109 cfu/mL samples were incubated in the sol-gel
capillaries for 45 minutes, washed, then measured in 1 minute.
Task 3 – Determine Sensitivity & Selectivity
Both pathogens could be detected at 105 cfu/mL, the equivalent of 300 cells within the
focus of the laser. This included a 2 minute centrifugation to concentrate the cells.
Discrimination was at least 3-orders of magnitude at this concentration (the non-
specific pathogen had to have a concentration of >108 cfu/mL to be detected).
22. 22
The Results: Technology Comparison
RTA-2012 Culture Growth
Culture Growth /PCR is measured after
the stationary phase is reached.
The SERS-FBPD will be measured after
1.5 and 2.5 hours of lag and log phase
Salmonella growth for Salmonella and Listeria,
respectively.
Listeria
23. 23
Future Work
Task 4 – Achieve Required Cell Detection.
Task 5 – Design and Build Lab-on-Chips.
Task 6 – Test Lab-on-Chips.
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Providing Chemical Information When & Where You Need It
Mission:
To provide superior chemical analyzers
(faster, portable, easy to use, rugged, more sensitive, less expensive)
To meet specific needs of
Department of Defense (Fuel Analysis, IED Identification)
Homeland Security (CWA, BWA, IED Identification)
Chemical Manufacturing Industry (Process Control)
Medical (Drugs, HIV, TB)
General Information:
Launched: September 1, 2001
Experience: >75 years of Raman, >40 years SERS, >40 years analyzer design
Products: RamanPro, RamanID, Portable Fuel Analyzer, Chemical Residue
Analyzer, Simple SERS Sample Vials, SERS Capillaries, SERS Microplates
