Post on 09-Jun-2015
Food Borne Pathogen Analysis bySurface-Enhanced Raman Spectroscopy
Atanu Sengupta, Chetan Shende, Hermes Huang,Stuart Farquharson and Frank Inscore
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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
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The Need/Problem3
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)
The GoalDetect 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!)
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The Goal: FeasibilitySERS? Culture Growth
Salmonella
Listeria
Culture Growth /PCR is measured after the stationary phase is reached.
Goal, can SERS be used to detect cells long before the stationary phase. . . within 2 to 8 hours if possible.
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hνTransmitted
Absorbed
Scattered
Rayleigh
Raman
HH
H H
H H
Raman
How it works: Raman
(IR)
Laser light directed at a chemical generates Raman light.
o hνvib
hνvib
hνo hνscat
vib0
vib1
virt
hνscat
Light Chemical
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How it Works: SERS
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
30-80 nm diameter
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Laser
Grating
CCD
Filter
Sample
Bin Columns
Grating acts like a prism separating light into component colorsCCD is just like a digital camera
500 750 1000 1250 1500 1750Raman Shift (cm-1)
O
3+ NH CH
2
_
CH
COPhenylalanine
Ram
an In
tens
ity
How it Works: Instrument8
3.4x5x10”, 5 pounds
How it Works: RTA’s SERS-ID AnalyzerA Portable, Field Usable Analyzer
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The Solution: SERSSpecificity: 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.
Raman: Pure
Dipicolinic Acid
SERS: 1 ppm Dipicolinic Acid
Dipicolinic Acid
Farquharson, Maksymiuk & Inscore
Appl Spec, 58, 351 (2004)
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SERS-Active Substrates: Benzenthiol
10-3M
10-5M
10-8M(~10 ppb)
102
104
107
Concentration EnhancementFactor
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How it Works: RTA SERS Sampling Systems
Metal Particle
Sol-Gel Matrix
AdsorbedMolecules
Moleculesin Solution
Laser
RamanScattering
2001: Simple SERS Sample Vials
2001
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2004: SERS-Active Capillary2007: SERS LOCs
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 AnalysisThe 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 CaptureMeasure 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
Target SpecificMolecular
Recognition Elements
Ag Nanoparticles
Sol-Gel Layer
Glass Surface
Pathogens
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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-).
L. monocytogenes
Gold
Silver
SERS of and 109 cfu/mL L. monocytogenesusing gold-doped and silver-doped sol-gels. Spectral Conditions: 80 mW of 785 nm laser excitation, 1 minute acquisition.
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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
Salmonella
MRE2 on Gold
SERS of MRE2 functionalized gold for Listeria and Salmonella.
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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
Salmonella
MRE2 on Gold
Listeria
Salmonella
SERS of 109 cfu/mL L. monocytogenes and S. typhimurium using MRE1 functionalized 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.
SERS of L. monocytogenes and S. typhimurium measured on Listeria assay using MRE1 functionalized silver.
105 cfu/mL Salmonella
109 cfu/mL Listeria
105 cfu/mL Listeria
109 cfu/mL Salmonella
SERS of S. typhimurium and L. monocytogenes measured on Salmonella assay using MRE1 functionalized silver.
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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
Salmonella
SERS of and 105 cfu/mL (300 cells) L. monocytogenes and S. typhimurium using MRE1 functionalized gold.
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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 CaptureSurface-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).
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The Results: Technology ComparisonRTA-2012 Culture Growth
Salmonella
Listeria
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 growth for Salmonella and Listeria, respectively.
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Task 4 – Achieve Required Cell Detection.
Task 5 – Design and Build Lab-on-Chips.
Task 6 – Test Lab-on-Chips.
Future Work 23
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Mission:To provide superior chemical analyzers
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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, 2001Experience: >75 years of Raman, >40 years SERS, >40 years analyzer designProducts: RamanPro, RamanID, Portable Fuel Analyzer, Chemical Residue Analyzer, Simple SERS Sample Vials, SERS Capillaries, SERS Microplates
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