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

(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, 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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