Food Borne Pathogen Analysis by Surface-Enhanced Raman Spectroscopy Atanu Sengupta, Chetan Shende, Hermes Huang, Stuart Farquharson and Frank Inscorewww.rta.biz Visit our Booth
2 OutlineThe Need - Detection of Foodborne PathogensThe Solution - Surface-Enhanced Raman Spectroscopy • Basic Theory and Instrumentation • Proposed Assay Concept • Proposed Field Analysis • Previous (Relevant) RTA SuccessesThe Results
3 The Need/ProblemDetection 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/YearExamples • 2010: Salmonella contaminated eggs • 2009: Salmonella in peanut butter • 2008: Salmonella in peppers • 2007: E. coli in meat (Topps Meat Co. closes)
4 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!)
5 The Goal: FeasibilitySERS? 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 How it works: RamanLight Chemical virt H H Transmittedhνo 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 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 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 colorsCCD is just like a digital camera
9How it Works: RTA’s SERS-ID Analyzer A Portable, Field Usable Analyzer 3.4x5x10”, 5 pounds
10 The Solution: SERSSpecificity: Every chemical produces a unique Raman spectrumallowing unequivocal identification.Sensitivity: Silver and gold nanoparticles increase Raman signalsby 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)
13 The Proposal: The Analysis The proposed SERS-FBPD will extract and identifythe presence of ~1-10 cells of Salmonella and Listeria on surfaces in 2.5 and 3.5 hours from sample collection, respectively.
14 The Proposal: FeasibilityTask 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.
15The Proposal: SERS-Active Capture Assay Pathogens Target Specific Molecular Recognition Elements Ag Nanoparticles Sol-Gel Layer Glass Surface
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
17 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
18 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
19 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 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 The Results: FeasibilityTask 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).
22The 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
24 Providing Chemical Information When & Where You Need ItMission: 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