Detection of Foodborne

Pathogens via an

Integrated Spectroscopy &

Biosensor Approach

PIs: Irudayaraj, J.; Mauer, L.; and *Debroy, C.

Purdue University; *Penn State University

ARS-USDA and Purdue Center for Food Safety Engineering

Objectives

Develop nanoparticle biosensors for

pathogen detection

Develop surface enhanced-Raman

spectroscopic approaches for direct and

sensitive fingerprinting of pathogens

Advance portable infrared biosensor

Optimize biosensor platform

Appropriate sampling methods and testing

Pathogens tested

• E.coli E. coli O26, E. coli O103, E. coli O111, E. coli O157:H16, E. coli0157:H5, E. coli O157:H19 .

E. coli O157:H7 (Acc No: 5.2262, 99.0874, 0.1292, 99.0894, 0.0027, 0.1288, 0.1304, 7.3853, 7.3860)

• SalmonellaS. typhimurium, S. enteritidis

• Listeria

L. innocua, L. monocytogenes

• Shigella flexneri, Staphylococcus aureus

4

Raman Spectroscopy

The Raman system typically consists of four major

components:

1.Excitation source (Laser).

2.Sample illumination system and light collection optics.

3.Wavelength selector (Filter or Spectrophotometer).

4.Detector (Photo diode array, CCD or PMT).

• Typically, a sample is illuminated with a laser beam. Light

from the illuminated spot is collected with a lens and sent

through a monochromator. Wavelengths close to the laser

line, due to elastic Rayleigh scattering, are filtered out while

the rest of the collected light is dispersed onto a detector.

•The main difficulty of Raman spectroscopy is separating the

weak inelastically scattered light from the intense Rayleigh

scattered laser light.

FTIR vs Raman

Raman spectrum (red) is more highly resolved than the

FTIR spectrum (purple).

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Escherichia Coli O157:H7

Raman

FTIR

LOD: 103-104 CFU/ml

Raman and FTIR discrimination

Differentiation of five different species of pathogenic bacteria based on the canonical variates

FTIR

Raman

Tentative assignment of peaks from the SERS spectra of E. Coli O157:

H7, S. Typhimurium and S. Aureus

Strain level discrimination by Raman and FTIR

Discrimination of five different E.coli O157:H7 strains

obtained from different sources.

FTIR

Raman

October 27, 2010

Surface-Enhanced Raman Scattering (SERS)

Enormous Raman enhancement is

observed for molecules adsorbed

on special metallic surfaces, called

SERS

Analyte plasmon interaction

The technique of using SERS with

analytes which has resonant

chromophores is called SERRS

Charge transfer or chemical

enhancement

Excitation is through transfer of

electrons from the metal to molecule

and back to the metal again

Chem Comm (2007)

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Surface Enhanced Raman Spectroscopy using Silver Nanospheres

a) b)

c) d)

Wang and Irudayaraj. 2009. Ultrasensitive SERS fingerprinting and detection of bacteria using silver

nanospheres. (J. Physical Chemistry)

SERS fingerprinting of bacteria using novel AgNSs

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Inte

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Raman Shift (cm-1)

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A) SERS spectra of S. aureus on the as-prepared AgNSs with different concentrations from

a, 106; b, 105; c, 104; d, 103; e, 102 and f, 10 cfu/mL; B) Comparation of SERS spectra of E.

Coli O157:H7#5.2262 (a), S. Typhimurium (b), and S. aureus (c) at 785 nm excitation [102

cfu/ml]

CVA for species and strain level differentiation

Incorporating a separation step: Multifunctional nanoprobes for separation and detection

Small Journal (2007, 2009), Angew Chemie (2009)

Multiple pathogens detection, separation and photothermal ablation

Wang, C. and Irudayaraj, J. 2009. Multifunctional nanoprobes for separation, detection, and photothermal

ablation of foodborne pathogens. Small. (In Press)

UV-vis absorbance spectra after addition of a mixture of E. coli and S. typhimurium to anti- E. coli and S. typhimurium antibody-conjugated amine modified gold nanorods ofaspect ratios 2.0 and 3.2, respectively. The concentrations of E. coli and S. typhimuriumwere 1-10 to 106 cfu/mL.

Spectroscopy integrated

Biosensor

GOLD COATED SILICON WAFERS (10nM)

Spectral Resolution comparison

• Cost: $9000 vs $125000 (Benchtop)

• Wt: 3.5lbs; Operation: 150C - 600C

Traping of E. coli using Magnetic Nanoparticles and FTIR detection

Biosensor concept validation in a Portable system

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P-O in P-O-C

Nucleotide "fingerprints"

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(b)1058 cm-1

1005 cm-1

956 cm-1

Rapid formation of Nanoparticle mediated bacteria

clusters – an indirect signal enhancement

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Portable Mid-IR Biosensor for pathogen detection

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Nucleic Acids

related peaks

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Sensitivity : ~ 104 CFU/mlDetection time: Less than 30 minutesSamples: Skimmed Milk, 2% Milk, Spinach, etc.

Protein related peaks

Ravindranath et al. 2008. Biofunctionalized magnetic nanoparticle integratedmid-infrared pathogen sensor for food matrices. Analytical Chem, 81(8):2840-2846.

Summary

• Direct fingerprinting of pathogens using nanomaterials and Raman Spectroscopy

• Multiplex detection of pathogens using gold nanorods

• Multiplex detection in food matrices with a separation step

•Portable mid-infrared and SERS biosensor assay for detection in food matrices

Selected Publications1) Sandeep, R., Mauer, L., Debroy, C. and Irudayaraj, J. 2008. A portable spectroscopic

biosensor for pathogen detection in complex matrices. Analytical Chemistry.

2) Wang, C. and J. Irudayaraj. 2008. Gold nanorod probes detects multiple pathogens. SmallJournal.

3) Irudayaraj, J. 2009. “Pathogen Sensors”, Editor, special issue of Sensors.

4) Wang, C. and J. Irudayaraj. 2009. Multifunctional nanoprobes for separation, detection, and photothermal ablation of multiple foodborne pathogens. Small Journal.

5) Sandeep, R., Mauer, L., Debroy, C. and Irudayaraj, J. 2010. A cross platform biosensors approach to detect pathogens. Sensors and Actuators.

6) Wang, Y. and Irudayaraj, J. 2010. Silver nanocrystals for direct fingerprinting of pathogens. J. Physical Chemistry.

This work was supported through a cooperative agreement with the Agricultural Research Service of the U.S. Department of Agriculture project number 1935-42000-035 and the Center for Food Safety Engineering at Purdue University.

Acknowledgement