Optical Bio Sensors 07

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Optical biosensors for foodpathogen detection: fiber opticsensor

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, , -Center for Food Safety and Engineering,Purdue UniversityERRC, Wyndmoor, PA

CFSE Annual Review Oct 30-31, 2007

Team members : Viswaprakash Nanduri (PD), BK Hahm (PD),Hyochin Kim (GS) and Angela Valadez

Objectives

Develop and optimize fiber optic sensor foreach food pathogen: Listeria monocytogenes,E. coli O157:H7, Salmonella Enteritidis

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Develop multi-pathogen fiber optic sensor fordetection of three target pathogenssimultaneously

Development of a Multi-Pathogen FiberOptic Biosensor

Fluorescence

Detection AbBacteriaCapture AntibodyBiotin

F

Food

PED-

pump

3

Streptavidin

Waveguide

Inlet

Outlet

multipathogenenrichment (SEL)

Lm EC Sal Cont

Summary: Individual fiber opticsensor for each pathogen

Pathogen Detection limit Detectiontime

Publications

Listeria monocytogenes

10 3 cfu/ml10 4-10 5 cfu/ml

hotdog

23 h Geng et al. 2004. AEM. 70:6138Nanduri et al 2006. Sensors,

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bologna:

Kim et al. 2007. Fd. Sci.Biotechnol.16:337

E. coli O157: H7Stx

10 3 cfu/mlground beef

0.5 ug/ml

4-6 h Geng et al. 2006. Sensors,6:796

Tu et al. 2006. SPIE

SalmonellaEnteritidis

10 4 cfu/mlChicken

Egg

4-6 h Morgan et al. 2006. KeyEng. MaterialsValadez et al (unpublished)

Antibodies for use with fiber opticbiosensor

Pathogen Capture antibody Detection antibody

Listeria monocytogenes Anti-Listeria PAbAnti-P66 PAb

MAb C11E9

- -

5

. .O157:H7

.O157:H7

Salmonella Enteritidis KPL anti-Salmonella(CSA-1)

MAb-2F11

Analysis of sensitivity of the sensor with differentconcentrations of S . Enteritidis (PT4) cells suspended in PBS

1000

1200 SE

Control

Fiber optic sensor for Salmonella

6

-200

0

200

400

600

800

0 1 2 3 4 5 6 7 8 9

SE dilutions

S i g n a l p

1=10 9 cfu/ml8=10 2 cfu/ml

Detection limit = 1x10 3 cfu/ml

Valadez et al (unpublished)


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Specificity of the fiber-optic sensor

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Bacterial cells were used at 10 8 cfu/ml

Specificity of the Salmonella fiber opticsensor in presence of mixed bacterial

cultures

400500600700800900

n a l

( p A

8

0100200300

S. Enteritidis,E. faecalis,P. aeruginosa

E. faecalis,P. aeruginosa

S. Enteritidis,P. vulgarius,C. gallinarum

P. vulgarius,C. gallinarum

Mixture I Mixture II

S i

S. Enteritidis at 1x10 5 cfu/mlOthers at 1x10 6 cfu/ml each

Sensitivity of S . Enteritidis PTs byimmunomagnetic bead-time resolved

immunofluorescence (IMB-TRF) assay

40000

50000

60000

70000

n s

i t

PT 1PT 4PT 4-13PT 6PT 7

9

Detection limit = 1x10 5 cfu/ml

0

10000

20000

30000

1 E 9 1 E 6 1 E 5 1 E 4 1 E 3 1 E 2 1 E 1 Control

cfu/ml

T R F I n t

PT 8

Selectivity tests of IMB-TRF

15000

20000

25000

30000

I n t e n s i

t10 E 6 cfu/ml10 E 5 cfu/ml

10

Values are an average of two wells each tested with two differentconcentrations of cells

0

5000

10000

S. Typhimurium P. vulgaris E. faecalis E. coliO157:H7

SE PT 4

Bacterial species

T R F

Detection of S . Enteritidis grown in foodsamples and tested every 2 h intervals

using fiber optic biosensor.

400

600

800

1000

1200

1400

1600

p A

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

E n t e r

i t i d i s C o n c e n

t r a t

i o n

( c f u / m l )TSB Broth

500

1000

1500

2000

2500

3000

3500

p A

1 00E+011.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

S .

E n t e r i t i d i s

C o n c e n t r a t i o n ( c f u / m l )

Egg

11

0

200

Control 2 4 6 8

Time (hours)

1.00E+00

1.00E+01 S .

E

0

200

400

600

800

1000

1200

1400

1600

1800

Control 2 4 6 8

Time (hours)

p A

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

S .

E n

t e r i

t i d i s C o n c e n

t r a

t i o n

( c f u / m l )

Chicken breast

0

Control 2 4 6 8

Time (hours)

1.00E+00

. E+

Bars: Fiber optic signals; Lines: Growth curves

0

20000

40000

60000

80000

100000

120000

Control 2 4 4.5 6 8

Time (Hours)

T R F I n t e n s

i t yEggChicken

IMB-TRF

Multi-Pathogen Assay Using a Fiber opticWaveguide Sensor System

TasksOptimize immobilization conditionsScreen different combination of capture and detection

antibodies for optimum signal for Listeria monocytogenes,

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. co : , an amone a n er sRun simultaneous assay for each pathogenTest with food samples in presence of natural competitive

microorganismsProduce a multi-pathogen cartridge with waveguides pre-

coated with the capture antibodies


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Antibodies to be used for multi-pathogenfiber optic sensor

Pathogen Capture antibody Detection antibody

Listeria monocytogenes Anti-Listeria PAbAnti-P66 PAb

MAb C11E9

PAb404

13

PAb407

E. coli O157: H7 KPL anti-E. coliO157:H7

PAb Ec 8378

pET32a

Salmonella Enteritidis KPL anti-Salmonella(CSA-1)

MAb-2F11

50

100

150

200

250

300

350

400

S I G N A L , p

A

SIGNAL,pA

E. coli O157:H7Multi-pathogen

detection: Initial trial

50

100

150

200

250

300

350

400

450

S I G N A L , p

A

Signal, pA

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1 2 3 40

CHANNELS

Capture Ab: KPL (anti E.coli Detection Ab: pET32a (Cy5)Channels: 1: Control; 2-4: Exp

1 2 3 40

CHANNELS

C o n t r o l

S .

E n t e r i t i d i s

E . c

O 1 5 7 : H 7

L .

m o n o c y

t o

Listeria Salmonella E. coli

Capture PAb-P66 Anti-Sal (CSA-1) Anti-E.coli (KPL)

Detection MAb C11E9 MAb 2F11 pET32a

Conclusions

Sensitivity of fiber optic sensor for Salmonella Enteritidis is 1x10 3 cfu/ml with pure cultureFiber optic assay is highly selectiveSalmonella can be detected from spiked (10 2cfu/g) chicken and egg samples only after 4 h of

Need work!

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enrichmentSpecific antibodies are available for multi-pathogen fiber optic assay and initial trial showpromising result in simultaneous detection of allthree pathogens if present in a sample

Next steps for multi-pathogen testingusing fiber optic sensor

Optimize assay for simultaneousdetection of 3 pathogens suspended inbuffer/growth media

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Determine assay sensitivity/selectivity Validate the assay with spiked food

samples following enrichment in SEL Application of PED for sample

preparation

Thank you

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Optical Bio Sensors 07