Automated Sample Processing for Pathogen Detection Systems

Automated Sample Processing for Pathogen Detection Systems Eduardo Ximenes, Hunter Vibbert, Amy Fleishman-Littlejohn, Linda Liu, Kirk Foster, Arun Bhunia, Rashid Bashir, Michael Ladisch Center for Food Safety Engineering Laboratory of Renewable Resources Engineering Agricultural and Biological Engineering Food Science Biomedical Engineering Electrical and Computer Engineering (U. Illinois) Purdue University 1 LORRE, Laboratory of Renewable Resources Engineering

Acknowledgments Dr. Jim Lindsay, Dr. Shu-I Tu USDA Cooperative Agreement OSQR Eastern Regional Research Center Center for Food Safety Engineering Dr. Jaeho Shin, Dr. Eduardo Ximenes Dr. Mira Sedlak, Dr. Nathan Mosier, LORRE, ABE Bruce Applegate, Lisa Mauer, Department of Food Science

Co-founder of Biovitesse: Rashid Bashir; Arun Bhunia, Consultant

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Outline Automated Sample Processing for Pathogen Detection Systems 1. The Need and Goal 2. Distribution of Microorganisms 3. The Science Behind the Cell Concentration and Recovery

(CCR) Process 4. Hollow Fiber Membrane CCR Instrument: The Engineer 5. Applications

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The Need and Goal Rapid Detection of Food Pathogens as Well as the Source: reduce public health risks

Microbial concentrations need to be brought to detectable level

Enrichment Culture

Cell Concentration and Recovery 4

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The Need and Goal Cell Concentration and Recovery

Enrichment Culture

Time Consuming

Shorter Time

Goal: t < 4 h 5

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Distribution of Microorganisms The binding affinity is measured by using the formula below: Kd= Log (CS/CW)

Cs = Cs = Cw = Cw = Kd =

a measure of concentration of bacteria bound on the specific substrate; measured in cfu/g, where the average mass of the samples was used to determine a weight to substitute in for the mass; a measure of concentration of the bacteria in the unbound phase; measured in cfu/mL; measured on a log scale to enhance larger differences.

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Distribution of Microorganisms Buffer

Substrate Sample & Bacteria in Buffer Bacteria Sample in Buffer Substrate Method: & Buffer 1. Prepare ligand and calculate volume; 2. Vegetable or meat substrate sprayed with 70 % (v/v) ethanol and let drying; 3. Substrates placed in labeled microplate (low binding affinity) under hood; 4. In a hood, dilute bacteria to appropriate concentration; 5. Place 10 mL of buffer (PBS, PBS + 0.1 % Tween or Buffered Peptone Water) in each microplate well; 6. Inoculate appropriate wells with bacteria; 7. Place in ice bath shaker at 120 RPM for 1 hour; 8. Plate Samples (Selective Medium: LB-amp for E. coli and Chromo agar for Salmonella). 7

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Distribution of Microorganisms Non-Pathogenic E. coli GFP (First test) Binding to vegetables : Potato (Skin, Fresh and both) 2.0

Kd (mL/g)

1.0

Flesh Both 0.0

Skin PBS

PBS + 0.1 % Tween Solvents

Buffered Peptone Water

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Distribution of Microorganisms Non-Pathogenic E. coli GFP Binding to meat (Hot dog)

Kd (mL/g)

1 0

cells/mL 102 103 104 105

-1 -2

Buffered Peptone Water (some contamination) Buffered Peptone Water (no contamination) Water (no contamination) Sample 9 LORRE, Laboratory of Renewable Resources Engineering

The Science Behind the CCR Process

MAJOR CHALLEGES TO BE ADDRESSED Separation of Food Samples and Bacteria

Membrane Fouling Recover Viable Cells

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First CCR Instrument Flat Membrane CCR Process (1st Prototype) Effective for concentrating microbial cells for microbiological analysis of water, dairy, and food products*

Challenge 1. Fouling of the membrane and the need for removing and handling it. 2. Achieving semi-continuous, hands-off operation

*Chen et al. 2005. Biotechnol Bioeng. 89:263-273. 11 LORRE, Laboratory of Renewable Resources Engineering

Hollow Fiber Membrane CCR Process Advantages Over Flat Membranes: High surface area to volume ratio; Higher flux per unit volume of the membrane module; Continuous operation that avoids manual handling of the membrane 200 μM and sample; Cross section view of Easily back flushed to recover concentrated cells of interest a hollow fiber

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First Hollow Fiber System (2nd Prototype ) The concentration of cells utilizing hollow fibers in an integrated system has been prototyped and run Lessons applied to development of devices

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Dead End HF Microfiltration Dead-End Filtration Feed

Permeate Liquid solution passes through the HF membrane. Particles retained on the membrane surface and module – inner LiquidHF solution passes through the HFsurface. membrane. Particles retained on the inner HF membrane surface and module surface. Permeate flux decreases rapidly. – Permeate flux decreases rapidly. A fouling layer build-up causes the system to plug up. – A fouling layer build-up causes the system to plug up 14 LORRE, Laboratory of Renewable Resources Engineering

Hollow Fiber Membrane CCR Process: (3rd Prototype) Pump

Pressure Gauge

Valve Sample Solution Hollow Fiber

Volume (ml)

250 200

Permeate

150 100 50 0 0

50

100 150 Time (min)

200

250

Homogenized Hot Dog Experiment: Permeate Volume Retentate Volume 15

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Hollow Fiber Membrane CCR Process: (4th Prototype) Key Components

Fiber module

0.2 µm hollow fiber 11 inch, Polysulfone

Pressure Transmitter

60 PSI max

2 Peristaltic Pumps Rainin Rabbit Plus Flow Meter

0-50 mL/min

Software

Labview 2009f3

Second pump passes liquid through the permeate side of the membrane in order to achieve a constant pressure gradient and increase transmembrane flux. 16 LORRE, Laboratory of Renewable Resources Engineering

CCR Box Front Panel Display

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Testing 4TH Prototype Monoflow STEP Chicken* Extract + 3 X 104 cfu/mL S. enteriditis Chicken Extract 3 X104 cfu/mL S. enteriditis

1

2

Membranes Glass Microfiber Filters (2.7m)

Hollow fiber (CCR) (0.2 m)

Time for Filtration

Volume Applied

Volume Recovered

1 min

200 mL

~ 200 mL

60 min

~200 mL

~ 2.5 mL 2 X106 cfu/mL S. Enteriditis

*100 g of chicken legs was mixed with 500 mL water in a stomach bag. The chicken legs in water were finger massaged for 2 min, few times, and then incubated at room for 2.5 h. The liquid was collected for further work. 18 LORRE, Laboratory of Renewable Resources Engineering

Testing 4TH Prototype Monoflow Maximum Sustainable Pressure

Membrane Fouling: Minimized, but still an issue

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Testing 4TH Prototype Monoflow 1st to 3rd Quartile Process Control

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Testing 4TH Prototype Dual Flow

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Testing 4TH Prototype Dual Flow

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Testing 4TH Prototype Flow rate

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Work in Progress 1- Optimization of Pre- and Post-Filtration Steps;

1.1 Pre-Filtration: Addition of one step using glass microfiber filter (1.6 m);

1.2 Post-Filtration Steps (Washing solution): Testing Individually or Combined: Enzymes (Lipases, Proteases) Surfactants Buffers

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Work in Progress 2- Additional tests to confirm cleaning and sanitization of membranes for re-using;

Exhaustive tests under progress to test efficiency of 70% (v/v) ethanol; Use of 10% bleach also to be tested for comparison.

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Work in Progress Testing 5th Prototype: Used for Demonstration Here New feature: smaller pumps were added to the instrument Reduction of Dead Volume by 5 TIMES: from 2.5 mL (Prototype #4) to 0.5 mL Translates to 5  Increase in Cell Concentration Further Optimization in Progress: Addition and test of small diaphragm pumps; Addition and test of level and turbidity sensors 26 LORRE, Laboratory of Renewable Resources Engineering

Applications Concentrate Cells (Salmonella sp, Listeria sp, E.coli sp) Against a Background of Microorganisms Identification by Different Methods Multifluidic Detection

Antibody PCR

Ramon Light Bacteriophage Spectroscopy Scattering Reporter

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