Andy PageInnovaPrep LLC, Drexel, MOMay 10, 2013 11:30 am San Diego, CA

Development of a Novel Membrane-Based System for Fractionation and Concentration of Biological Particles from Complex Environmental Matrices

DHS PHASE II SBIR

• Development of an automated, integratable sample fractionation/concentration system for use autonomous biodetection systems

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Team3

ROY J. CARVERBIOTECHNOLOGY CENTERUNIVERSITY OF ILLINOIS

Autonomous Biodetection SystemsAerosol

Collection

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Sample Preparation Detection

• 5 to 20 mL• Inhibitors• Clutter

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• 0.001 to 0.2 mL• Bacteria

• Culture• Nucleic Acids• Proteins• Other

• Viruses• Nucleic Acids• Proteins

• Free Nucleic Acids• Free Proteins

• Remove environmental debris and clutter• Separate into 5 fractions

• Whole bacteria• Nucleic Acids from bacteria & viruses• Proteins from bacteria• Free Nucleic Acids• Free Proteins

• Concentrate from ~10 mL into 100 μL fractions• Perform these operations in less than 15 minutes• With high efficiency >70%• Operation for up to 30 days (~240 samples)

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Sample Preparation Need for Autonomous Biodetection Systems

• Aerosol• Metagenomics• Hospital Acquired Infection• Animal Production

• Water• Drinking Water• Environmental Water• Pharma Water

• Life Sciences Research• Food• Clinical• Forensics• Other?

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Commercial Applications?

Technical Approach

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8 membranes + lysisCartridge 1

• 1 – Remove Humics• 2 – Remove Environmental Debris• 3 – Concentrate Whole Bacteria• 4 – Concentrate Free Nucleic Acids• 5 – Concentrate Free Proteins

Lysis• Lyse Whole Bacteria

Cartridge 2• 1 – Concentrate Cellular Debris• 2 – Concentrate Nucleic Acids• 3 – Concentrate Proteins

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Membrane Filter Fractionation & Concentration

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30%

70%

Lysis

Input Sample

Stage A.1

Debris/Inhibitor Removal

Debris/Inhibitors/ Spent Media

Stage A.2

Whole Viable Organism Fractionation

and Concentration

Stage A.3

Nucleic Acid Fractionation and

Concentration

Whole Viable Organism

Concentrate

Permeate

Permeate

Permeate

Free Solution Nucleic Acid Concentrate

Stage A.4

Protein Concentration

Permeate/ Waste

Free Solution Protein

Concentrate

Stage B.2

Nucleic Acid Fractionation and

Concentration

Whole Cell Nucleic Acid Concentrate

Permeate

Stage B.3

Protein Concentration

Permeate/ Waste

Whole Cell Protein

Concentrate

Archive

Whole Viable Organism

Concentrate

Free Solution Fractions

Whole Cell Fractions

PVPP Column

Humic Removal Debris/Inhibitors/ Spent Media

Cartridge B

Cartridge A

Stage B.1

Cellular Debris Fractionation and

Concentration

Cellular Debris Concentrate

Complex Process

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Complex System

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•Environmental debris

•Whole bacteria

•Nucleic acids

•Proteins

Improved “Cartridge” Design

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• “Dead-end” filtration• Particles larger than the filter pore size are

retained• A viscous, expanded wet foam is pushed

tangential to the filter collection surface -capturing the particles

The InnovaPrep Concentration Process

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Wet Foam Elution• Mode of Operation

• “Expanded Liquid”• Increased Viscosity – reduced channeling (Yan 2006)• Moves as rigid body w/ narrow (<10 µm) lubricating layer

(Briceno 2003; Tisne 2004)• Bursting bubbles

• Improved Elution• Lower minimum elution volumes• Higher elution efficiencies• Largely unaffected by sample matrix

• Quickly Breaks Down into a Liquid

System Design

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Membrane Filter Cascade Cartridge• Stack of 5 membranes

• Environmental Particle Debris• Humics• Whole Bacteria• Free Nucleic Acids/Viruses• Free Proteins

• Flow Sequentially through Each Membrane

• Wet Foam Elution

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5-Stage Membrane Filter Cascade Cartridge• Alternating layers of fluidics, gaskets with integrated valves, & membranes.• Structures manufactured by three dimensional laser engraving.• Cartridge Dimensions 11.9 cm L x 5.5 cm W x 1.7 cm thick

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5-Stage Membrane Filter Cascade Cartridge• 5 membranes in series

• Humic removal• Prefilter• Bacteria filter• Virus and DNA filter• Protein filter

• 21 total layers• 8 fluidic layers• 8 gasket/valve layers• 5 membrane layers

• 20 pneumatic valves• Valve diameter 3 mm• Valve holdup volume <2uL

• Total internal volume 2.6 mL• 86 vertical fluid channels• 66 horizontal fluid channels

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Manifold• Manifold remains connected to instrument• Gasketed cartridge allows for relatively quick change• Quick swap cartridge in development

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Fraction Output• Delivery of concentrated 100 uL fractions

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Prototype Instrument with 5-Stage Cartridge

• Less than 10 minute run time for 10 mL sample• 100 μL concentrate volume• Programming

•Automated run•Automated backflush•Automated storage•Automated filter check

Test Data and Final Thoughts

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3-Stage System –1.0 μm Microspheres• Stage 1 – Prefilter• Stage 2 – Whole Bacteria• Stage 3 – Free Nucleic Acids

3 PSI System Pressure 20 PSI System Pressure

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3-Stage System – 0.05 μm Microspheres• Stage 1 – Prefilter• Stage 2 – Whole Bacteria• Stage 3 – Free Nucleic Acids

3 PSI System Pressure 20 PSI System Pressure

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5-Stage System –• Stage 1 – Humic Removal• Stage 2 – Prefilter• Stage 3 – Whole Bacteria• Stage 4 – Free Nucleic Acids• Stage 5 – Free Proteins

Status• Process refinement near completion• Humic removal stage

• Demonstrated passage of up to 1.0 μm PSMs• Demonstrated capture of up to 50% of Humics (at 30 ppm)• Demonstrated automated recovery

• Refined system/process ready for biological testing this month

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• Higher-Performance Membranes• Very High Flux• Highly Selective

• Membrane Fouling• Particles• Humics

• Flux/Transmembrane Pressure vs. Transmission• Particle Interactions• Long term operation• System Complexity

Technical Hurdles

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InnovaPrep Technical Team

Zach PackinghamSteve GrahamPam Murowchick

Michael Hornback, Ph.D.Tyler Davis

Stephanie Cantrell

Alec Adolphson JD Birkenholz

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Contact Information

Andy Pageapage@innovaprep.com

Phone: 816.868.6204

InnovaPrep LLC132 E. Main St.

Drexel, Missouri 64742

www.innovaprep.com