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
2
Autonomous Biodetection SystemsAerosol
Collection
4
Sample Preparation Detection
• 5 to 20 mL• Inhibitors• Clutter
?
• 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)
5
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?
6
Commercial Applications?
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
8
Membrane Filter Fractionation & Concentration
9
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
12
• “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
13
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
15
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
16
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
17
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
18
Manifold• Manifold remains connected to instrument• Gasketed cartridge allows for relatively quick change• Quick swap cartridge in development
20
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
22
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
23
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
24
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
25
• 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
26
InnovaPrep Technical Team
Zach PackinghamSteve GrahamPam Murowchick
Michael Hornback, Ph.D.Tyler Davis
Stephanie Cantrell
Alec Adolphson JD Birkenholz
27
Contact Information
Andy [email protected]
Phone: 816.868.6204
InnovaPrep LLC132 E. Main St.
Drexel, Missouri 64742
www.innovaprep.com
Top Related