Transcript Sample preparation for point-of-care infectious disease diagnostics Ian M. White Don L. DeVoe Jeffrey Burke Kunal Pandit Eric Kendall John Goertz Fischell Festival 10/16/2014

Sample preparation for point-of-care
infectious disease diagnostics
Ian M. White
Don L. DeVoe
Jeffrey Burke
Kunal Pandit
Eric Kendall
John Goertz
Fischell Festival
10/16/2014
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Bacterial pathogen identification
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Infectious disease diagnostics for bacterial pathogens.
– Gold standard: cultures.
– Requires 1-2 days for infection confirmation.
– Requires additional 1-5 days for species identification.
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Cost of waiting:
– Patient in isolation.
– Broad spectrum antibiotics (emerging superbugs).
– Further spread of the pathogen in the community.
– Patient health deterioration.
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There is an urgent need for rapid identification of pathogens and the correct
course of treatment.
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PCR for bloodstream infection diagnosis
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Polymerase chain reaction (PCR) can be used to identify pathogens.
Commercial products available for post-culture analysis.
– Too late.
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Challenges with using PCR for rapid diagnosis:
– Initial sample may be complex (worst case: bloodstream infections).
– Must pre-concentrate bacterial genomic DNA.
– Standard sample prep for PCR is labor intensive as well.
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Requirement for intensive sample prep procedures prohibits the use of PCR
for near-patient BSI diagnosis.
– Need exists for automated sample prep.
Automated microfluidic sample prep for diagnosis of
bloodstream infections
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Sample prep steps performed in microsystems to enable sample-to-answer
genetic diagnostics.
– Recovery and pre-concentration of cells from whole blood.
– Extraction of genomic DNA via cell lysis.
– DNA purification and release.
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Design goals:
– Process mLs of blood in minutes.
– No manual steps or intervention.
– Low-cost (enable single-use “reimbursable” devices).
Microfluidic
PCR
Cell purification
and concentration
Cell lysis
DNA
purification
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Cell extraction and pre-concentration
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Extraction of a certain cell type from whole blood has a range of diagnostic
applications.
– Circulating tumor cells for cancer metastatis.
– Circulating neutrophils for bloodstream infections.
• Neutrophils may contain pathogen DNA.
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Challenges:
– Process blood at a high rate (~1 mL/min).
– Reduce volume and flow rate for downstream microfluidic steps.
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Spiral Inertial Filtration (SIFT)
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Inertial microfluidic separation in curved
structures – high speed sorting.
– Toner, et al., Di Carlo, et al., Papautsky, et al.
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Our goals:
– Apply to whole blood.
– Achieve high level of concentration.
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SIFT microsystem.
– Spiral for cell sorting.
– Balanced filtration channels for
concentration of recovered cells.
• Side channels on outer spiral.
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Cancer cell separation from blood
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Methods:
– Partial red blood cell lysis is necessary because whole blood is non-Newtonian.
• Osmotic lysis utilized.
– Flow rate of ~1 mL/min.
– WBC cells fluorescently stained for imaging.
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Results:
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– ~95% recovery of white blood cells.
– >13X concentration factor.
Inlet
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Focused WBCs
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Bacterial genome separation from blood
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Methods:
Whole blood
Stain WBCs
Incubate with
fluorescent E. coli
Off-chip
RBC lysis
• Results:
Smaller white blood cells
Larger white blood cells,
including engulfed
bacteria.
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Cancer cell separation from blood
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Methods:
– Partial red blood cell lysis is necessary because whole blood is non-Newtonian.
• Osmotic lysis utilized.
– Flow rate of ~1 mL/min.
– MCF7 cells stained and spiked into blood; WBC cells pre-stained.
• Results:
– ~100% recovery of MCF7 cells.
– ~14X concentration factor.
Burke, et al., Biomicrofluidics, 8, 024105 (2014)
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Continuous-flow cell lysis
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After cell recovery, must access DNA for PCR.
Challenges:
– Millions of cells have been recovered.
– Flow rate has been reduced by concentration, but is still high.
– Must lyse cells quickly to meet design goals.
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Cannot use conventional chemical cell lysis or many other methods.
Microfluidic
PCR
Cell purification
and concentration
Cell lysis
DNA
purification
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Simple continuous-flow cell lysis
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Mechanical lysis enables high-throughput.
Cells are forced through a series of nozzles.
– 3-5 mm, depending on target cell.
– Simple to fabricate through replication.
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Massive energy dissipation at nozzle entrance/exit results in cell lysis.
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Lysis with soft lithography devices
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Low-cost, rigid chip is desired for mechanical cell lysis.
– i.e., thermoplastics.
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However, rapid prototyping in thermoplastics is challenging.
– Not many labs are equipped.
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Rapid prototyping with soft lithography is ideal.
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However, soft materials (PDMS) do not work for mechanical functions under
high flow rates (high pressures).
– Expansion of small lysis nozzles under high pressure.
No pressure
PDMS
nozzles
100 mL/min
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Continuous-flow microfluidic lysis in OSTE
microsystem
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Off-stoichiometry thiol-ene (OSTE):
– Casts soft.
– UV cures as a rigid material.
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Simple bonding:
OSTE
OSTE
Si mold
SiO2
– Partial cure on replica mold.
– Peel from mold and assemble layers.
– Complete UV cure to form a rigid device.
Bonded OSTE device
Continuous-flow microfluidic lysis in OSTE
microsystem
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White blood cells:
– Essentially 100% lysis of white blood cells in OSTE device.
– 3 mm nozzles.
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Metastatic cancer cells:
– MDA-MB-231 – infamously flexible.
– 5 mm nozzles.
– >80% lysis in OSTE (<40% lysis in PDMS).
Lysis of MDA-MB-231 cells
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Microfluidic DNA Recovery and Purification
DeVoe, et al.
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Conventional method:
– Solid phase extraction (SPE) in a spin column.
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The steps of SPE have been duplicated in a microfluidic channel.
– Several steps.
– PCR inhibitors are used.
Charge switching:
– Electrostatic capture of DNA with pH-modulated material.
– Implemented in microfluidics with chitosan.
• Landers, et al.
Charge switching
Microfluidic
PCR
Cell purification
and concentration
Cell lysis
DNA
purification
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Charge-switching DNA purification with a microfluidic
monolith
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Our goals:
– Increase the DNA loading capacity of charge-switched microfluidic devices.
– Simplify fabrication of high-surface-area microfluidic structures.
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Microfluidic polymer monoliths.
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DeVoe, et al.
Extremely high surface area.
Embedded within microfluidic channels.
Can be functionalized with biomaterials, including chitosan.
• Expoxide chemistry.
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Fabrication of microfluidic monolith
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In-situ UV photo-patterning within a microfluidic channel.
– Simple fabrication.
– However, not extendible to batch process, mass production.
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Fabrication of microfluidic monolith
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Batch-process fabrication invented by the DeVoe group.
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DNA capture/release performance
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Performance is comparable to conventional technique.
– Recovery of 54.2% +/- 14.2% (n=3).
High surface area leads to superior DNA loading capacity.
– Demonstrated a break-through capacity of 120 ng capture.
– 0.3 mL monolith.
Low pH
DNA measured
at the outlet
High pH
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Summary
New sample prep technologies for microfluidic PCR.
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Automated cell recovery and concentration with inertial microfluidics.
– Circulating tumor cells.
– White blood cells.
– 100% recovery, ~14X concentration factor, ~1 mL/min.
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Continuous-flow sample lysis.
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Rigid but low cost microfluidic device.
Nozzle-based mechanical lysis.
100% lysis of white blood cells, >80% lysis of metastatic cancer cells.
New fabrication protocol for rigid devices using soft lithography.
DNA purification and release.
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High surface area polymer monolith grafted with chitosan.
Loading capacity of 120 ng DNA in 0.3 mL monolith.
100% DNA capture, >50% recovery.
New batch fabrication method to enable low-cost mass production.
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Acknowledgments
Microfluidic cell separation
Dr. Jeffrey Burke
Microfluidic cell lysis
Dr. Jeffrey Burke
Kunal Pandit
Hiroshi Inoue
Dr. Shulin Zeng
Dr. Alex Blake
Dr. Ivor Knight.
Microfluidic monolith
Prof. Don DeVoe
Dr. Eric Kendall
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Backup
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Cell recovery and purification
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Channel correction to maintain focusing after side-channel waste removal
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Particle sorting with SIFT
Near perfect recovery
Sorting:
4.8 mm beads, 8 mm beads
>13X concentration factor
Burke, et al., Biomicrofluidics, 8,
024105 (2014)
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Microfluidic mechanical cell lysis
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Mechanical cell lysis is the only method that enables continuous-flow cell
lysis at high flow rates.
– Millions of cells/min.
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Mechanical lysis has previously been reported in
silicon microsystems.
– Too expensive for single-use reimbursable devices.
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Large-scale, replication-based fab is preferred.
Di Carlo, et al., Lab Chip, 2012, 12, 2914–2921
Continuous flow cell lysis – energy dissipation
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Energy dissipation in rigid vs. expandable materials.
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DNA recovery
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Performance is comparable to conventional technique.
– Recovery of 54.2% +/- 14.2%.
– N = 3.
DNA measured at the outlet
Low pH
High pH
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Charge switching - control
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Bare monolith vs. chitosan monolith.