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Presentation Slides
A non-amplification molecular probe approach
John Gerdes, Ph. D.
Direct Molecular Detection
Advantages
Challenges
 Rapid turnaround since
no amplification step
 Avoids sample inhibitors
of amplification enzyme
 No enzyme = lower cost
 Direct molecule
detection for quantitation
 On-site no-instrument
testing methods possible
 no cross-contamination
Achieve adequate levels
of detection / sensitivity
 Hybridization specificity /
detection of dead cells
 Interferents TBD
 Nucleic Acid release or
probe access within cell
 Correlate detection with
culture CFU counts / ?
Viable non-culturable
Strategy : Fluid flow NA capture,
hybridization, and wide field detection
 Capture and concentrate from large volume input upon
gravity flow through a microfluidic chip
 Target rRNA with high copy per cell /7 DNA, 6800 rRNA
stationary, 72,000 rRNA exponential copies per cell
 Cell lysis buffer suppresses nucleases but promotes
binding to solid phase (Al2O3) material
 Hybridization buffer for capture of fluorescent beads
conjugated with target specific probes upon flow through
the chip
 Fluorescent beads detected and enumerated using a Cell
phone microscope
 Detection sensitivity enhanced using wide field of view
 Results in 30 minutes uploaded to cloud by cell phone
Al2O3 properties permit flow chip strategy
 RNA or DNA binds to Al2O3 in certain bacterial cell lysis
buffers (US 6,291,166, expires April, 2018 and prior work at
Xtrana Inc) / viruses can bind directly from water
 Other buffer conditions block nucleic acid binding but are
compatible with probe hybridization
Binding can occur even with rapid fluid flow across the
Al2O3 matrix
Capture of nucleic acid onto Al2O3 is essentially
irreversible which permits aqueous washes
 Binding is due to positive surface charge of Al2O3 that
enhances capture of negatively charged molecules such as
nucleic acid or viruses
Capture by deflective flow
microfluidic design
Flow directed to ensure
contact of negative
charged RNA with positive
Al2O3 pillars and
subsequent hybridization
capture of fluorescent
microparticles
enumerated by wide field
of view cell phone
fluorescence reader
• Microfluidic modeling predicts 99.68% capture
of 154 base pair molecule
• Large channel diameter permits flow through of cells / debris
Integrated platform with Four Simple Steps
1) Add water that flows through capture chip (5 min)
2) Connect microparticle vial and wicking pad
3) Wait for wicking pad to turn blue (15 min)
4) Insert chip into reader to record results
6
VisuGen Global LLC
“Fish on Chips” Prototype
 On site molecular detection from 100 mL fluid
 Results in 30 minutes
 Cell phone reader result documentation and upload
VisuGen US Patent applications 62/463,447 & 62/500,302
7
Microparticle chip detection of E. coli
/ rRNA captured at 20 ml/min
Left: negative control, microparticles flow through the chip if no target
Middle: E. coli probe conjugated fluorescent microparticles are captured
within the channels of the chip by hybridization following lysis and 20
milliliter per minute flow through of 30 milliliters water
Right: fluorescent-only view of middle panel
VisuGen Proprietary US Patent 62/463,447
8
Prototype reader (CellMic)
Cell phone reader particle detection
Chip Image E coli : Read on CellMic Prototype Detector (with optical enhancement)
100 copies of E coli on test chip
Enterococcus detection microscopy vs chip
Bottom center
Top Center
Side Center
Top Center
Bottom
Center
Side Center
In Conclusion
Direct molecular detection methods potentially
could enable rapid on-site testing with cell
phone data transmission
 Adequate sensitivity could be accomplished by
targeting rRNA that is released from large
volume samples, captured upon flow through
microfluidic chips, and detected using
fluorescent bead hybridization and detection
using a cell phone reader using a wide field of
view (“Fish on Chips”)

Thank You
Funding provided in part by the National
Science Foundation Phase I SBIR no 1621593
Technical support provided by
Kirsten L. Nelson, Senior Scientist at VisuGen Global LLC
Funding provided in part by USDA
SBIR phase I no 2016-33610-25358

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