1. A non-amplification molecular probe approach John Gerdes, Ph. D.

2. 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

3. 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

4. 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

5. Capture by deflective flow microfluidic design
Microfluidic modeling predicts 99.68% capture
of 154 base pair molecule
Large channel diameter permits flow through of cells / debris

6. 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

7. “Fish on Chips” Prototype
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

8. 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

9. Prototype reader (CellMic)

10. Cell phone reader particle detection

11. Enterococcus detection microscopy vs chip

12. 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”)

13. Thank You
Funding provided in part by the National Science Foundation Phase I SBIR no
Technical support provided by
Kirsten L. Nelson, Senior Scientist at VisuGen Global LLC
Funding provided in part by USDA
SBIR phase I no