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

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

2. Direct Molecular Detection Advantages Challenges 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 • 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

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. 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 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