Detection of Pathogens Using Electrochemical DNA Sensors for Resource-Limited Settings

1. Detection of Pathogens Using Electrochemical DNA Sensors for Resource-Limited Settings Sarah Ghanbari, Nicholas Giustini, Cameron Mar, Pankti Doshi, Unyoung Kim Santa Clara University October 15th, 2011 SCHOOL OF ENGINEERING

2. Overview • Problem Statement • Current Technological Solutions • Key Components of Design • High throughput concentrator • Lysis Chamber • DNA Sensor Chamber • Concentrator • Fabrication • Analysis • Sensor Chamber • Analysis • Summary of Work

3. Problem Statement • Approximately one in eight people lack access to safe water.1 • The water and sanitation crisis claims more lives through disease than any war claims through guns (with more than 3.5 millions deaths each year).2 • Diarrhea is the second leading cause of death in children under five. It kills more young children than AIDS, malaria, and measles combined.3 1. Special Focus on Sanitation. UNICEF, WHO. 2008. 2. 2006 United Nations Human Development Report. 3. Diarrhea: Why children are still dying and what can be done. UNICEF, WHO 2009.

4. Problem Statement Populations without access to safe drinking water 26% - 50% 51%-75% No Data 1% - 25% 76% - 100% The World’s Water The Biennial Report on Freshwater Resources (Gleick 1998)

5. Research Question • Can we make a device that is: • Small and portable • Reduces reagent and power consumption • More accurate • Provides faster diagnosis • User friendly • Yes, by utilizing a microfluidic platform • Our method is to create a microfluidic platform combining an inertial concentrator and electrochemical DNA sensor.

6. Problems with Current Solutions Traditional Tests Developing Microfluidic Tests2 • Time consuming • Expensive • Tests only indicate possibility of contamination • Complicated fabrication and architecture • Expensive and time consuming preparatory procedures • Requires non-portable equipment for full functionality 1 1.Potatest water test kit by Wagtech WTD 2. Sengupta, Shramik , et. al, Microfulidic Diagnostic Systems.

7. Key Components of Design • High-throughput concentrator • Cell lysis chamber • Electrochemical DNA sensor Inlet Low current * Lysis Chamber High current Rf = 2ra2/Dh3 Outlet Rf: Inertial Force Ratio r : Radius of Curvature a : Particle Size Dh: Hydraulic Diameter Schematics of Electrochemical DNA Sensor Schematics of High-throughput Concentrator *Di Carlo, Dino D., et al, PNAS Vol. 104, pp. 18892-18897 (2007)

8. Fabrication: Traditional Methods Contact Liquid Polymer Process (CLiPP) * Coat Substrate Photolithography Fill Chamber with Monomer Mixture Etching Align Photomask Resist Removal Expose with UV Wafer Bonding Remove Uncured Monomer *Hutchison, J. Brian, et. al., Lab on a Chip 4.6 (2004): 658-62.

9. Concentrator Results Flow Rate: 0.2 mL/min Flow Rate: 0.1 mL/min Flow Rate: 1.6 mL/min Flow Rate: 1.1 mL/min

10. Chemistry of the DNA Sensor Methylene Blue Low current High current TargetSequence SensorProbe Au Working Electrodes Pt Counter & Reference SensorProbe Self Hybridization Region Methylene Blue Thiol attachment to gold Thiol attachment to gold

11. Low current High current

12. Summary • Key components • Concentrator: achieved focusing for 10 μm particles at a flow rate of 1.1 mL/min to concentrate the pathogens in a water sample • DNA Sensor Chamber: confirmed the specificity of DNA sensor strands toward an identified target (E. coli) at a concentration of 500 nM • Ongoing and Future Work • Improve concentrating efficiency for 0.5-2 µm particles and asymmetrical particles • Integrate concentrator, lysis chamber, and sensor chamber into a monolithic chip

13. Acknowledgements • Dr. Ashley Kim • Dr. Teresa Ruscetti • Dr. Steven Suljak • Mr. Daryn Baker • Dr. Cary Yang • Dr. Dan Strickland • Dr. Hohyun Lee • Stanford Nanofabrication Facilities • Roelandts Fellows • School of Engineering • Biomedical Engineering Society • OAI