1. SCHOOL OF ENGINEERING 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 15 th, 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. SCHOOL OF ENGINEERING 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. SCHOOL OF ENGINEERING Problem Statement Populations without access to safe drinking water No Data 1% - 25% 26% - 50% 51%-75% 76% - 100% The World’s Water The Biennial Report on Freshwater Resources (Gleick 1998)

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

7. SCHOOL OF ENGINEERING Key Components of Design  High-throughput concentrator  Cell lysis chamber  Electrochemical DNA sensor Schematics of High-throughput Concentrator Schematics of Electrochemical DNA Sensor Inlet Outlet *Di Carlo, Dino D., et al, PNAS Vol. 104, pp. 18892-18897 (2007) R f = 2ra 2 /D h 3 High current Low current Lysis Chamber R f : Inertial Force Ratio r : Radius of Curvature a : Particle Size D h : Hydraulic Diameter *

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

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

10. SCHOOL OF ENGINEERING Chemistry of the DNA Sensor Thiol attachment to gold Methylene Blue Self Hybridization Region Sensor Probe Target Sequence Au Working Electrodes Pt Counter & Reference Thiol attachment to gold High current Low current

11. SCHOOL OF ENGINEERING High current Low current

12. SCHOOL OF ENGINEERING Summary  Key components – Concentrator : achieved focusing for 10 μ m 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. SCHOOL OF ENGINEERING 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