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

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

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 Special Focus on Sanitation. UNICEF, WHO United Nations Human Development Report. 3. Diarrhea: Why children are still dying and what can be done. UNICEF, WHO 2009.

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)

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.

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

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 (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 *

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): Traditional Methods Contact Liquid Polymer Process (CLiPP) *

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

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

SCHOOL OF ENGINEERING High current Low current

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 µm particles and asymmetrical particles –Integrate concentrator, lysis chamber, and sensor chamber into a monolithic chip

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