Instrumented NanoPhysiometer for High Throughput Drug Screening D. Michael Ackermann, Jon Payne, Hilary Samples, James Wells

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Big Picture Applications: A Research Tool Target Population: Target Population: Protoype of research tool Protoype of research tool Private research of BioMEMS group of VUSE BME dept Private research of BioMEMS group of VUSE BME dept Market Demand: Market Demand: Custom project for specific research Custom project for specific research Future implications to broad market Future implications to broad market High throughput screening Pharmaceutical Testing Toxicology

Motivation Limited study of cell life, activity, and volumes Previous methods: Previous methods: Single phase, stationary state Microliter scale & volumes Microliter scale & volumes  Nanophysiometer Nanoliter Scale Nanoliter Scale Real Time Monitoring Real Time Monitoring Decrease: Reagents (if any!) Decrease: Reagents (if any!)ProcessingTime 128 Well Plate Assays

Project Goals Develop nanoliter sized cell culture volume Develop nanoliter sized cell culture volume On-chip pumps for low flow perfusion and drug administration. On-chip pumps for low flow perfusion and drug administration. Thin film microelectrodes for monitoring of various analytes such as pH, oxygen, glucose and lactose in the media. Thin film microelectrodes for monitoring of various analytes such as pH, oxygen, glucose and lactose in the media. Optimize cell culture conditions to maintain cell viability over long periods of time. Optimize cell culture conditions to maintain cell viability over long periods of time. Develop a Labview based user interface for mircofluidic control of the NanoPhysiometer Develop a Labview based user interface for mircofluidic control of the NanoPhysiometer

Develop On-Chip Drug Delivery Systems To Achieve Desirable Low Flow Profiles Using Peristaltic Pumping Providing Ideal Parameters for Cell Viability The NanoPhysiometer 800 um Goals:

Physiometer Mask Design Microfluidics Pneumatics Electrodes 800 um

Physiometer Design Concerns Filter Size Filter Size 3 um, 5 um, 8 um 3 um, 5 um, 8 um Channel Aspect Ratio Channel Aspect Ratio Space between fluidic and pneumatic layers Space between fluidic and pneumatic layers

Peristaltic Pumps/Valves [1] S.R. Quake and A. Scherer, "From Micro to Nano Fabrication with Soft Materials", Science 290: (2000). [2] M.A. Unger, H.-P. Chou, T. Thorsen, A. Scherer, and S.R. Quake, "Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography", Science 288: (2000). Fluidics Layer Flexible PDMS Membrane (Valve) STATUS: Working! Currently just optimizing.

Electrochemical Monitoring Use integrated thin film microelectrodes to monitor physiological parameters Use integrated thin film microelectrodes to monitor physiological parameters pH, glucose, etc. pH, glucose, etc. Electrodes coated with a substrate specific oxidase Electrodes coated with a substrate specific oxidase Catalyze reaction producing H 2 O 2 Catalyze reaction producing H 2 O 2 H 2 O 2 then detected H 2 O 2 then detected STATUS: Will be integrated once fluidics/pneumatics are performing optimally: hard to make!

Optimize Cell Culture Conditions Determine minimal flow rates for maintaining vitality & sufficient perfusion Allow for physiological measurements Allow for physiological measurements Low flow for detectable pH and electrochemical differential Low flow for detectable pH and electrochemical differential STATUS: We seeded and imaged fibroblast in devices of various sized filters for observation, testing of cell attachment, and minimal survival. FIBROBLASTS *3-8  m when spherical, (flat, dendrite-like when attached) *1-2 day doubling time *Robust *Medium- antibiotics, vitamins, essential AA

Atmospheric Cell Culture Conditions Cells demand optimal temperature and CO 2 /O 2 levels Cells demand optimal temperature and CO 2 /O 2 levels PDMS is gas permeable PDMS is gas permeable Plexiglas enclosure  Contained, humidified incubator environment of 5% CO 2 Heated Microscope stage  Maintains optimal heated environment of 37 C

LabView programming User control of nanophysiometer system User control of nanophysiometer system Program Presets based on experimental needs Program Presets based on experimental needs Manual Control of Pumps and valves Manual Control of Pumps and valves Measurements & Data acquisition Measurements & Data acquisition Show parameter measurements Show parameter measurements Time-Lapse Image Capture Time-Lapse Image Capture Qualitative analysis Qualitative analysis STATUS: Nearly complete.

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Schematic LabView D/A Converter Camera Nanophysiometer Pneumatic Controller Electrode

JANFEBMARAPR Literature & Patent Search Define Goals & Project Approach Design Mask Prototype/Device Fabrication Programming Cell Culture Operation Assessment & Evaluation Final Paper & Presentation

Budget Mask of device design- $600/mask Mask of device design- $600/mask PDMS kit - $15 PDMS kit - $15 Cell culture supplies- $300/month Cell culture supplies- $300/month Tubing, wiring, etc.- ~$10 Tubing, wiring, etc.- ~$10 Electrodes- $500 (owned by lab) Electrodes- $500 (owned by lab)

References Unger, Quake, et. al. Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography. Science. Vol April 7, 2000 Unger, Quake, et. al. Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography. Science. Vol April 7, 2000 Ho, Chih-Ming. Fluidics – The Link Between Micro and Nano Sciences and Technologies / IEEE Ho, Chih-Ming. Fluidics – The Link Between Micro and Nano Sciences and Technologies / IEEE Arik, Zurn, et. al. Design, Fabrication and Experimental- Numerical Study of PZT Sensors. MSM Puerto Rico. Arik, Zurn, et. al. Design, Fabrication and Experimental- Numerical Study of PZT Sensors. MSM Puerto Rico. Gonzalez, Moussa. Simulation of MEMS Piezoelectric Micropump for Biomedical Applications Algor Incorporated; Technical Document. Gonzalez, Moussa. Simulation of MEMS Piezoelectric Micropump for Biomedical Applications Algor Incorporated; Technical Document. Bar-Cohen, Chang. Piezoelectrically Actuated Miniature Peristaltic Pump. March Proceeding of 2000 SPIE Smart Structures and Materials Symposium. No Bar-Cohen, Chang. Piezoelectrically Actuated Miniature Peristaltic Pump. March Proceeding of 2000 SPIE Smart Structures and Materials Symposium. No

Acknowledgements Dr. Franz Baudenbacher Dr. Franz Baudenbacher David Schaffer David Schaffer Andreas Andreas Nanodelivery, Inc. Nanodelivery, Inc.