1. Microfluidic Glucose Sensor Senior Design Group 4 Kristen Jevsevar Jason McGill Sean Mercado Rebecca Tarrant Advisor: Dr. John Wikswo, Dr. David Cliffel Graduate Advisor: Jennifer Merritt

2. Problem Statement Primary Objective Primary Objective Quantify glucose consumption/production on a microliter scale Quantify glucose consumption/production on a microliter scale Design a glucose electrode interface that will measure micro-scale concentrations while maintaining affordability Design a glucose electrode interface that will measure micro-scale concentrations while maintaining affordability Long Term Goal Long Term Goal Extend the design to measure lactate, oxygen, and pH Extend the design to measure lactate, oxygen, and pH

3. Diabetic Applications Inexpensive glucose monitors are regularly used by diabetics. Inexpensive glucose monitors are regularly used by diabetics. Our device will not be used in diabetic diagnostics. Our device will not be used in diabetic diagnostics. However, biological researchers may use similar techniques to study cellular metabolism and toxicology. However, biological researchers may use similar techniques to study cellular metabolism and toxicology.

4. Applications Currently, researchers measure extracellular metabolites, such as glucose, on a millileter scale. Microfluidics work on micro and nanoliter scales. Microfluidics work on micro and nanoliter scales. Smaller volumes yield faster and more accurate results. Smaller volumes yield faster and more accurate results. Less expensive and easily reproducible Less expensive and easily reproducible Provides near real time results Provides near real time results

5. Similar Systems Specially designed electrodes already exist that are able to measure glucose concentrations. To date, there are very few ways to simultaneously measure glucose, lactate, oxygen, pH and other metabolites easily and inexpensively. Using a readily produced electrode, it is possible to easily measure glucose levels on a small scale. This technology can then be extended to multiple metabolites.

6. Performance Criteria Must be able to measure glucose concentrations within a biologically relevant range, between 0mM and 6mM Must be able to measure glucose concentrations within a biologically relevant range, between 0mM and 6mM Must be affordable – less expensive than current research technology, more expensive than the disposable strips for diabetics Must be affordable – less expensive than current research technology, more expensive than the disposable strips for diabetics Needs to work for at least 24 hours Needs to work for at least 24 hours Should recalibrate automatically to account for electrochemical drift Should recalibrate automatically to account for electrochemical drift

7. Design Concept Map

8. Our Design Our design utilizes a commercially produced Pine Instruments electrode that is much larger. Our design utilizes a commercially produced Pine Instruments electrode that is much larger. This electrode is interfaced with a microfluidic pumping device that allows small volumes, on the microscale, to be studied. This electrode is interfaced with a microfluidic pumping device that allows small volumes, on the microscale, to be studied.

9. Design Components Electrode Electrode Channel System Channel System Electrode Housing Electrode Housing Pumping System Pumping System Bioreactor Bioreactor Electrochemical workstation Electrochemical workstation Computer software – A/D converter Computer software – A/D converter

10. Design: the Electrode Cellular glucose sensors consist of an electrode that utilizes a chemical reaction to determine glucose concentrations. Cellular glucose sensors consist of an electrode that utilizes a chemical reaction to determine glucose concentrations. An enzyme film is cast onto the electrode. An enzyme film is cast onto the electrode. The electrode consists of three “contacts” The electrode consists of three “contacts” Working electrode Working electrode Reference electrode Reference electrode Counter electrode Counter electrode

11. Chemical Reaction This reaction takes place on the electrode. Platinum Electrode Nafion Glucose Oxidase (GOx) GOx Glucose + O2O2 O 2 (+ H 2 O) Gluconolactone + H 2 O 2 e-e- Nafion

12. Potentiostat An instrument that controls the electrical potential between the working and reference electrodes. Keeps the potential of the working electrode at a constant level with respect to the reference electrode Controls the potential across the electrochemical cell by sensing changes in its resistance, and changing the current supplied to the system accordingly

13. Design: Channel System Using a CNC, a PDMS mold was made to create uniform channels. Using a CNC, a PDMS mold was made to create uniform channels. Solution containing glucose is run through these channels, passing over the electrode. Solution containing glucose is run through these channels, passing over the electrode. Channel Electrode Shape

14. Channel Fabrication CNC mold PDMS CNC mold electrode channel

15. Design: Electrode Housing Plexiglass plates are placed on each side of the electrode to clamp the PDMS in place, sealing the system from leakage. Plexiglass plates are placed on each side of the electrode to clamp the PDMS in place, sealing the system from leakage. Clamping pressure can be manually adjusted. Clamping pressure can be manually adjusted. Holes are drilled in the plates in order to run tubing to the channel. Holes are drilled in the plates in order to run tubing to the channel.

16. Electrode Housing

17. Design: the Pumping System Tubes are run through holes drilled in the plates. Tubes are run through holes drilled in the plates. These tubes are attached to the Harvard Apparatus pumping system. These tubes are attached to the Harvard Apparatus pumping system. The pumping system is controlled using LabView. The pumping system is controlled using LabView.

18. LabView Pump controller

19. Design: Bioreactor The bioreactor cultures a small amount of cells. The bioreactor cultures a small amount of cells. The Harvard Apparatus pumps media and glucose, in respective tubes, through the bioreactor to the electrode housing. The Harvard Apparatus pumps media and glucose, in respective tubes, through the bioreactor to the electrode housing.

20. Design: Electrochemical workstation The glucose concentrations are measured using a CH Instruments electrochemical workstation. The glucose concentrations are measured using a CH Instruments electrochemical workstation. This workstation consists of a Picoamp Booster and Faraday cage. This workstation consists of a Picoamp Booster and Faraday cage.

21. Design: Computer software The CH Instruments workstation is an amperometric sensor that measures a current at a fixed applied voltage. CH Instruments computer software is responsible for converting this analog signal to a digital format.

22. Design A/D converter Bioreactor Pumping systemElectrode and housingElectrochemical workstation Pumping system

23. Optimization: Electrode and Tubing We are using profilometry to characterize the electrode We are also testing to determine the most efficient tubing length

24. Troubleshooting: Uniformity In order to have a uniform channel height between experiments, we have designed a PDMS torque wrench. This uniformity will increase consistency between experiments.

25. Previous Experiments Calibration curve in beaker 1mM 2mM 4mM 3mM 5mM 7mM 8mM 9mM 6mM

26. Previous Experiments Calibration curve in microfluidic device Linear Trend

27. Recent Experiments

28. Current Experiments Cytosensor glucose concentrations

29. Current Experiments Cytosensor, 1 mM, and 3 mM glucose concentrations

30. Expenses Pine Instruments Electrode: $30 Tubing & electrode housing: ~$15 Harvard apparatus: $2,000 Bioreactor: $20 Electrochemistry workstation: $2,000 Computer: $1,500 Chemicals (i.e. glucose oxidase): $50

31. Current Work Working on new design for new electrode Working on new design for new electrode Optimize channel height and tubing length Optimize channel height and tubing length Running manual experiments with cytosensor Running manual experiments with cytosensor Stop-flow Stop-flow Continuous Continuous

32. Future Work Test LabView pump controller Get a new mold made for the new electrode Find a smaller pumping system

33. Future Applications Obtain results for other metabolites Configure on chip peristaltic pump Interface with nanobioreactor