Design Team 8: Fluorescent Detection using Optical Fibers with Cardiac Myocytes

1. Design Team 8:Fluorescent Detection using Optical Fibers with Cardiac Myocytes Team Members: Paul Clark Martin Garcia Chris Gorga John Ling III Giordano Lo Regio Advisors/Assistants: Dr. Franz Baudenbacher Raghav Venkat Tobias Meyer

2. Introduction • At the end of the last progress report our goals were too: • Get clean room certified • Begin casting optical fibers in PDMS device • Prepare flourescein concentrations and begin data acquisition • Adapt Device to incorporate cells

3. Current Status • Everyone in group is now clean room certified • We have started building our setup to be used for device testing • Stock solutions of flourescene are made up and ready for the initial tests

4. Work Completed • Initial LabView program has been written • It will be continually updated as needed throughout project • Optical box has been acquired, but it is not in full working order • 4 different protocols have been written • Creating a master, Integrating Optical Fiber, Preparing Stock Solutions, Testing Fura-2 Excitation Levels

5. Development of a 3-Channel Master on Silicon Wafer • Apply Su-8 to silicon wafer w/ spinner • 500 rpm at 100 rpm/s & 1600 rpm at 300 rpm/s • Soft bake w/ hot plate (65ºC & 95ºC) • Expose w/ UV light around 365 nm • Post Exposure Bake (65ºC & 95ºC) • Develop by immersing in ethyl acetate • Rinse w/ isopropyl alcohol & dry w/ O2 • Remove substrate using 70ºC bath of Remover PG

6. Production of PDMS Device w/ Optical Fiber • Mix curing agent and base in 1:15 ratio • Pour PDMS giving thickness of 200m • Cure w/ oven at 65ºC for 20 min. • Insert fiber optic cable on top of channels using a micromanipulator • 3 channels  3 micromanipulators • Replace PDMS in 65ºC oven for 40 min. • Plasma bond PDMS to a glass slide

7. Diagram of 3-Channel PDMS Device w/ 3 Optical Fibers

8. Preparation of a Calcium Stock Solution • Measure 1g CaCO3 w/ an analytical balanc • Add CaCO3 to 700mL DI water while stirring • Add 1 mL 12M HCl to aid dissolving • Titrate solution to pH of 7.5 by adding 3M NaOH • Add DI water to bring total volume to 1L

9. Testing Base Fura-2 Excitation Levels • Place inlet and outlet holes on PDMS device • Mount the device on inverted microscope • Connect optical fiber to computer • Begin pumping Fura-2 solution through device • Begin fluorescence data acquisition • Allow mixing of Ca2+ w/ Fura-2 solution

10. Configuration of Optical Components for Fluorescent Measurement Sipido & Callewaert (1995), Cardiovascular Research [Modified (2007)]

11. Magnified Cell Chamber Displaying Fura-2 and Ca2+ Pumps **Magnified Cell Chamber from Previous Slide

12. Goals Revisited • Get clean room certified • Begin casting optical fibers in PDMS device • Prepare flourescene concentrations and begin data acquisition • Adapt Device to incorporate cells

13. Timeline for Future Work • Wednesday Feb. 14 • Meet with Prof. Baudenbacher to obtain necessary materials • Create master and complete our setup in the lab • Monday Feb. 19 - Monday Feb. 26 • Cast optical fibers in PDMS device • Wednesday Feb. 28 • Begin collecting data with flourescene, LabView, and PDMS device • Start date of this task depends on length of time required to complete previous task

14. References Sipido, Karin R., Callewaert, Geert (1995). How to measure intracellular [Ca2+] in single cardiac cells with fura-2 or indo-1. Cardiovascular Research, 29, 717-726. Negative Tone Photoresist Formulations 2002-2025. Micro Chem Website, www.microchem.com. Min-Hsien Wu, Haoyuan Cai, Xia Xu, Jill P.G. Urban, Zhan-Feng Cui, and Zheng Cui. A SU-8/PDMS Hybrid Microfluidic Device with Integrated Optical Fibers for Online Monitoring of Lactate. Biomedical Microdevices 7:4, 323ﾐ329, 2005. Fura-2 and Indo-1 Ratiometric Calcium Indicators. Molecular Probes, Invitrogen Detection Technologies. June 21, 2005.