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/ O 2  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 CaCO 3 w/ an analytical balanc Add CaCO 3 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 Ca 2+ 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 Ca 2+ 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 [Ca 2+ ] 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.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.