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

Team Members:Team Members:Paul ClarkPaul ClarkMartin GarciaMartin GarciaChris GorgaChris GorgaJohn Ling IIIJohn Ling IIIGiordano Lo RegioGiordano Lo Regio

Advisors/Assistants:Advisors/Assistants:Dr. Franz BaudenbacherDr. Franz BaudenbacherRaghav VenkatRaghav VenkatTobias MeyerTobias Meyer

IntroductionAt the end of the last progress report our

goals were too:Get clean room certifiedBegin casting optical fibers in PDMS devicePrepare flourescein concentrations and

begin data acquisitionAdapt Device to incorporate cells

Current StatusEveryone in group is now clean room

certifiedWe have started building our setup to be

used for device testingStock solutions of flourescene are made up

and ready for the initial tests

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

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

Production of PDMS Device w/ Optical Fiber

Mix curing agent and base in 1:15 ratioPour PDMS giving thickness of 200mCure w/ oven at 65ºC for 20 min.Insert fiber optic cable on top of channels

using a micromanipulator3 channels 3 micromanipulators

Replace PDMS in 65ºC oven for 40 min.Plasma bond PDMS to a glass slide

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

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

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

Configuration of Optical Components for Fluorescent Measurement

Sipido & Callewaert (1995), Cardiovascular Research [Modified (2007)]

Magnified Cell Chamber Displaying Fura-2 and Ca2+ Pumps

**Magnified Cell Chamber from Previous Slide

Goals RevisitedGet clean room certifiedBegin casting optical fibers in PDMS

devicePrepare flourescene concentrations and

begin data acquisitionAdapt Device to incorporate cells

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

ReferencesSipido, 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.