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Supplementary Material for Applied Physics Letters
Probing the physiology of ASH neuron in Caenorhabditis elegans using
Electric Current Stimulation.
Trushal Vijaykumar Chokshi1, Daphne Bazopoulou2 and Nikos Chronis2,3
1
Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, MI
48109-2121, USA.
2
Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
3
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
A. e-chip Microfabrication:
The microfabrication process consists of three steps:
Step 1: Fabrication of ITO (Indium Titanium Oxide) electrodes on a glass coverslip.
A layer of ITO is first sputtered to a thickness of 150 nm on a glass coverslip (#1.5). A positive
photoresist is then spun over the ITO and patterned using photolithography to define the electrodes. The
electrodes are formed by wet etching the exposed ITO for approximately 1 minute in a 1:1 solution of
HCL:H20. The remaining photoresist is stripped using acetone and the coverslip is cleaned using
isopropanol.
Step 2: Fabrication of the PDMS microfluidic chip.
The PDMS microfluidic chip is fabricated using soft lithography. A three-layer SU-8 mold is first created
using a three-step photolithography process. The first SU-8 layer contains the microfluidic channel that
connects the flush-channel with the worm trap. The second SU-8 layer contains the worm trap, while the
third layer includes the worm and buffer inlet channels, outlet channel and the flush channel. PDMS is
poured onto the SU-8 mold and allowed to cure at 65 ºC for approximately 3 hours. The PDMS layer is
[1]
then peeled off from the SU-8 mold and punched using a sharpened 19-gauge needle (0.031 inch I.D.,
0.042 inch O.D.; Kahnetics) to form the fluidic inlets and outlets.
We fabricated three different SU-8 molds for conducting experiments with Day 1, 3 and 5 worms
respectively. For each SU-8 mold, the Table I lists the thicknesses of the three SU-8 layers and the type of
SU-8 used to pattern them. In all cases, we processed the SU-8 photoresist according to the recipe
provided by MicroChem on its website.
Supplementary Table I: Information about the thicknesses of the different layers in the SU-8 mold and
the type of SU-8 used to fabricate them. Different SU-8 molds were fabricated for conducting
experiments on Day 1,3 and 5 worms.
Day 1
SU-8 Layer 1
Thickness
Type
(µm)
7
SU-8 2007
SU-8 Layer 2
Thickness
Type
(µm)
28
SU-8 2015
SU-8 Layer 3
Thickness
Type
(µm)
36
SU-8 2015
Day 3
7
SU-8 2007
32
SU-8 2015
40
SU-8 2015
Day 5
7
SU-8 2007
42
SU-8 2050
50
SU-8 2050
Worm Age
Step 3: Assembly of the e-chip.
The e-chip is assembled by aligning and bonding the PDMS chip to the ITO-patterned glass coverslip
using air plasma (50 W, 250 mTorr, 35 s). As PDMS does not bond very strongly to ITO, we used an
epoxy glue to secure the PDMS chip on the glass coverslip. In order to access the ITO electrodes, we
glued copper wires to them using a conductive silver epoxy (8331-14G from www.mgchemicals.com).
B. Experimental setup of the Automated Platform:
The automated platform consists of the e-chip, an epifluorescent microscope (IX71, Olympus) equipped
with a z-moving stage, a dual-color imager for FRET imaging [DV2 from MAG Biosystems, equipped
with a dichroic (505dcxr) and two emission filters (470/30 nm and 535/30 nm)] attached to a back-
[2]
illuminated CCD camera (QUANTEM:512SC, Photometrics), a current source (Keithley 2400
Sourcemeter) and a valve manifold connected to the buffer and worm-containing solutions
(Supplementary Figure 1).
Supplementary Figure 1: Experimental setup of the automated platform for acquiring ASH calcium
transients in response to electrical current stimulation.
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