Download Microchip with an Open Tubular Immobilized pH Gradient for

Supporting Information
Microchip with an Open Tubular Immobilized pH Gradient for UV
Whole Column Imaging Detection
Hulie Zeng, Tomasz Glawdel, Carolyn L. Ren
Department of Mechanical and Mechatronics Engineering, University of Waterloo,
Waterloo, Ontario, Canada N2L 3G1
Materials and methods
Reagents
Poly(dimethylsiloxane) (PDMS) prepolymer and its curing agent were purchased from Dow
Corning (Midland, MI, USA). Negative photoresist, SU-8, and propylene glycol monoether
acetate (PGMEA) developer were obtained from MicroChem (Newton, MA, USA). Maleic acid,
surfuric acid compounds with N-allylimidodicarbonimidic diamide, N,N’-methylenebisacrylamide,
acrylamide,
3,-(trimethoxysilyl)
propyl
methacrylate
(γ-MAPS),
acetic
acid,
2-isopropylthioxanthone, sodium hydroxide, phosphoric acid, and urea were from Sigma-Aldrich
(St. Louis, MO, USA). Standard pI markers (3.59, 4.65, 6.14, 7.05, 8.18, 8.79) were from
Convergent Biosciences (Toronto, ON, Canada, later acquired by Protein Simple). Human
hemoglobin control AFSC was purchased from Helena (Beaumont, TX, USA). Water was purified
with an ultra-pure water system (Barnstead/Thermolyne, Dubuque, IA, USA) and was used to
prepare all of the solutions. Quartz slides (3 inch × 1 inch) were from SPI Supplies (West Chester,
PA, USA). Nickel slit sheets (3 mm × 6.5 cm; slit size: 65 µm × 5 cm) were tailor-ordered

To whom correspondence should be addressed. [email protected]. Phone: +1-519-888-4567, x
33030. Fax: +1-519-885-5862.
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products from StenTech (Mississauga, ON, Canada).
Experimental Setup
The UV detection system is a whole column imaging detection (WCID) system, made in-house.
As shown in Figure 1(a), it consists of a deuterium (D2) lamp from Hamamatsu Photonics
(Hamamatsu, Shizuoka, Japan), a narrow band optical filter (270-290 nm) form eSource Optics
(MA, USA), an optical fiber bundle and a Pixis 256 CCD camera from Princeton Instruments
(Trenton, NJ, USA). A high-voltage power supply from Labsmith (Livermore, CA, USA) was
used to provide adjustable high voltages (0-3000V) for the separation experiments of IPG
microchip.
Fabrication of PDMS-quartz chip for IPG
PDMS microfluidic chips were fabricated using standard soft lithography techniques. Details
about the fabrication procedures are illustrated in Figure S1. Briefly, two photo masks that
contained the design of a main channel and the design of four sets of diffusion channels (each set
consists of 10 parallel diffusion channels), respectively, were printed (Colorado Springs, CO,
USA). The designs were then patterned into SU-8 photoresists (also called master) that were
coated on a silicon wafer. PDMS replicas were made using the master and then bonded with
quartz slides via oxygen plasma treatment at a dosage of 29.6 W for 30s to make enclosed
channels.
To prepare the master with different channel heights, the silicon wafer was first spincoated with
SU-8 2005 at 1500 rpm for 60 s, giving rise to a thickness of 10 μm for the diffusion channels.
The coated silicon wafer was baked at 65oC for 1 min and then 95 oC for 2 min, and cooled down
to room temperature slowly. The design of the diffusion channels was then transferred into the
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coated SU-8 2005 photoresist via UV exposure at a dosage of 500 mJ cm-2. The silicon wafer was
baked again at 65oC for 2min and then 95oC for 3.5min and cooled down slowly to room
temperature. To fabricate the main channel with the shallow diffusion channels on the same wafer,
SU-8 2075 was spincoated on the top of the previously coated SU-8 2005 at 1800 rpm for 60 s,
giving rise to a thickness of 100 μm. The wafer was baked at 65oC for 5min and at 95oC for 18min,
and cooled down to room temperature slowly. The design of the main channel was then transferred
into the two layers of SU-8 photoresists (100 µm SU-8 2075 and 10 µm SU-8 2005) via UV
exposure at a dosage of 810 mJ cm-2 after carefully aligning the main and diffusion channels.
After post exposure baking at 65oC for 4 min and 95oC for 9 min and cooling down slowly to
room temperature, the pattern was developed by dipping the silicon wafer into the PGMEA
developer for 10min. The developed master was rinsed by pure water and blown dry by nitrogen.
To make replicas using the master, PDMS prepolymer was thoroughly mixed with the curing
agent at a weight ratio of 10:1 and degassed for 30 min under vacuum. The mixture was then
poured onto the master and cured at 80oC for 1 h. After curing, the PDMS substrate with a
thickness of 0.5 mm was peeled off from the master, and four 0.5 mm diameter holes were
punched at the reservoir locations of the diffusion channels with a set of two reservoirs at each
end.
Development of IPG chip
The main channel and diffusion channels were all pre-treated with 0.4% (w/w) γ-MAPS in 0.4%
(w/w) acetic acid aqueous solution for 1 h for assisting the bonding between the gel and channel
walls. Then the microchannels were rinsed by pure water. 2% (w/w) 2-isopropylthioxanthone
ethanol solution which is the initiator for polymerization was then pumped into the microchannels
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and kept for 10min to ensure its adsorption to the channel walls. The un-adsorbed
2-isopropylthioxanthone was then rinsed out by pure water. For the development of pH gradient,
the neutral prepolymer solution which contained 5% (w/w) acrylamide and 2.5% (w/w)
N,N’-methylenebisacrylamide was filled into the main channel first. As shown in Figure S1, the
base prepolymer solution containing additive 11 mM N-allylimidodicarbonimidic diamide was
loaded via the two sets of diffusion channels at one end of the main channel while the acid
prepolymer solution containing additive 11 mM maleic acid was loaded from the other end of the
main channel via the nearby two sets of diffusion channels at the same time. After 6 hrs, the
microchip was put under a UV light source with a central wavelength of 365 nm (using an I-line
filter) for 8 min to immobilize the pH gradient via photo-polymerization reaction.
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Figure S1: The procedure to fabricate IPG microchip consists of: (a) Fabrication of
the master for replica molding, (b) fabrication of the microchip, and (c) development
of IPG microchip.
NH
N
H
NH
N
H
O
NH2
O
HO
N-allylimidodicarbonimidic diamide
OH
Maleic acid
Figure S2: The structures of the basic and acidic chemicals used in the development of pH
gradient in IPG microchip.
5
10
9
8
pH
7
6
5
4
3
2
-3
-2
-1
Maleic acid (mM)
0
1
2
3
4
N-allylimidodicarbonimidic diamide (mM)
Figure S3: The titration curve of maleic acid and N-allylimidodicarbonimidic diamide monomers
in pre-polymer solutions excluded acidic and basic monomers.
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