Supplementary material
Protein patterning utilizing region-specific
control of wettability by surface modification
under atmospheric pressure
Donghee Lee1, Min-Sung Kwon2, Ji-Chul Hyun3,
Chang-Duk Chun2, Euiheon Chung1,3, Sung Yang1,3,*
1
Department of Medical System Engineering, 2School of Life Science, 3School of
Mechatronics, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712,
Republic of Korea
1
SUPPLEMENTAL MATERIAL
 Surface modification by applying AP-PECVD with TEOS or TEOS-O2
(100) Silicon wafers were washed successively for 15 min each in acetone, methanol, and
deionized (DI) water using an ultrasonic cleaner and were subsequently rinsed with running
DI water and dried with nitrogen. An AP-PECVD system operating at a radio frequency (RF)
of 13.56 MHz was used to implement region-specific control of wettability onto the Si wafers.
TEOS (vaporized using Ar at a flow rate of 1 slm) was used as the precursor to deposit less
hydrophilic than bare Si substrate and a mixture of vaporized TEOS and O2 (100 sccm) to
deposit superhydrophilic layer on Si substrate. He (15 slm) was used in common as the
carrier gas for the deposition processes. An RF power of 200 W was used to generate plasma.
The distance between the nozzle head of the plasma source and the sample was adjusted to
0.5 mm for TEOS-treated surface and to 1 mm for TEOS-O2-treated surface. The samples
were mounted on a moving stage positioned below the plasma source; the stage moved at 20
mm/s orthogonally to the plasma-source head. Each substrate was passed back and forth 20
times across the plasma-head region.
 Fabrication process for a region-specific control of wettability on Si substrate
After depositing either a TEOS-O2-treated layer (for 2D hydrophobic/hydrophilic pattern)
or a TEOS-treated layer (for 3D hydrophobic/hydrophilic pattern) was deposited on Si
substrate, standard photolithography and lift-off were applied on the substrate. Layers of the
photoresist (AZ1512) were deposited by briefly the coated substrates at 3000 rpm for 30 s. A
mask aligner was used to selectively expose the layers to ultraviolet (UV) light (15 mJ•cm2 s)
for 10 s. The exposed photoresist layers were subsequently developed using the developer
2
MIF-300 for 40 s. Either a TEOS-treated layer or a TEOS-O2-treated layer was deposited on
top of the PR-patterned Si substrate, depending on whether the 2D or 3D region-specific
control of wettability were to be formed, and the inessential AP-PECVD-deposited layers and
photoresist patterns were stripped using acetone. The resulting PR-patterned Si substrates
were then rinsed in methanol and DI water. 3D Si structures were etched in the PR-patterned
Si substrates using a deep reactive-ion etching (DRIE) system (AMS200, Alcatel Co.) under
2300 W of RF power and SF6 and C4F8 mixed with O2 as the etching gases.
 Variation of wettability depending on the number of treatment in AP-PECVD process
To investigate the change of contact angle by the number of treatments in AP-PECVD
process, four conditions of the number of treatment were selected such as 5, 10, 15 ,20 times
respectively for four kinds of modified surfaces such as TEOS/Si, TEOS-O2/Si, TEOSO2/TEOS/Si, and TEOS/TEOS-O2/ Si. FIG. S1 shows the variation of static contact angle
depending on the number of treatment of the sort of precursors. Both the TEOS-O2/Si and
TEOS-O2/TEOS/Si showed superhydrophilic property regardless of the number of treatment.
But, TEOS/Si presented smooth changes to be less hydrophilic. On the other hand,
TEOS/TEOS-O2/ Si showed abrupt increase in wettability from hydrophilic (C.A.= 47 ) to
hydrophobic (C.A.=132.3) when 10 times of AP-PECVD treatment was applied respectively
using TEOS-O2 and TEOS sequentially. However, the contact angle of TEOS/TEOS-O2/Si
decreased if more than 10 times of treatments were applied. Considering the change of
roughness and chemical composition, it can be inferred that the wettability of AP-PECVDtreated surface is highly dependent on the number of treatment as well as the sort of precursor.
3
Static contact angle (Unit: Deg.)
150
TEOS/Si
TEOS-O2 /Si
120
TEOS-O2 /TEOS/Si
TEOS/TEOS-O2 /Si
90
60
30
0
0
5
10
15
20
Number of treatment by AP-PECVD
FIG. S1. Variation of wettability depending on the number of treatment and precursors in APPECVD process.
4
 Evaluation of region-specific control of wettability on Si substrate
To evaluate the region-specific control of wettability on Si substrate, a superhydrophilic
circular sport-array deposited by AP-PECVD using TEOS-O2 was patterned with a layer
using TEOS and the boundary of receding water droplet was investigated. Supp. FIG. S2
shows that a receding water droplet placed on such an array was separated into individual
microdroplets, which formed on the superhydrophilic spots by TEOS-O2 treatment, or
became irregularly shaped. As the water droplet shrank owing to evaporation, its boundary
was affected by the controlled wettability of the substrate. FIG. S2(a) shows a water droplet
of100 µm in diameter on a circular-spot array, and FIG. S2(b) shows a water droplet of 20
µm in diameter on such an array. The shape and dimension of region-specific control of
wettability on Si substrate corresponded to that of pattern in mask film used in
photolithography for lift-off process.
(a)
(b)
(c)
200 m
200 m
200 m
FIG. S2. Evaporation of a water drop places on the wettability patterns on a Si wafer. (a)
Water droplets formed from the drop on the array of superhydrophilic circular spots on the
wettability patterning. (b) Change in boundary of water drop owing to the contrasting
wettabilities of the different regions of the wettability-patterned surface. (c) Change in the
boundary of the water drop and in the shape of the individual water droplets formed on the
array of circular spots on TEOS-treated substrate. (Light blue: silicon substrate; dark blue:
water droplets).
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 Quantitative analysis to evaluate the coefficient variation (CV) in protein patterning
TABLE SI. Quantitative analysis result of 2D protein patterning
Mean,
Standard deviation,
Coefficient of variation[b],
Location [a]
/ (%)


#1
60.7
3.8
6.26%
#2
60.6
2.7
4.46%
#3
60.6
3
4.95%
#4
59.5
2.7
4.54%
#5
64.1
4.1
6.40%
#6
65.8
2.4
3.65%
#7
65.5
2.7
4.12%
#8
64.9
2.8
4.31%
#9
64.9
2.9
4.47%
#10
66.1
3.7
5.60%
#11
65.3
2.2
3.37%
#12
65.1
3.7
5.68%
Mean of means
63.59
Standard deviation of means
2.46
Coefficient of variation of means [c]
3.87%
[a] Twelve spots were randomly selected from the square spot array in FIG. 3(a) (Right)
[b] CV values presenting intra-spot fluorescence homogeneity
[c] CV values presenting inter-spot fluorescence repeatability.
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TABLE SII. Quantitative analysis result of 3D protein patterning
Mean,
Standard deviation,
Coefficient of variation,
Location [a]
/ (%)


#1
54.5
4.2
7.71%
#2
54.6
4.6
8.42%
#3
56.3
3.9
6.93%
#4
57.3
3.6
6.28%
#5
56.5
4.2
7.43%
#6
56.5
3.6
6.37%
#7
58.8
4.5
7.65%
#8
59
3.5
5.93%
#9
58.1
4.9
8.43%
#10
61.3
4.1
6.69%
#11
60.6
4.6
7.59%
#12
60.6
3.6
5.94%
Mean of means
57.84
Standard deviation of means
2.29
Coefficient of variation of means
3.95%
[a] Twelve spots were randomly selected from the square spot array in FIG. 3(b) (Right)
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 Bioactivity of the immobilized antibodies on region-specific control of wettability
The nonfluorescent mouse antihuman antibody LFA-1 IgG (TS2/4) was dissolved in PBS
at a concentration of 100 µg/mL, and the entire patterned region of the substrate was covered
with the solution containing the proteins. After incubation for 1 hour at 37 C, the entire
surface was rinsed with PBS, and then it was covered with a solution containing the FITClabeled secondary antibody for TS2/4 in a concentration of 50 µg/mL, and followed by the
incubation again for 1 hour at 37 C. The sample was then rinsed again with PBS. As a
second step, the CMRA-labeled Jurkat T cells were incubated on the patterned surface for 1
hour at 37 C. After the incubation, any unbound secondary antibody molecules and T cells
were washed away with PBS.
FIG. S3(a) shows a fluorescence image of the FITC-labeled anti-mouse antibody IgG
imaged through a green filter (460-495 nm exciter and 510 nm emitter; U-MWIB3, Olympus).
FITC-labeled anti-mouse antibody IgG was selectively immobilized on the TEOS-treated
region of the surface and was suppressed to be bound on the TEOS-treated region of the
surface. FIG. S3(b) shows the fluorescence image of the CMRA-labeled Jurkat T cells
obtained through a yellow filter (530–550 nm exciter and 575 nm emitter; U-MWIG3,
Olympus), further confirmed the conservation of the bioactivity of the immobilized TS2/4.
8
(a)
(b)
500 m
500 m
FIG. S3. Fluorescence images to evaluate the biofunctional activity of the immobilized TS2/4
mouse antibody by immunoreaction with FITC-labeled anti-mouse IgG and cell adhesion test
with Cell-tracking dye (CMRA; orange cell tracker)-labeled Jurkat T cells. (a) A green
fluorescence signal by the FITC-labeled anti-mouse IgG and (b) A orange fluorescence signal
by the CMRA-labeled Jurkat T cells were restricted in the region where TS2/4 was
immobilized only.
9