Supplemental material for
Photothermolysis of Immobilized Bacteria on Gold Nanograil arrays
Soo Kyung Kim,1 Chul-Joon Heo,1 Jong Young Choi,2 Su Yeon Lee,1 Se Gyu Jang,1 Jae Won
Shim,1 Tae Seok Seo,2 and Seung-Man Yang1a
1
National Creative Research Initiative Center for Integrated Optofluidic
Systems and Department of Chemical and Biomolecular Engineering, KAIST
335 Gwahangno, Yuseong-gu, Daejeon, 305-701, Korea.
2
Institute for BioCentury and
Department of Chemical and Biomolecular Engineering, KAIST
335 Gwahangno, Yuseong-gu, Daejeon, 305-701, Korea.
a)
Author to whom correspondence should be addressed. Electronic mail: smyang@kaist.ac.kr.
A. Fabrication of Gold Nanograils. Silica particles were synthesized by the seededgrowth method.1 Seeds were made by the sol-gel reaction in a biphasic oil-water
mixture using basic amino acid.2 First, a polystyrene (PS) film (7 wt%) was spin-cast
onto a silicon wafer (100) at 3000 rpm for 20 sec, then silica particles (1.2 m) were
self-assembled hexagonally by spin-casting onto the PS film. In the next step, these
particles were embedded into the PS polymer film at 115 ℃. The annealing temperature
was set sufficiently high above the glass transition temperature (Tg) of the PS. To etch
polymer and silica particles anisotropically, reactive-ion etching (RIE) (VSRIE-400A,
Vacuum Science) with CF4 and O2 gases were followed under 6.9×10-2 torr and 9.1×10-2
torr, respectively. Anisotropic etching of the PS polymer templates was achieved at 80
W, 60 sccm (standard cubic centimeters per minute). The resulting structure was coated
with gold (~ 90 nm thick) by sputtering and ion milling with argon gas was applied to
remove the gold coated on the top surfaces of the colloids and substrate. The DC bias
for the Ar ion milling was 400 V, and the Ar pressure and substrate temperature were
kept below 1×10-2 torr and 7 ℃, respectively. After exposure to Ar milling for 10 min,
two more steps were necessary: HF treatment and RIE with O 2. HF (5 wt%) was used to
remove silica particles, and RIE with O2 removed residual PS pillars (See Figure S1).
S1
FIG. S1. Schematic of the process for fabricating the gold nanograils via colloidal lithography.
S2
B. Controlling Processing Parameters. We controlled the process parameters to match
the ring size to the bacteria size, and to tune the LSPR peak in the laser controlling
range (780 ~ 920 nm). The ring size was controlled by varying the embedding and RIE
times. The embedding time was varied from 5 to 120 sec at 115 ℃. To check the
diameter of the dimple resulting from embedding, silica particles were removed by HF
and then were observed by SEM imaging (Figure S2). The dimple diameters in the array
were measured by SEM for each embedding procedure. Dimple diameter increased
approximately linearly with increasing embedding time. To achieve the first goal of
matching the ring size to the bacteria size, a 10 sec embedding time was selected
because to yield a bacterium mean size of 800 nm. Similarity of ring size and bacteria
size made it possible to capture one bacterium into each gold nanograil. After
embedding the silica particles, RIE with first CF4 then O2 was applied to prepare the
templates for gold nanograil fabrication. In this step, CF4 RIE etches silica particles, and
O2 RIE etches the PS film masking the silica particles. The RIE time for CF4 RIE was
varied from 6 to 10 min with a fixed O2 RIE time of 5 min. After RIE, a mushroom-like
template was fabricated. As shown in Figure S3, the diameter of silica particles
decreased with increasing RIE time. The top ring size was required to be bigger than the
size of bacteria in order to capture the bacteria easily. Thus, 8 min of RIE was deemed
optimal.
S3
FIG. S2. SEM images of the dimple arrays indicating embedded silica particles into PS film at
115 ℃ for (a) 5 sec, (b) 10 sec, (c) 20 sec, (d) 40 sec and (e) 60 sec. 10 sec is the optimal
embedding time. The scale bars are 2 m.
S4
FIG. S3. SEM images of mushroom-like structures in top view after CF4 and O2 RIE under 8
W and 60 sccm. CF4 RIE was carried out for (a) 6 min (b) 8 min and (c) 10 min with a fixed O2
RIE time of 5 min. (b) is the optimal condition. The scale bars are 2 m.
S5
C. Finite-difference time-domain (FDTD) simulation. A FDTD simulation was
performed as shown in Figure S4. At first, the designed gold nanograils S4(a, b) were
modeled, and the plasmon band was calculated with FDTD. A plasmon band in the NIR
region (830 nm) was calculated, which matched the measured LSPR, although the
calculated reflectance was higher than the experimental value. The next simulation step
assumed that the irradiation wavelength was 830 nm, and the electric field intensity
distribution after irradiation was calculated. The maximum electromagnetic field
enhancement was calculated to be 150×, and the edges of the upper and lower ring had
high electric field intensities. Thus, the calculated resonance energy and field
enhancement were predicted to photolyse bacteria efficiently. In conclusion, gold
nanograils were successfully simulated and fabricated, achieving the two goals
necessary to achieve photothermal cell lysis: optimal shape and optical properties for
successful capture and lysis, respectively.
S6
FIG. S4. FDTD simulation of the gold nanograil in (a) top view and (b) cross-sectional view. (c)
Electric field intensity distribution calculated by FDTD simulation under irradiation at the dip
wavelength, 830 nm. The reflectance spectrum calculated from FDTD simulation (d) and the
measured reflectance spectrum (e). The purple box indicates that the dips of these two spectra
exist within the same range of wavelength, ~ 830 nm.
S7
D. Functionalization of Gold Nanograils Surface with self-assembled monolayer
(SAM). Many researchers have investigated bacterial adhesion to various types of
functionalized surfaces with SAMs, because adhesion is an important step to during host
infection. Cooper et al.3 investigated bacterial adhesion on various SAM gold surfaces.
Their results showed that, among the various SAM treatments, methyl-terminated
SAMs yielded the highest level S. aureus adhesion. Gold nanograils were immersed into
a 1 mM ethanolic solution of 1-dodecanethiol for 8 hrs. The functionalized gold surface
was washed with ethanol immersed into a bacteria suspension for 1hr with shaking.
Finally, gold nanograil templates were washed twice with phosphate buffered saline
(PBS, pH 7.4). As a result, comparing with SAM surface and without SAM (Figure S5),
no difference in bacterial adhesion was observed between surfaces coated with SAMs
and those not coated with SAMs.
S8
FIG. S5. Confocal microscope images of the captured bacteria in the gold nanograils (a)
without SAM treatment and (b) with SAM treatment. Green fluorescence signals represent live
cells. The scale bars are 20 m.
S9
FIG. S6. Confocal microscope images of bacterial viability before and after heating at 90 ℃
for 1 min. The scale bar is 20 m.
S10
FIG. S7. Confocal microscope images of bacterial viability before and after irradiation at 60
mW/cm2 and 120 mW/cm2 with different irradiation times.
S11
References
1
W. Stöber, A. Fink, and E. Bohn, J. Colloid Interface Sci. 26, 62 (1968).
2
K. D. Hartlen, A. P. T. Athanasopoulos, and V. Kitaev, Langmuir 24, 1714
(2008).
3
V. A. Tegoulia and S. L. Cooper, Colloids and Surfaces B-Biointerfaces 24,
217 (2002).
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