of 9
Copyright WILEY-VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 2016.
Supporting Information
for
Adv. Healthcare Mater.,
DOI: 10.1002/adhm.201601356
Biocompatible Multifunctional Black-Silicon for Implantable
Intraocular Sensor
Jeong Oen Lee
, Vinayak Narasimhan
, Juan Du
, Blaise
Ndjamen
, David Sretavan
, and
Hyuck Choo
*
Submitted to
1
Supporting Information for
Biocompatible Multifunctional Black
-
Silicon
for Implantable Intraocular Sensor
Jeong Oen Lee
+
, Vinayak Narasimhan
+
, Juan Du, Blaise Ndjamen, David S
re
t
a
van and Hyuck
Choo
*
[*] Prof. H. Choo, Corresponding
-
Author
hchoo@caltech.edu
[+] These authors contributed equally to this work
Dr. J.
O.
Lee, V.
Narasimhan,
Dr. B. Ndjamen,
Prof. H.
Choo
Department of Medical Engineering,
California Institute of Technology, Pasadena, CA, 91106, (USA)
Dr. J.
O.
Lee, Prof. H.
Ch
oo
Department of Electrical Engineering,
California Institute of Technology, Pasadena, CA, 91106, (USA)
Dr. J.
Du, Prof. D.
Sretavan
Department of Ophthalmology,
University of California, San Francisco, San Francisco, CA,
Submitted to
2
Device Working Principle
B
-
Si Surface Profile
Figure S1
.
(a)
The sensor primarily consists of an optomechanical cavity with a deformable
transparent top layer and a fixed reflective bottom layer. The initial air gap
(or cavity gap)
between the two layers
,
L
o
,
is 4 μm
. When the ambient pressure
changes
the top layer
deforms
,
thereby altering the air gap to L
f
. (b) Upon optically exciting the cavity with a NIR
tungsten light source, the reflected resonance spectrum is captured by a spectrometer. A
change in gap size results in a shift in the refle
cted resonance spectrum which can
subsequently be correlated to IOP values.
Figure S2
. SEM image at 6500x magnification of b
-
Si texturing with annotations indicating structure
height, width and spacing. Through SEM image processing, the RMS surface roughness was estimated
to be 4.63
μ
m. Scale: 5
μ
m.
Submitted to
3
Absolute Reflecti
vity Measurements
Figure
S
3
.
T
he
absolute
reflectiv
ity
of Si and
five
b
-
Si
samples
(categorized by process
time)
were characterized
. b
-
Si
samples were optimized to an
absolute reflectivity as low as
3.5% in the wavelength range of
interest.
A
NIR
high
-
reflectivity
mirror (R
avg
> 97%, Edmund
Industrial
Optics)
was used
as a
reference.
Submitted to
4
Fabrication Process Flow
The
top and bottom substrates that
form
the b
-
Si IOP sensor
were fabricated individually
.
Initially
the top structure consisting of a flexible
Si
3
N
4
membrane and a hollow circular opening in the center
was processed
. 2
μm
thick silicon dioxide
(SiO
2
) and 300
nm
thick silicon nitride (Si
3
N
4
) layers
were deposited o
n the top and bottom surfaces of a double
side
polished Si
wafer (thickness: 300
μm)
using thermal oxidation and low pressure chemical vapor deposition (LPCVD), respectively
(
Fig. S
4
T1
)
.
Then
the Si
3
N
4
and SiO
2
layers on the top surface of the wafer were completely
removed using reactive ion etching (RIE) (Plasmalab System 100 RIE/ICP, Oxford Instruments,
Inc.) and buffered hydrofluoric (BHF) acid.
Next, a 300
nm
thick aluminum oxide (Al
2
O
3
) layer
was deposited
using an e
-
beam evaporator (FC
-
1800 E
-
Beam Evaporator, Temescal) and patterned
the surface
using photolithography and plasma etch
ing (
Fig. S
4
T2
)
.
Using the patterned Al
2
O
3
layer
as a hard mask, the wafer
was
etched down via a Bosch
process (Plasmalab System 100 RIE/ICP,
Oxford Instruments, Inc.) to the SiO
2
etch stop at the bottom surface of the wafer
(
Fig. S
4
T
3)
.
Figure
S4
.
Illustration of the b
-
Si sensor
fabrication process flow. The sensor consists of
two
parts
(top and bottom)
individually
batch
-
fabricated and bonded
together
using medical
grade epoxy
to produce
a hermetically sealed
active core cavity. The diagram also illustrates
the seamless integration of the room
-
temperature b
-
Si process
in
to the sensor
fa
brication.
Submitted to
5
After removing the Al
2
O
3
hard mask by soaking
in
BHF
(
Fig. S
4
T
4
)
, the b
-
Si process (
SF
6
/O
2
mixed
-
mode plasma process
for
21
min
) described in the previous section was conducted to create
an anti
-
reflective surface on the inactive region of the hollow disk
(
Fig. S
4
T
5
)
.
During the b
-
Si
process, the Si
3
N
4
membrane located at the bottom of the wafer was protected by the sti
ll
-
present
SiO
2
etch
-
stop layer. After soaking in
BHF
in order to
remov
e
this SiO
2
, the Si
3
N
4
membrane was
released from the wafer
(
Fig. S
4
T
6
)
.
Individual parts were then diced from the processed wafer
using a through
-
wafer deep RIE (DRIE) process
(
Fig. S
4
T
7)
.
Next, the bottom part of the sensor was fabricated using another Si wafer
(
Fig. S
4B1
)
.
The mirror in
the center was obtained using a precisely controlled etch process that creates a 4 μm
-
deep recessed
surface
(
Fig. S
4B2, S4B3
),
and the concentric circ
ular trenches were patterned using another timed
DRIE run
(
Fig. S
4B4
)
.
The
through
-
wafer DRIE
was used again
to dice the processed wafer into
individual
parts
(
Fig. S
4B5, S4B6
)
.
Finally, a pair of top and bottom parts
were
aligned under a
microscope and assembled using a water
-
proof medical
-
grade epoxy
(
Fig. 2g
, Fig. S4
)
.
Submitted to
6
Slit
-
Lamp Measurement Setup
Variation in Peak Detection
a
b
Time (sec)
0
5
10
W
a
v
e
l
e
n
g
t
h
(
n
m
)
800
850
900
950
1000
IOP (mmHG)
6
8
10
W
a
v
e
l
e
n
g
t
h
(
n
m
)
860
880
900
920
940
960
980
Figure S
5
.
a)
Photograph of
a
slit
-
lamp
readout
setup with
a spectrometer and a CCD camera
connected to the slit
-
lamp ex
pansion ports. b) Photograph of the slit
-
lamp being used for
in
-
vivo
IOP measurements on an awake rabbit. The inset shows slit
-
lamp illumination in the
rabbit’s eye.
Figure S
6
.
(a)
Fluctuations of the major
peaks
during the 10
-
sec
in
-
vivo
measurement. Only
the top 30 highest SNR spectra were selected and plotted. The spectral fluctuation in the peak
locations is
within ±
1 nm
.
(b)
The
corresponding
IOP
for the spectrum was
7.3
mmHg and
the peak
-
to
-
peak fluctuation was less than
0.4 mmHg.
Submitted to
7
Immunofluorescence Analyses
Sample Preparation Protocol
1)
Paraformaldehyde (PFA) Fixation:
-
Upon explanting, rinse the sensor in 1xPBS/0.02% NaN
3
.
-
Incubate in PFA for at least 30 min at room temperature (RT) overnight (O/N) or longer at 4
o
C.
2)
Quenching:
-
Rinse sample in sterile 1x PBS (gently shaking for 5 min)
x3
.
-
Incubate sample in freshly prepared 1xPBS/0.02% NaN
3
/50 mM NH
4
Cl (quenching buffer) for 10
min at RT.
-
Rinse sample in sterile 1xPBS/0.02% NaN
3
(gently shaking for 5 min)
x3
.
3)
Blocking:
-
Incubate sample in 1xPBS/0.02% NaN
3
/2% BSA/0.25% TritonX100 (blocking buffer) at 4
o
C with
gentle shaking O/N.
4)
Staining:
-
Incubate sample in 1mL blocking buffer/DAPI 405 1:2000/CD62L 488 1:500/Phalloidin 555 1:1000
(staining reagent) at RT with shaking for
1 hr.
-
Incubate in staining reagent at 4
o
C O/N.
5)
Preparation for imaging:
-
Transfer sample into blocking buffer.
-
Wash in blocking buffer
x3
.
-
Incubate in blocking buffer at 4
o
C O/N.
-
Rinse sample in sterile 1xPBS/0.02% NaN
3
(gently shaking for 5 min)
x3
.
-
Soak
sample in sterile 1xPBS/0.02% NaN
3
for imaging.
Submitted to
8
Imaging Methodology and Biofouling
Immune Response Comparison
Figure
S7
.
(a)
Illustration showing imaging depth (shown in red) of 300
μ
m starting from the
top of the rigid hollow disk to the top of the
Si
3
N
4
membrane.
(b)
Extent of sensor biofouling
was evaluated through assessing
the percentage of
the sensor active surface area (
highlighted
by the
white
dashed
circle)
in merged images (DAPI 405 and Phalloidin 555)
covered by
tissue for the Si
-
only
and b
-
Si sensors.
Figure
S8
.
Immune response comparison that indicates the presence of potential
inflammation
in the Si
-
only sensor
. The CD62L 488 marker is overlapped with DAPI 405 to
ascertain the location of immune cells.
Multiple instances of inflammation were observed in
the case of the Si
-
only sensor (indicated with white arrows). No inflammation was detected in
the b
-
Si sensor.