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Multifunctional biophotonic nanostructures inspired by longtail
glasswing butterfly for medical devices
Vinayak Narasimhan
1,†
,
Radwanul Hasan Siddique
1,†
,
Jeong Oen Lee
1,2
,
Shailabh Kumar
1
,
Blaise Ndjamen
1
,
Juan Du
3
,
Natalie Hong
1
,
David Sretavan
3,*
, and
Hyuck Choo
1,2,*
1
Department of Medical Engineering, California Institute of Technology, 1200 E. California Blvd.,
MC 136-93, Pasadena, California 91125, USA.
2
Department of Electrical Engineering, California Institute of Technology, 1200 E. California Blvd.,
MC 136-93, Pasadena, California 91125, USA.
3
Department of Ophthalmology, University of California, San Francisco, San Francisco, CA 94143,
USA.
Abstract
Numerous living organisms possess biophotonic nanostructures that provide coloration and other
diverse functions for survival. While such structures have been actively studied and replicated in
the laboratory, it remains unclear whether they can be used for biomedical applications. Here we
show a transparent photonic nanostructure inspired by the longtail glasswing (
Chorinea faunus
)
butterfly and demonstrate its use in intraocular pressure (IOP) sensors
in vivo
. We exploit the
phase separation between two immiscible polymers (poly(methyl methacrylate) and polystyrene)
to form nanostructured features on top of a Si
3
N
4
substrate. The membrane thus formed shows
good angle-independent white light transmission, strong hydrophilicity and anti-biofouling
properties that prevent adhesion of proteins, bacteria, and eukaryotic cells. We then developed a
microscale implantable IOP sensor using our photonic membrane as an optomechanical sensing
element. Finally, we performed
in vivo
testing on New Zealand white rabbits and show that our
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.
*
These authors are co-corresponding authors. hchoo@caltech.edu, David.Sretavan@ucsf.edu.
These authors contributed equally to this work.
Author Contributions
V.N., R.H.S., and H.C. conceived the study. V.N. and R.H.S. designed the analyses while supervised by H.C. R.H.S. conducted the
microscopy and spectroscopy of the longtail glasswing butterfly. R.H.S. conducted the simulations and numerical analysis. V.N. and
R.H.S. fabricated and characterised the nanostructured Si
3
N
4
-membrane samples. V.N., R.H.S., S.K. and N.H. conducted the
in vitro
tests. V.N., J.L. and R.H.S. fabricated and characterised the benchtop IOP sensors. V.N., J.L. and J.D. performed the
in vivo
experiments under the supervision of D.S. V.N. and B.N. conducted the biocompatibility experiments of the
in vivo
IOP sensors. V.N.,
R.H.S. and H.C. co-wrote the manuscript with assistance from D.S. All authors discussed the results and commented on the
manuscript.
Data availability
The data that support the plots within this paper and other finding of this study are available from the corresponding author upon
reasonable request.
Supplementary information is available in the online version of the paper.
Competing financial interests
The authors declare no competing financial interests.
HHS Public Access
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Published in final edited form as:
Nat Nanotechnol
. 2018 June ; 13(6): 512–519. doi:10.1038/s41565-018-0111-5.
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device reduces the mean IOP measurement variation compared to conventional rebound tonometry
without signs of inflammation.
An estimated 8–10% of Americans and 5–6% of people in other developed nations depend
on implantable medical devices to support or rebuild organs and other functions of the body
during their lifetime
1
,
2
. Consequently, efforts to develop medical implant technologies are
increasing. A major deterrent to these efforts, however, has been the requirement to
incorporate multiple functionalities within a tightly constrained footprint while ensuring
acceptable
in vivo
performance and reliability
3
6
.Inspiration for engineering multifunctional
surfaces is often drawn from nature, which boasts a plethora of nanostructures with a wide
array of desirable properties
4
8
. For example, vertically-tapered needle-like nanostructures
found on the wings of insects exhibit multifunctionality including omnidirectional
antireflection, self-cleaning, antifouling, and bactericidal properties
9
13
. Such properties
may prove to be advantageous for biomedical applications such as
in vivo
sensing, imaging,
and stimulation.
Herein, we seek inspiration from the multifunctional biophotonic nanostructures found on
the transparent wings of the longtail glasswing (
Chorinea faunus
) butterfly to advance the
versatility of micro-optical implants whose practical use is often limited by the angle
dependency of sensing and readout processes
14
,
15
as well as short- and long-term
biofouling
15
17
. We characterised in detail the surface and optical properties of the short-
range-ordered nanostructures found on the
C. faunus
butterfly wings that could overcome
the shortcomings of micro-optical implants. We reveal that
C. faunus
relies on relatively
moderate-aspect-ratio (aspect-ratio
1) chitin nanostructures to produce (1) transparency
that is a unique combination of wavelength-selective anti-reflection and angle-independent
transmission resulting from isotropic Mie scattering, and (2) antifouling properties through
disruption of cellular growth similar to that observed on high-aspect-ratio (aspect-ratio > 1)
structures found in nature
12
,
13
. Drawing our inspiration from the
C. faunus
nanostructures,
we created low-aspect-ratio (aspect-ratio < 1) bio-inspired nanostructures on freestanding
Si
3
N
4
-membranes using a highly-scalable phase-separation-based polymer-assembly
process. Unlike previous high-aspect-ratio bio-inspired nanostructures replicating
antireflection
9
,
12
,
13
, we engineered the pseudo-periodic arrangement and dimensions of
nanostructures to control isotropic scattering and enhance omnidirectional optical
transmission, which could benefit sensing and readout processes of micro-optical implants.
In addition, improving from the anti-biofouling properties of high- and moderate-aspect-
ratio nanostructures that typically rely on physical cell lysis
12
,
13
,
18
, we engineered the low-
aspect-ratio nanostructures to generate strong nanostructure-mediated hydrophilicity and
anti-adhesion barrier for proteins and cellular fouling without inducing cell lysis and
inflammation.
To demonstrate the multifunctionality of bio-inspired nanostructures in medical use, we
combined the bottom-up nanofabrication approach with top-down microfabrication
processes and made a nanostructured micro-optical implant that senses intraocular pressure
(IOP) for diagnosis and management of glaucoma, which is a leading cause of irreversible
blindness globally
19
21
. We confirmed significant improvement in the bio-inspired
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nanostructured sensor’s optical readout angle, pressure-sensing performance, and
biocompatibility during a one-month
in vivo
study conducted in rabbits.
Multifunctional nanostructures of
C. faunus
butterfly
The
C. faunus
butterfly (Fig. 1a) belongs to the Riodinidae family found in South America.
We have discovered that the
C. faunus
wings are distinct from most other transparent wings
in nature
9
,
11
,
22
. They have a rare combination of two transparent regions that transmit light
differently: (1) the basal transparent areas close to thorax (indicated by a blue arrow in Fig.
1a); and (2) the postdiscal transparent areas further away from the thorax (indicated by a red
arrow in Fig. 1a) of both the forewing and hindwing. High-resolution scanning electron
microscopy (SEM) of the postdiscal transparent area (Fig. 1c) reveals dome-shaped
nanopillars with moderate aspect-ratios (1.090 ± 0.041; Fig. 1b and Supplementary Fig. S1),
compared to other natural transparent wings with higher aspect-ratios (>1)
10
12
.
Interestingly, the basal transparent area is composed of similarly shaped nanostructures at a
lower density (Fig. 1d). The two-dimensional fast Fourier transforms of the SEM images
showed ring-shaped distributions (insets of Fig. 1c and 1d), which confirmed their short-
range-ordered arrangements
11
,
23
. The finite diameters of the rings in
k
-space quantified the
average periods of the nanostructures as 140–180 nm and 200–300 nm for the postdiscal and
basal areas, respectively.
The zone-dependent variation in average inter-structural periods on the
C. faunus
wing plays
an important role in the extent of light scattering on the wing. The postdiscal area with an
average period below 200 nm remains scattering-free and anti-reflective in the ultraviolet
(UV)-visible (VIS)-near-infrared (NIR) regime, and this is well-explained by the effective
medium theory and the transfer-matrix model as in other anti-reflective subwavelength
nanostructures found in nature
11
,
24
(Supplementary Fig. S2). The basal area with an average
period exceeding 200 nm comparable to light wavelengths shows forward narrow-angle
scattering due to more sparsely-located, moderate-aspect-ratio low-index
nanostructures
25
,
26
. Our finite-difference time-domain simulations performed on both
groups of nanostructures for 420-nm wavelength produced matching results that confirmed
the scattering mechanism of the basal area (Fig. 1e–f). Although we used the same structural
height and diameter for both groups in the simulations, the nanostructures with a 150-nm
period (similar to the postdiscal area) did not alter the transmitted field (Fig. 1e), whereas
nanostructures with a 300-nm period (similar to the basal area) showed forward scattering of
the transmitted light (Fig. 1f). The scattering phenomenon of the basal region is moreover
confirmed by the difference observed between the specular transmittance and total
transmittance in the VIS-NIR range shown in Fig. 1h.
To further analyse the transmissive scattering properties of both areas, we have performed
the angle-resolved scattering spectroscopy in the VIS range and varied both the incident and
detection angles (Fig. 1i–j). Having almost identical specular and diffuse transmissions in
the VIS-NIR range (Fig. 1g), the postdiscal area exhibited specular transmittance with a low
scattering angle of ±3° (Fig. 1i). On the other hand, the basal area scattered light in the
forward direction with scattering angles up to ±12° and showed negligible changes with
incident angle variation, demonstrating its potentially very useful angle-independent
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scattering property (Fig. 1j). This scattering property could ameliorate the difficulty of
detecting optical signals at wide angles, a commonly observed challenge among many light-
based devices
14
such as implantable IOP sensors
15
(See Supplementary Section SI1 and Fig.
S3–S4 for more details on the biological significance of the multifunctional transparency
and its dual nano-structural basis present on
C. faunus
wings.)
Additionally, the periods of the nanostructures on
C. faunus
wings also influence the wetting
properties, and the static contact angles in the postdiscal and basal areas measured 105° and
85°, respectively. The contact angle in the postdiscal area is larger due to the higher surface
roughness
27
. In our experiments, these nanostructures with moderate aspect-ratios, similar to
nanostructures with high aspect-ratios, resist microbial and cellular growth (Supplementary
Fig. S5–S6).
Development of bio-inspired nanostructured membranes
Inspired by the nanostructures on the basal area of the
C. faunus
wings, we implemented
short-range-ordered nanostructures on Si
3
N
4
-membranes (Fig. 2a) by utilizing a highly
scalable bottom-up fabrication process based on polymer-phase separation
28
,
29
. Si
3
N
4
was
chosen for its ease of fabrication on Si and proven performance as an optically transparent
and mechanically robust freestanding membrane in microdevices
30
,
31
as well as for its
intrinsic hydrophilicity that is crucial to the antifouling property of the nanostructures as
discussed in more detail later in this work.
We created disk-shaped nanostructures with aspect-ratios ranging from 0.15 to 0.90 and
performed parametric studies to determine optical and anti-biofouling properties
(Supplementary Fig. S7). Nanostructures with an aspect-ratio of 0.45 (Fig. 2b), which was
smaller than the aspect-ratio of
1 observed in the nanostructures found on the
C. faunus
wings, were found to provide an optimal balance between the anti-biofouling and angle-
independent optical properties best-suited for optical implants (see Supplementary Section
SI2). Henceforth, the aspect-ratio of nanostructures integrated on the membrane is 0.45
unless stated otherwise.
The SEM image of the nanostructured Si
3
N
4
-membrane is shown in Fig. 2c. The 2D-fast
Fourier transform of the SEM image shown in the inset indicates a short-range order with a
mean period of 445 ± 60 nm, similar to the periodicity of the basal area. The contact angle
on the surface of the nanostructured Si
3
N
4
-membrane was 17°, suggesting an increase in
hydrophilicity compared to 38° measured on flat Si
3
N
4
without nanostructures
(Supplementary Fig. S8).
We characterised the optical properties of the nanostructured Si
3
N
4
-membrane using angle-
resolved transmission spectroscopy in the VIS-NIR range and compared the results to a flat
Si
3
N
4
-membrane without nanostructures (Fig. 2d and e). Using the nanostructures, the angle
independence of the Si
3
N
4
-membrane transmission was improved by 50%. 3D simulation of
the fabricated structures (Supplementary Fig. S9b) further confirms the improved angle-
independent transmittance. This angle-independent transmission results from the isotropic
nature of the forward scattering caused by the short-range-ordered nanostructures, which is
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irrespective of the incident angle (Fig. 1j). As the total transmission is the combination of
ballistic (specular) transmission through the thin membrane and scattered transmission
caused by the nanostructures (Supplementary Fig. S10)
32
, the angle-independent property of
the scattered component decreases the overall angle dependence of the total transmission.
Biophysical properties of the nanostructured surface
In vitro
testing compared the adhesion of representative proteins, prokaryotes, and
eukaryotes on nanostructured and flat Si
3
N
4
surfaces with lysine-coated glass slides as
positive controls. Flat Si
3
N
4
is moderately hydrophilic (contact angle: 35–40°) and known to
vigorously promote cell adhesion and proliferation due to increased adsorption of proteins
when compared to more hydrophilic surfaces (contact angle: <20°)
33
,
34
. Hence, we further
increased the hydrophilicity of the Si
3
N
4
surface by varying the aspect-ratios of the
nanostructures from 0.15 to 0.90 and systematically controlled surface hydrophilicity
(Supplementary Fig. S8). Once strong hydrophilicity is achieved (contact angle: <20°), a
nanostructure-mediated aqueous barrier forms on the surface and limits protein adsorption
and cell adhesion to provide an anti-adhesion property (Supplementary Section SI3,
Supplementary Fig. S11 and S16)
6
,
35
,
36
.
We initially investigated the surface adhesion of two representative proteins: (1) fluorescent-
labelled bovine serum albumin for its cardinal role in blood–material interactions
4
and high
non-specific binding affinity to the surfaces of biomaterials
37
; and (2) streptavidin for its
specific binding affinity to Si
3
N
4
surfaces
38
. Fluorescence-intensity-based quantification of
the adhesion force (Fig. 3a, 4b, and Supplementary Fig. S11) demonstrated adhesion on flat
Si
3
N
4
surfaces was three and two times greater compared with nanostructured Si
3
N
4
surfaces for albumin and streptavidin, respectively.
We then quantified bacterial adhesion using
E. coli
transformed with the green fluorescent
protein (Supplementary Fig. S12). In addition to being a popular prokaryotic model,
E. coli
was chosen for its pathogenic potential to cause gram-negative and often antibiotic-resistant
infections on and around implants
39
,
40
. Bacteria on each surface were quantified through a
measure of colony-forming units (CFU) (Fig. 3c) and fluorescence-intensity measurements
(Supplementary Fig. S13). Both results indicated significantly lower bacterial adhesion on
the nanostructured surface compared to flat Si
3
N
4
. Additionally, the SEM image of
individual bacterial cells on the nanostructured surface shows no disruption to their shape,
indicating no physical lysis (Supplementary Fig. S14).
The HeLa cell line was chosen as a representative eukaryote for its proven robustness,
aggressive growth rate, and adherent nature that prompts its frequent usage in adhesion and
cytotoxicity assays
41
,
42
. After 72 hours, the adherent cell density on the flat Si
3
N
4
was eight
times greater than that on the nanostructured Si
3
N
4
surface (Fig. 3d & 4e). Next, a mortality
ratio, the number of dead cells to the number of living cells, was computed for each surface
every 24 hours over a 72-hour period. The difference in the mortality ratios of the two
surfaces after 72 hours was not statistically significant (Supplementary Fig. S15), which
suggested the nanostructured surfaces inhibited eukaryote adhesion and proliferation without
inducing cell death.
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These results highlight the advantage of the anti-biofouling approach based on strong
hydrophilicity and anti-adhesion properties (Supplementary Fig. S11–S16). High or
moderate aspect-ratio nanostructures either with tapered sharp tips or dome-shaped tips as in
C. faunus
display potent geometry-dependent bactericidal properties that induce large
stresses and deformation on cell walls regardless of their surface chemical composition
43
and actively promote autogenous lysis when placed in contact with mammalian cells
44
. Such
anti-biofouling approaches relying on physical lysis could undesirably damage tissues
surrounding implants and elicit inflammation. Supplementary Table S1 shows physical lysis
occurs on either natural or synthetic nanostructured surfaces if the aspect-ratio of the
nanostructures is 1 or greater. Hence, by keeping the aspect-ratio of the nanostructures at
0.45, the anti-adhesion property was leveraged to prevent biofouling without causing any
physical lysis. Additionally, the hydrophilicity of the nanostructured surface originates from
surface topology, which may provide better long-term reliability over chemical-treatment
methods. (See Supplementary Section SI3 and Fig. S17.)
Use of nanostructures in intraocular pressure sensing
To demonstrate a medical application of multifunctional nanostructures, we used the
nanostructured Si
3
N
4
-membrane as an opto-mechanical sensing element in a microscale
implantable IOP sensor, which is a hermetically-sealed, pressure-sensitive, Fabry-Perot (FP)
resonator
15
. A flat-surfaced or nanostructured flexible Si
3
N
4
-membrane forms the top
surface of the FP-resonator while a mirror-like rigid Si forms the bottom surface. The sensor
is optimised in the NIR range for minimum absorption in tissue and water. If the ambient
pressure or IOP changes, the membrane will deflect accordingly, and the resulting shift in
the resonance wavelength will be captured remotely in reflection (Fig. 4a, Supplementary
Fig. S19). In
in vivo
testing, FP-based IOP sensors suffer from narrow readout angles that
severely limits sensor practical usability (Supplementary Fig. S20) and biofouling that
shortens sensor lifespan
15
.
To study the dependence on readout angle
θ
, we compared the measurements from a
nanostructured and a flat-surfaced IOP sensor at 1 atm (Fig. 4b–d). The flat-surfaced sensor
produced a maximum resonance shift of 16 nm at incident angle of 12° (Fig. 4b). In contrast,
the nanostructured sensor produced shifts of 2 nm at 12° and 5 nm at 30°. Decay in the
intensity of reflected resonance was also measured as a function of the incident angle (Fig.
4c). For the flat-surfaced sensor, the intensity decayed to zero when the incident angle
reached 12° while the signal from the nanostructured sensor remained detectable until 30°.
The IOP-measurement error of the flat-surfaced sensor reached 4.59 mmHg at 12° (Fig. 4d),
which is approximately 46% of the physiological IOP range observed in humans (10–20
mmHg) and exceeds the ±1.2 mmHg error range of existing clinical tonometers
45
,
46
. On the
other hand, the IOP-measurement error of the nanostructured sensor was 0.07 and 0.92
mmHg at 12° and 28°, respectively. These results highlight the wide-angle performance of
the nanostructured sensor. The nanostructured sensor showed excellent linearity (correlation
factor: ~1.00) over the clinical range of interest range from 0 to 30 mmHg when tested in a
pressure-controlled chamber interfaced with a digital pressure gauge (Fig. 4e). The
maximum readout error was 0.26 mmHg, approximately four times lower than that of the
flat-surfaced sensor (1 mmHg).
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A nanostructured and a flat-surfaced sensor were implanted individually inside the anterior
chambers of two New Zealand white rabbits to investigate
in vivo
optical performance and
biocompatibility (Fig. 5a). To examine the stability of sensor measurements, the shift
Δ
λ
of
the most prominent peak in each spectrum of the set was computed with respect to the mean
of the set (Fig. 5b). The standard deviation (s. d.) of
Δ
λ
of the nanostructured sensor was 0.6
nm as opposed to 1.3 nm observed for the flat-surfaced sensor (Fig. 5c). Additionally, the s.
d. of IOP measurements produced using the nanostructured sensor was 0.23 mmHg as
opposed to 0.64 and 1.97 mmHg calculated from measurements concurrently obtained using
the flat-surfaced sensor and tonometry, respectively (Fig. 5d). The angle independence
enhanced by the nanostructure integration improved the stability and accuracy of the optical
measurements against potential error sources such as respiratory movements, subtle eye
motions, and detector misalignment. Furthermore, indirect IOP measurement techniques
such as tonometry are influenced by various factors such as corneal thickness, curvature and
biomechanics and are in general more error-prone compared to direct IOP measurement
techniques such as implantable sensors
15
,
47
.
Both sensors were retrieved after one month of implantation to quantify cell growth on
surface and to assess biocompatibility. We used confocal fluorescence microscopy to
determine the extent of tissue growth and cellular viability at the time of retrieval. DAPI was
used to localize all constituent cells while Phalloidin, which selectively binds to actin, was
used as an indicator of cellular processes and health
48
. Additionally, matrix
metalloproteinases-2 (MMP-2) was used as an indicator of inflammation for its role in
various inflammatory and repair processes
49
.
Fig. 5e and 5f show the top views of the z-stacked multi-channel immunofluorescence
images of the flat-surfaced and the nanostructured sensors, respectively. Approximately 59%
of the flat-surfaced sensor was covered by tissue, and the presence of a vast filamentous F-
actin network (Fig. 5e, in green) indicates healthy tissue growth at the time of extraction.
Additionally, MMP-2 (Fig. 5e, in red) was observed over the membrane of the flat-surfaced
sensor, which could have triggered the extensive cell migration towards this region. In
comparison, approximately 5% of the nanostructured surface was covered by tissue, which
was a 12-fold improvement over the flat-surfaced sensor, and there was no detectable
MMP-2 signal, suggesting the cell signalling and migration patterns present on the flat-
surfaced sensor were absent on the nanostructured sensor. This indicates no inflammation
occurred post-implantation and highlights the promising role of the nanostructures towards
significantly improving
in vivo
biocompatibility of medical implants.
Conclusions
Inspired by the short-range-ordered nanostructures found on the wings of the transparent
longtail glasswing
C. faunus
butterfly, we engineered the biophotonic nanostructures
optimised for use in medical implants. By tuning the key physical dimensions of the
nanostructures, we have engineered structurally-induced scattering that expands an optical
readout angle and improved antifouling with supressed inflammation suitable for IOP-
sensing implants. In Glaucoma, accurate IOP monitoring is the only mainstay of disease
diagnosis and management
47
, and optical sensing approaches for IOP monitoring have been
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promising in terms of miniaturization, energy efficiency and frequency of monitoring
15
;
however they also require improvement in readout angle and biocompatibility for practical
use. The integration of the nanostructures on an IOP-sensing implant significantly expanded
its detection range while reducing mean
in vivo
IOP error by 3-folds. Further, the
nanostructures effectively suppressed biofouling and inflammation by 12-folds, resulting in
a highly practical implant for long-term IOP monitoring. Further development of our bio-
inspired work, including continuous IOP monitoring using mobile devices with an
integration of features such as memory-based tracking
50
, will improve glaucoma treatment
outcomes and lower the risk of visual impairment and blindness. With these promising
results, we envisage numerous medical technologies and devices will benefit greatly from
the multifunctionality of biophotonic nanostructures.
Methods
a. High resolution imaging
Dried wings of
C. faunus
, bought from Bicbugs LLC were coated with a 15 nm gold layer
(Lesker Labline E-beam Evaporator, Kurt J. Lesker Company®, USA) before examination
by scanning electron microscopy (SEM) (FEI Nova 200 NanoLab Dualbeam, USA)
operated at 5 kV.
b. Topographical analysis
ImageJ (
https://imagej.nih.gov/ij/
), a public domain and Java-based image processing
program tool, was used to perform the statistical analysis of the nanostructure size on the
wing membrane and fabricated nanostructured Si
3
N
4
samples. Every pixel in the images was
turned into either black or white using a threshold value that was obtained by calculating the
mean intensity value of all the pixels of the same image. Subsequently, the diameters and
areas of the nanostructures were computed. The 2-D Fourier power spectra were obtained
from SEM images and calculated with a fast Fourier transform algorithm in MATLAB.
c. Optical simulation
The thin film simulations of flat Si
3
N
4
-membrane and the postdiscal area were calculated
analytically using MATLAB
11
. The developed multilayer thin-film calculator is based on
matching the boundary conditions for Maxwell’s equations. Calculations were first done for
individual polarizations and by taking the average afterwards to consider the unpolarized
light ((TE+TM)/2) condition.
The optical properties of the basal area nanostructures were numerically simulated with the
2D finite-difference time-domain software (Lumerical Solutions Inc.®, Canada). The
transmittance of nanostructured membrane were numerically simulated with the 3D finite
element method (FEM) (COMSOL Multiphysics®, USA). In order to simulate exact optical
properties of the fabricated nanostructured samples, the measured 3D patterning profile was
directly imported from SEM in the simulated model. Periodic boundary conditions in the
lateral directions (
x
and
y
) were applied for the calculations. An incoming plane wave
impinging the structures under normal and oblique incidence was used and all calculations
were performed with a spatial resolution of 5 nm. In the simulation process, the boundary
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conditions of the electromagnetic fields in the vertical (z) direction were set on the perfect
matching layer (PML) for the model. The total transmittance was calculated for individual
geometries for unpolarised light ((TE + TM)/2) at normal and oblique incident angles by
integrating the near-zone scattered power (Poynting vector) over a surface before the bottom
PML and afterward normalizing with incident intensity.
d. Optical spectroscopic analysis
Specular transmission and scattering spectra of the
C. faunus
wings were measured using a
custom-built optical goniometric setup. A stabilized Tungsten-Halogen light source
(SLS201, ThorLabs, USA) was collimated to form a 500 μm wide parallel incident beam
that illuminates the sample at a fixed angle. The specular transmission and forward scattered
light was detected at fixed and different angles, respectively, with an angular resolution of 2°
and coupled into an optical fibre connected to the spectrometer (Flame, Ocean Optics,
USA). All measurements were recorded with an unpolarized light.
The diffuse transmittance measurements were performed using a commercial Cary 5000
Vis/NIR with integrating sphere. All measurements were recorded with unpolarised light.
The samples were placed in the middle of the integrating sphere using a vice-type centre-
mount and the sample holder was rotated around the vertical axis for angle-resolved
measurements. Transmission measurements were normalised to that of the uncovered area of
the underlying glass slide.
e. Biomimetic phase-separation through polymer blends and nanostructure texturing
Poly (methyl methacrylate) (PMMA,
M
w
= 9590, Polymer Standards Service GmbH,
Germany) and polystyrene (PS,
M
w
= 19100, Polymer Standards Service GmbH, Germany)
were dissolved in methyl ethyl ketone (MEK, Sigma-Aldrich Co. LLC, USA) with a mass
ratio of 65% and 35%. The concentration of the solutions was kept fixed to 25 mg/ml.
Solutions were spin-coated on the substrates with a speed of 3500 rpm and acceleration of
2000 rpm/s for 30 seconds. Relative humidity was maintained between 40% and 50% during
the spin coating. The de-mixing of the blend components out of the smooth surface occurred
during spin coating itself due to the difference in relative solubility of PS and PMMA in
MEK. Schematics of this complete 3D lateral phase separation process in humid
condition
28
. When the sample begins to spin, water condensation begins at humidity levels
above 35%. A layer of water-rich solution is formed at the air solution interface due to the
difference in evaporation rate between water and MEK. Water starts to condense from the air
into the solution because of the evaporation of MEK, which decreases the temperature on
top below the dew point. Because of the high water concentration, a 3-dimensional phase
separation occurs between PS/MEK and PMMA/MEK/water. When the film is completely
dried, a purely lateral morphology is formed and the PS islands end with an ellipsoidal
shape. The samples were then rinsed in cyclohexane for 2 minutes and dried in a stream of
N
2
to remove the PS islands. Using the PMMA layer as a template, a 30 nm Al
2
O
3
hard
masking layer was deposited via e-beam evaporation (CHA Industries Mark 40). After lift-
off, the Si
3
N
4
was textured through RIE (Oxford PlasmaLab 100 ICP380) resulting in the
nanostructured surface.
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f. Nanostructured membrane and sensor fabrication
The fabrication process flow of the nanostructured membrane and IOP sensor is provided in
Fig. S18. The sensor consists of two parts (top and bottom) that were individually batch-
fabricated and bonded together using a medical grade epoxy to produce a hermetically
sealed miniaturized Fabry-Perot (FP) cavity. (T1) The top substrate fabrication begins with 2
μm-thick SiO
2
and 400 nm thick Si
3
N
4
layers deposited on the top and bottom surfaces of a
double-side-polished (DSP) Si wafer (thickness: 300 μm) using thermal oxidation and low
pressure chemical vapour deposition (LPCVD), respectively. (T2) 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 oxide etch
(BOE). Next, a 300 nm-thick Al 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 BOE. (T3) Using the patterned Al
2
O
3
layer as a hard mask, the wafer
was etched using a Bosch process (Plasmalab System 100 RIE/ICP, Oxford Instruments,
Inc.) down to the SiO
2
etch stop at the bottom surface of the wafer. (T4) The SiO
2
layer was
removed through BOE to create freestanding Si
3
N
4
-membranes. The nanostructuring
process described in the previous section was used to structure the Si
3
N
4
-membrane. (T5)
Individual nanostructured membranes were released from the substrate through
photolithography and RIE from the backside. (B1) The bottom substrate fabrication begins
with a double-side-polished Si wafer (thickness: 300 μm). (B2) Using a photoresist mask, a
precisely controlled 4 μm recess was created through RIE to generate the FP cavity gap.
(B3) Next, a 300 nm-thick Al
2
O
3
layer was deposited using an e-beam evaporator and
patterned. (B4) Using a single Bosch process, concentric shallow trenches and a deep trench
were etched. The shallow trenches were created to serve as reservoirs during the epoxy
bonding process to prevent any of the adhesive from overflowing into the FP cavity. The
deep trenches were created for the easy separation of bottom chips (bottom parts of the
sensors) from the wafer. (B5) The Al
2
O
3
masking layer was removed in BOE. Finally, a
medical grade epoxy was applied along the sides of the top nanostructured membrane chip
and the bottom chip to create a hermetically sealed FP sensor implant.
g. Nanostructured sensor characterisation
The sensors were placed on a tilt stage, which allows for variation of the incident angle, and
were probed by a 20× objective lens interfaced with an NIR light source (HL-2000, Ocean
Optics), a mini-spectrometer (Maya200 Pro), Ocean Optics), and a CCD camera (Thorlabs,
Inc). For linearity measurements, the sensors were placed in a custom-build pressure-
controlled chamber. The hydrostatic pressure was between 0 to 32 mmHg and increased at
steps of 0.2 mmHg. The output from the sensors were referenced against a digital pressure
gauge (1210 Pressure Sensor, TE Connectivity Ltd.) with an accuracy of ± 0.5%. An in-
house IOP detection algorithm on MATLAB was used to rapidly calculate the IOP based on
the peak locations of the captured reflection spectra (Supplementary Fig. S15).
h. Protein adhesion assay
Fluorescently labelled bovine serum albumin (A13100, Thermo Fisher Scientific Inc.,
Waltham, MA, USA) protein with molecular weight of 66 kDa and streptavidin (S21375,
Narasimhan et al.
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