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Gaining Micropattern Fidelity in an NOA81 Microsieve Laser Ablation Process

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We studied the micropattern fidelity of a Norland Optical Adhesive 81 (NOA81) microsieve made by soft-lithography and laser micromachining. Ablation opens replicated cavities, resulting in three-dimensional (3D) micropores. We previously demonstrated that microsieves can capture cells by passive pumping. Flow, capture yield, and cell survival depend on the control of the micropore geometry and must yield high reproducibility within the device and from device to device. We investigated the NOA81 film thickness, the laser pulse repetition rate, the number of pulses, and the beam focusing distance. For NOA81 films spin-coated between 600 and 1200 rpm, the pulse number controls the breaching of films to form the pore’s aperture and dominates the process. Pulse repetition rates between 50 and 200 Hz had no observable influence. We also explored laser focal plane to substrate distance to find the most effective ablation conditions. Scanning electron micrographs (SEM) of focused ion beam (FIB)-cut cross sections of the NOA81 micropores and inverted micropore copies in polydimethylsiloxane (PDMS) show a smooth surface topology with minimal debris. Our studies reveal that the combined process allows for a 3D micropore quality from device to device with a large enough process window for biological studies.
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micromachines
Article
Gaining Micropattern Fidelity in an NOA81 Microsieve Laser
Ablation Process
Rahman Sabahi-Kaviani and Regina Luttge *


Citation: Sabahi-Kaviani, R.; Luttge,
R. Gaining Micropattern Fidelity in
an NOA81 Microsieve Laser Ablation
Process. Micromachines 2021,12, 21.
https://dx.doi.org/10.3390/mi12010
021
Received: 11 December 2020
Accepted: 23 December 2020
Published: 27 December 2020
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional claims
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affiliations.
Copyright: © 2020 by the authors. Li-
censeeMDPI, Basel, Switzerland. This
articleis an open accessarticle distributed
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Creative CommonsAttribution (CCBY)
license(https://creativecommons.org/
licenses/by/4.0/).
Neuro-Nanoscale Engineering, Mechanical Engineering Department and Institute of Complex Molecular Systems,
Eindhoven University of Technology (TU/e), 5600MB Eindhoven, The Netherlands; r.sabahi.kaviani@tue.nl
*Correspondence: r.luttge@tue.nl; Tel.: +31-40-247-5235
Abstract:
We studied the micropattern fidelity of a Norland Optical Adhesive 81 (NOA81) microsieve
made by soft-lithography and laser micromachining. Ablation opens replicated cavities, resulting in
three-dimensional (3D) micropores. We previously demonstrated that microsieves can capture cells
by passive pumping. Flow, capture yield, and cell survival depend on the control of the micropore
geometry and must yield high reproducibility within the device and from device to device. We
investigated the NOA81 film thickness, the laser pulse repetition rate, the number of pulses, and the
beam focusing distance. For NOA81 films spin-coated between 600 and 1200 rpm, the pulse number
controls the breaching of films to form the pore’s aperture and dominates the process. Pulse repetition
rates between 50 and 200 Hz had no observable influence. We also explored laser focal plane to
substrate distance to find the most effective ablation conditions. Scanning electron micrographs
(SEM) of focused ion beam (FIB)-cut cross sections of the NOA81 micropores and inverted micropore
copies in polydimethylsiloxane (PDMS) show a smooth surface topology with minimal debris. Our
studies reveal that the combined process allows for a 3D micropore quality from device to device
with a large enough process window for biological studies.
Keywords: microsieve; replica molding; NOA81; excimer laser micromachining
1. Introduction
Laser micromachining has gained popularity as a fabrication method for microsystems
that need specific materials to be processed [
1
5
]. Such a requirement can also be found
in the quest for advanced 3D tissue models [
6
11
]. Several studies have demonstrated
the importance of mechanical properties such as stiffness and curvature in the design of
in vitro
cell cultures [
12
14
]. The design of proper 3D microenvironments enhances cell
adherence and maintains the cells’ natural spherical 3D morphology [
12
,
15
,
16
]. Among
other authors in the literature [
17
,
18
], we have also shown that there is a difference in
neuronal differentiation and organization as well as total neurite length between 2D and
3D cell cultures [
19
21
]. In this work, we focus on the optimization of a microfabrication
process for a microsieve structure, i.e., arrayed 3D micropores. Originally, these microsieves
were made with a high pattern fidelity by silicon micromachining, as we demonstrated to
pair single cells to electrodes [
22
]. This technique was developed by Schurink et al. with a
high potency for cell capture yield and survival [
23
]. Additive manufacturing using photo-
curable polymers is an alternative for photo-lithography processes and can directly yield a
final product structure, which allows for the implementation of complex 3D-multilayer
patterns in a cost-accessible manner [
24
27
]. Such capabilities have been explored for
breast cancer cell culture [
28
]. Furthermore, fabrication of 3D multilayers patterns for
neuronal culture using photo curable materials has rapidly received attention [
29
,
30
].
Therefore, lately, we also demonstrated a microsieve structure that was similar to that
developed by Schurink et al. but was made from a photo-curable optical adhesive (NOA81),
and it showed an even higher cell survival than silicon [
31
]. Microsieves made from
polymer materials, specifically using NOA81, known as an optical adhesive, offer new
Micromachines 2021,12, 21. https://dx.doi.org/10.3390/mi12010021 https://www.mdpi.com/journal/micromachines
Micromachines 2021,12, 21 2 of 13
capabilities in this field of application. NOA81 micromachining took place via drop-casting
in double replica molding and subsequent excimer laser ablation, resulting in funnel-like
3D micropores. Generally, it is known that the material’s properties and specific geometries
of the microenvironment are important factors in cell differentiation [
32
]. In this paper, we
introduce the control of parameters of the replica molding and laser ablation processes for
3D micropores in more depth.
Consequently, we investigated polydimethylsiloxane (PDMS)-to-NOA81 replica mold-
ing via spin-coating and ultraviolet (UV) exposure to create NOA81 microcavities in the
polymeric foils of a range of defined thicknesses, and thus prepared patterned substrates
for ablation tests to control the specific 3D geometry of the pores. For all ablation tests, a
krypton fluoride (KrF) nanosecond excimer laser setup was used in order to investigate
how the 3D micropore’s geometry depends on the pulse repetition frequency, the number
of pulses, and the laser beam focus distance adjustment relative to the patterned substrate
surface. The new results found in this study resemble our previous laser ablation results
in NOA81 but improve upon the reproducibility of the microfabrication process. This
method of micromachining allows for a rapid prototyping approach to further explore
design features for microsieve-assisted cell capturing.
2. Materials and Methods
2.1. NOA81 Replica Molding from Silicon Microsieve Electrode Array
NOA81 (Norland Optical Adhesive 81) microsieves were fabricated by a double replica
molding procedure fully described by Moonen et al. [
31
]. In brief, the microsieve pattern of
an original micro-Silicon Electrode Array (
µ
SEA) fabricated previously by Schurink during
his PhD work at University of Twente [
33
] was transferred into polydimethylsiloxane
(PDMS) using standard soft lithography and NOA81 replica molding. In order to better
appreciate improvements yielded in this paper, the fabrication process is depicted in
Figure 1
for completeness. First, the
µ
SEA, here used as a master mold, was blow-cleaned
using an N
2
gun (Figure 1i). Afterwards, a PDMS base and cross-linking agents (SYLGARD
184, Dow Corning, Midland, MI, USA), i.e., the two liquid components of PDMS, were
mixed at a ratio of 10:1, and the mixture was placed inside a vacuum desiccator for 20 min
to degas. After removal of the air bubbles, the mixture was poured on the
µ
SEA with
a thickness of around 500
µ
m, and the PDMS was cured by placing the
µ
SEA on a hot
plate at 95
C for 10 min (Figure 1ii). The PDMS was peeled off from the master mold
and cut into a square of 2
×
2 cm
2
, acting as an inverted mold for the subsequent NOA81
replica molding step. Here, we investigated spin-coating instead of drop-casting to yield a
well-defined NOA81 film thickness for ensuing detailed laser ablation studies. Since the
PDMS mold needs to be hydrophilic to allow a wetting of NOA81 during spin-coating, a
plasma oxidation step at 10 W for 30 s was performed using a plasma asher (EMITECH
K1050X, Quorum, Laughton, UK) (Figure 1iii). NOA81 liquid was poured in excess on
the surface of the PDMS mold shortly after plasma activation covering the full area of the
mold. Next, spin-coating was performed in three consecutive steps: first at 500 rpm for
30 s with an acceleration of 200 rpm/s, then at a speed depending on the desired final
thickness for 60 s (Figure 1iv) and with an acceleration of 300 rpm/s, and finally with a
deceleration of 300 rpm/s until it stopped. The mold with the NOA81 film was placed
inside a UV-LED exposure system (IDONUS, UV-EXP 150R, Neuchatel, Switzerland) with
an intensity set to 15 mW/cm
2
. Pre-curing of the NOA81 films was achieved when they
received a UV dosage yielding 2100 mJ/cm
2
(Figure 1v). A higher amount of UV dosage or
higher intensity hampers the separation between PDMS and NOA81, and a lower dosage
will not sufficiently pre-cure it for peeling. Although UV dosage may also depend on the
thickness of photo-cured films, we have not observed this effect in the range of thicknesses
that we have produced with NOA81. After photo-curing, the NOA81 film was therefore
peeled off quickly (Figure 1vi). A delay in this step can lead to a strong bond and make
peeling off impossible. During the peeling-off step, the thin NOA81 can bend or roll over,
so the process should be practiced with caution. After peeling the film off the PDMS
Micromachines 2021,12, 21 3 of 13
mold, the resulting thin NOA81 foil contained the microcavities and was placed on a flat
substrate, such as on a paper tissue atop of a glass slide, with as little wrinkles as possible,
and put back inside the UV exposure system once more with the above-mentioned settings
to be completely cured. To assess how the thickness of patterned NOA81 foils depends
on spin-coating speeds, the thickness of the cured polymer foil was measured using a
profilometer (HEIDENHAIN, Traunreut, Germany). In this step, thickness measurements
were performed on a flat region close to the cavities. For an easier handling of the NOA81
foil during laser micromachining and cell culture, the foil was mounted on a ring made
of polymethyl methacrylate (PMMA) with a thickness of either 0.5 mm or 1 mm and an
inner and an outer diameter of 13 mm and 18 mm, respectively (Figure 1vii). The two
pieces were attached by transferring a small amount of NOA81 onto the PMMA ring using
a sharp tip and placing the foil on it. The two pieces were then assembled firmly after a UV
light curing step with the settings previously mentioned. It is important to keep in mind
which side of the polymer chip has the microcavity structures when mounting the foil on
the ring, which serves as a simple holder. The last step in the process was the laser ablation,
to yield the microsieve with its arrayed 3D micropores. This step is discussed in Section 2.2,
and all results per step are presented in Section 3. An example of a fabricated microsieve
mounted on the holder is shown in Figure 2a. A 3D SOLIDWORKS screenshot depicts
the assembly with 900 square pyramidal-shaped microcavities laying within a circular
area with a 2.4 mm diameter in the center in Figure 2b, and a zoom-in thereof shows the
pyramidal shape and spacing of the micropores at scale in Figure 2c. In our design, the
pyramidal structure extends at its tip into a uniform diameter through-hole. Its diameter
is indicative of the exit hole size, which corresponds to a 3D micropore geometry that is
expected to capture an individual cell.
2.2. Laser Ablation of the NOA81 Microsieve
The NOA81 foils containing the microcavities, which were mounted on a PMMA
holder, were placed inside the Optec MicroMaster KrF-Laser set-up (Optec S.A., Frameries,
Belgium) that produces ultraviolet light with a wavelength of 248 nm and a pulse duration
of 5–6 ns for laser ablation treatment (Figure 1viii) [
34
]. In this step, the laser beam is
positioned at the location of the tip of a pyramid, i.e., in the center of the microcavity, and
the laser generates a through-hole. Afterwards, this process is repeated until the required
number of through-holes forming the microsieve is achieved. The tool is equipped with
a servo motor control that can produce movements of 1
µ
m in all three coordinates, and
the so-called LightDeck system allows us to define the ablation position relative to the
surface of the foil within the center of the prepatterned microcavities. The pulse energy is
kept constant over time by the tool’s automatic laser voltage adjustment up to a maximum
limit, and the gas must be exchanged for consistency in the machining process thereafter.
Although in this class of lasers the thermal damage is minimal per pulse, ablation in
the thermal and the plasma regime can take place. The size of the generated laser beam
after passing the de-magnifying objective can be reduced to a diameter of at least 10
µ
m.
Given the size of neuronal cells, which is also around a diameter of 10
µ
m, the resultant
opening using this laser beam size depends also on the ablation depths and could be wider
than required for efficient capturing cells by passive capillary flow [31]. To explore which
parameters of the laser ablation process affect the geometry and quality of the resulting
feature dependent on the NOA81 film thickness, several different settings have been tested.
These include the pulse repetition frequency, the number of laser pulses, and the vertical
distance of the surface of NOA81 foil to the height where the beam is focused by visual
inspection. The recommended working range of frequency of the laser is up to 150 Hz;
however, to investigate the effect of this parameter, we performed experiments with 50,
100, 150, and 200 Hz. Furthermore, the number of laser pulses on each position can be
increased to even tens of thousands of pulses; however, only increasing this parameter will
not increase the ablated depth without refocusing the laser beam in z-direction. Our results
Micromachines 2021,12, 21 4 of 13
and discussion regarding the ablation parameters for gaining micropattern fidelity in the
fabrication process for microsieves are further refined in Section 3.
Micromachines 2021, 12, x 3 of 13
bond and make peeling off impossible. During the peeling-off step, the thin NOA81 can
bend or roll over, so the process should be practiced with caution. After peeling the film
off the PDMS mold, the resulting thin NOA81 foil contained the microcavities and was
placed on a flat substrate, such as on a paper tissue atop of a glass slide, with as little
wrinkles as possible, and put back inside the UV exposure system once more with the
above-mentioned settings to be completely cured. To assess how the thickness of pat-
terned NOA81 foils depends on spin-coating speeds, the thickness of the cured polymer
foil was measured using a profilometer (HEIDENHAIN, Traunreut, Germany). In this
step, thickness measurements were performed on a flat region close to the cavities. For an
easier handling of the NOA81 foil during laser micromachining and cell culture, the foil
was mounted on a ring made of polymethyl methacrylate (PMMA) with a thickness of
either 0.5 mm or 1 mm and an inner and an outer diameter of 13 mm and 18 mm, respec-
tively (Figure 1vii). The two pieces were attached by transferring a small amount of
NOA81 onto the PMMA ring using a sharp tip and placing the foil on it. The two pieces
were then assembled firmly after a UV light curing step with the settings previously men-
tioned. It is important to keep in mind which side of the polymer chip has the microcavity
structures when mounting the foil on the ring, which serves as a simple holder. The last
step in the process was the laser ablation, to yield the microsieve with its arrayed 3D mi-
cropores. This step is discussed in Section 2.2, and all results per step are presented in
Section 3. An example of a fabricated microsieve mounted on the holder is shown in Fig-
ure 2a. A 3D SOLIDWORKS screenshot depicts the assembly with 900 square pyramidal-
shaped microcavities laying within a circular area with a 2.4 mm diameter in the center in
Figure 2b, and a zoom-in thereof shows the pyramidal shape and spacing of the mi-
cropores at scale in Figure 2c. In our design, the pyramidal structure extends at its tip into
a uniform diameter through-hole. Its diameter is indicative of the exit hole size, which
corresponds to a 3D micropore geometry that is expected to capture an individual cell.
Figure 1. Replica molding and laser ablation process workflow for NOA81 microsieve. From a clean
silicon electrode microsieve array (µSEA), (i) a negative replica is created by casting polydime-
thylsiloxane (PDMS) (10:1 w/w ratio) followed by curing at 95°C and peeling off (ii). The PDMS
mold is then oxygen-plasma-treated with 10 W for 30 s to become hydrophilic and allowing for a
Figure 1.
Replica molding and laser ablation process workflow for NOA81 microsieve. From a
clean silicon electrode microsieve array (
µ
SEA), (
i
) a negative replica is created by casting poly-
dimethylsiloxane (PDMS) (10:1 w/wratio) followed by curing at 95
C and peeling off (
ii
). The
PDMS mold is then oxygen-plasma-treated with 10 W for 30 s to become hydrophilic and allowing
for a wetting of NOA81 during spinning (
iii
). An excess of NOA81 is poured onto the mold and
spin-coated at 500 rpm for 30 s followed by whatever speed results in the final thickness for 60 s (
iv
).
The NOA81 is then cured under ultraviolet (UV) light with an energy amount of 2100 mJ/cm
2
at an
intensity of 15 mW/cm
2
(
v
). NOA81 is then peeled off quickly (
vi
) and is mounted on a polymethyl
methacrylate (PMMA) ring (
vii
). The assembly is then submitted to an excimer laser station to
produce the through-holes in the pyramid structures by ablation (viii).
Micromachines 2021, 12, x 4 of 13
wetting of NOA81 during spinning (iii). An excess of NOA81 is poured onto the mold and spin-
coated at 500 rpm for 30 s followed by whatever speed results in the final thickness for 60 s (iv). The
NOA81 is then cured under ultraviolet (UV) light with an energy amount of 2100 mJ/cm2 at an in-
tensity of 15 mW/cm2 (v). NOA81 is then peeled off quickly (vi) and is mounted on a polymethyl
methacrylate (PMMA) ring (vii). The assembly is then submitted to an excimer laser station to pro-
duce the through-holes in the pyramid structures by ablation (viii).
Figure 2. (a) A fabricated NOA81 foil mounted on a PMMA ring for ease of handling. All the 900
pyramid-shaped microcavities are in the central circle with a diameter of 2.4 mm, of which a selec-
tion is subsequently ablated to form the arrayed 3D micropores. (b) A 3D SOLIDWORKS sketch of
the resulting microsieve chip. (c) Zoom-in of the anticipated 3D micropore design at scale.
2.2. Laser Ablation of the NOA81 Microsieve
The NOA81 foils containing the microcavities, which were mounted on a PMMA
holder, were placed inside the Optec MicroMaster KrF-Laser set-up (Optec S.A., Framer-
ies, Belgium) that produces ultraviolet light with a wavelength of 248 nm and a pulse
duration of 5-6 ns for laser ablation treatment (Figure 1viii) [34]. In this step, the laser
beam is positioned at the location of the tip of a pyramid, i.e., in the center of the micro-
cavity, and the laser generates a through-hole. Afterwards, this process is repeated until
the required number of through-holes forming the microsieve is achieved. The tool is
equipped with a servo motor control that can produce movements of 1 µm in all three
coordinates, and the so-called LightDeck system allows us to define the ablation position
relative to the surface of the foil within the center of the prepatterned microcavities. The
pulse energy is kept constant over time by the tool’s automatic laser voltage adjustment
up to a maximum limit, and the gas must be exchanged for consistency in the machining
process thereafter. Although in this class of lasers the thermal damage is minimal per
pulse, ablation in the thermal and the plasma regime can take place. The size of the gen-
erated laser beam after passing the de-magnifying objective can be reduced to a diameter
of at least 10 µm. Given the size of neuronal cells, which is also around a diameter of 10
µm, the resultant opening using this laser beam size depends also on the ablation depths
and could be wider than required for efficient capturing cells by passive capillary flow
[31]. To explore which parameters of the laser ablation process affect the geometry and
quality of the resulting feature dependent on the NOA81 film thickness, several different
settings have been tested. These include the pulse repetition frequency, the number of
laser pulses, and the vertical distance of the surface of NOA81 foil to the height where the
beam is focused by visual inspection. The recommended working range of frequency of
the laser is up to 150 Hz; however, to investigate the effect of this parameter, we per-
formed experiments with 50, 100, 150, and 200 Hz. Furthermore, the number of laser
pulses on each position can be increased to even tens of thousands of pulses; however,
only increasing this parameter will not increase the ablated depth without refocusing the
laser beam in z-direction. Our results and discussion regarding the ablation parameters
for gaining micropattern fidelity in the fabrication process for microsieves are further re-
fined in Section 3.
3. Results and Discussion
3.1. NOA81 Device Replication
Figure 2.
(
a
) A fabricated NOA81 foil mounted on a PMMA ring for ease of handling. All the
900 pyramid-shaped microcavities
are in the central circle with a diameter of 2.4 mm, of which a
selection is subsequently ablated to form the arrayed 3D micropores. (
b
) A 3D SOLIDWORKS sketch
of the resulting microsieve chip. (c) Zoom-in of the anticipated 3D micropore design at scale.
3. Results and Discussion
3.1. NOA81 Device Replication
The material used in this article for the fabrication of microsieves is NOA81. It is a
single component adhesive liquid that will cure within a few seconds to a few minutes to a
Micromachines 2021,12, 21 5 of 13
tough and hard polymer when exposed to ultraviolet (UV) light [
35
]. This polymer was
previously proposed as an alternative for PDMS in microfluidic chip applications [
36
] and
has been used for low-cost microfluidic and microchannel fabrication [
37
]. In this work,
the double replica molding procedure with PDMS as the negative copy of the original
µ
SEA and NOA81 as the final device material was improved to achieve a certain thickness
of the polymer microsieve structure. In a previous work reported by
Moonen et al. [31]
,
the final device thickness varies between 60 and 110
µ
m, which potentially makes a
difference in fluidic performance. To prevent this variation, we chose to spin-coat NOA81
on the PDMS mold instead of drop-casting and an application of capillary force to flatten
the film. By spin-coating, we assure that NOA81 results in a well-defined thickness on
the PDMS mold dependent on the spin-coating speed, yielding consistent final devices.
Additionally, given the limitation of our laser source being recently refurbished since
our preliminary work described in Moonen et al. [
31
], we found that making through-
holes in an NOA81 foil without a well-defined thicknesses was not reproducible enough
within the given ablation parameters of our preliminary work, which mainly focused
on the demonstration of the new biological application. Hence, a process window had
to be explored more thoroughly. Furthermore, using NOA81 films with well-defined
and equal thicknesses, laser ablation within a controlled parameter space must produce
micropores with highly reproducible features, including geometry and shape. Only devices
made based on defined design specifications will then also yield a well-defined micropore
geometry and result in controlled microfluidic and cell capture performance from device to
device. Having control over the microfluidic performance of the microsieves is essential,
and single cells aimed to be captured inside the pores initially should also be supported
throughout the culture time by the microsieve scaffold and kept alive. Single cells might
react differently to variant flow speeds and shear stresses. Therefore, it is essential to
keep the 3D micropore geometry consistent for repetitive sets of culture experiments by
providing devices within circumscribed error margins. To define quality measures in the
reproducible performance of the fabrication of NOA81 foils, we measured the resulting
foil thickness as a parameter of the spin-speed. Figure 3shows the data points as well
as the fitted spin-curve. These outcomes confirm that fabrication of microsieves with
a thickness close to what was reported by Schurink et al. [
22
] for the original
µ
SEA is
now also achievable for NOA81 microsieves. Therefore, spin-coating provides a better
condition for comparing the device performance across the fabrication of devices made
at different timeslots in the laboratory. Unfortunately, this method of fabrication relies
on a piece-by-piece process; hence, variations in the process are critical. One potential
challenge in the spin-coating step is that the PDMS mold carries a convex pattern. It is
known in the field of microfabrication methods that spin-coating over topography can
lead to non-uniform film formation [
38
,
39
]. With regard to the features in this work,
planarization of the convex patterns was achieved with the selected spin-coat protocol
described in the method
Section 2.1
. Still, minor deviations on film thickness could occur
and a cross-sectional study on 3D pore geometry across the rows and columns of the
entire microsieve could reveal error margins on film thickness in more detail. In addition,
spin-coating results in very flexible foils, which intrinsically lead to difficulties in handling
during laser micromachining and cell loading procedure. However, applying assembly to
a mechanically stable PMMA ring solves this issue and allows convenient handling and
alignment, as can be seen in Figure 2a. A cured NOA81 replica after laser treatment step
has been imaged, and the scanning electron micrograph (SEM) is shown in Figure 4. The
900 pyramidal microcavities from the original
µ
SEA have been faithfully replicated into
the NOA81 foil. The sharp edges of the pyramidal pores are an indication of a successful
replication with high precision. Moreover, the traces of electrodes in the original
µ
SEA
with a height of 220 nm were also transferred into the NOA81 foil, which is highlighted in
Figure 4with yellow arrows. This confirms that the added steps to the fabrication process,
including plasma treatment and spin-coating, do not negatively affect the replication of
even very fine details.
Micromachines 2021,12, 21 6 of 13
Micromachines 2021, 12, x 6 of 13
of a successful replication with high precision. Moreover, the traces of electrodes in the
original µ SEA with a height of 220 nm were also transferred into the NOA81 foil, which
is highlighted in Figure 4 with yellow arrows. This confirms that the added steps to the
fabrication process, including plasma treatment and spin-coating, do not negatively affect
the replication of even very fine details.
Figure 3. NOA81 foil thickness against spin-coating speeds. The curve is a quadratic fit to the data.
The function equation of the curve is        and the R-squared value for
the fitting curve is 0.978.
Figure 4. Fabricated NOA81 microsieve using spin-coating shows a successful replica with the
nanometer high features of the original SEA (yellow arrows).
3.2. Laser Ablation of NOA81
The next step in the fabrication procedure is laser ablation at the tip of the pyramid-
shaped microcavities. Achieving a specific 3D micropore geometry depends on the pulse
repetition frequency, the laser focus location, and the number of pulses. Pulse repetition
frequency refers to the number of pulses sent per second. The influence of the changing
pulse rate has not yet been observed in depth, but, in theory, it should affect the surface
morphology. Figure 5a shows the quality of the pores for an NOA81 foil spin-coated at
800 rpm, subsequently processed with excimer laser ablation at the center of the
microcavities, but at variant pulse repetition rates. The frequency is 50, 100, 150, and 200
Hz. There is not much difference in the quality of the pore geometry and the surface
properties in this range of frequencies when inspected by standard top-down SEM or
optical microscopy. Therefore, a solid conclusion regarding the influence of repetition
frequency on the ablation depth, shape, and produced debris in this laser process cannot
yet be drawn. Figure 5b presents the pulse repetition test to compare the exit hole size and
Figure 3.
NOA81 foil thickness against spin-coating speeds. The curve is a quadratic fit to the data.
The function equation of the curve is
y=
2.2
×
10
5x2
0.059
x+
53 and the R-squared value for the
fitting curve is 0.978.
Micromachines 2021, 12, x 6 of 13
confirms that the added steps to the fabrication process, including plasma treatment and
spin-coating, do not negatively affect the replication of even very fine details.
Figure 3. NOA81 foil thickness against spin-coating speeds. The curve is a quadratic fit to the data.
The function equation of the curve is 𝑦2.210

𝑥
 0.059𝑥  53 and the R-squared value for
the fitting curve is 0.978.
Figure 4. Fabricated NOA81 microsieve using spin-coating shows a successful replica with the na-
nometer high features of the original μSEA (yellow arrows).
3.2. Laser Ablation of NOA81
The next step in the fabrication procedure is laser ablation at the tip of the pyramid-
shaped microcavities. Achieving a specific 3D micropore geometry depends on the pulse
repetition frequency, the laser focus location, and the number of pulses. Pulse repetition
frequency refers to the number of pulses sent per second. The influence of the changing
pulse rate has not yet been observed in depth, but, in theory, it should affect the surface
morphology. Figure 5a shows the quality of the pores for an NOA81 foil spin-coated at
800 rpm, subsequently processed with excimer laser ablation at the center of the micro-
cavities, but at variant pulse repetition rates. The frequency is 50, 100, 150, and 200 Hz.
There is not much difference in the quality of the pore geometry and the surface properties
in this range of frequencies when inspected by standard top-down SEM or optical micros-
copy. Therefore, a solid conclusion regarding the influence of repetition frequency on the
ablation depth, shape, and produced debris in this laser process cannot yet be drawn.
Figure 5b presents the pulse repetition test to compare the exit hole size and geometry of
the ablated micropores of an NOA81 foil spin-coated at 800 rpm followed by ablation with
800 pulses at the four different frequencies. Although the exit hole (also called the bottom
hole or the aperture of the 3D micropore) results in an irregular shape, there is no other
0 200 400 600 800 1000 1200
Spin (rpm)
0
5
10
15
20
25
30
35
40
Thickness ( m)
Figure 4.
Fabricated NOA81 microsieve using spin-coating shows a successful replica with the
nanometer high features of the original µSEA (yellow arrows).
3.2. Laser Ablation of NOA81
The next step in the fabrication procedure is laser ablation at the tip of the pyramid-
shaped microcavities. Achieving a specific 3D micropore geometry depends on the pulse
repetition frequency, the laser focus location, and the number of pulses. Pulse repetition
frequency refers to the number of pulses sent per second. The influence of the changing
pulse rate has not yet been observed in depth, but, in theory, it should affect the surface
morphology. Figure 5a shows the quality of the pores for an NOA81 foil spin-coated at
800 rpm, subsequently processed with excimer laser ablation at the center of the microcavi-
ties, but at variant pulse repetition rates. The frequency is 50, 100, 150, and 200 Hz. There
is not much difference in the quality of the pore geometry and the surface properties in this
range of frequencies when inspected by standard top-down SEM or optical microscopy.
Therefore, a solid conclusion regarding the influence of repetition frequency on the ablation
depth, shape, and produced debris in this laser process cannot yet be drawn. Figure 5b
presents the pulse repetition test to compare the exit hole size and geometry of the ablated
micropores of an NOA81 foil spin-coated at 800 rpm followed by ablation with 800 pulses
at the four different frequencies. Although the exit hole (also called the bottom hole or
the aperture of the 3D micropore) results in an irregular shape, there is no other obvious
difference. However, the higher the frequency is, the quicker the production will be. This
is more critical for the ablation of an entire array of 10
×
10, which will be set as a standard
for our future cell capturing experiments or when even more holes should be opened, as
this parameter will influence production throughput. Since frequencies above 150 Hz are
Micromachines 2021,12, 21 7 of 13
not recommended for this laser tool, we used 150 Hz for our detailed experiments as an
upper limit.
Micromachines 2021, 12, x 7 of 13
obvious difference. However, the higher the frequency is, the quicker the production will
be. This is more critical for the ablation of an entire array of 10 × 10, which will be set as a
standard for our future cell capturing experiments or when even more holes should be
opened, as this parameter will influence production throughput. Since frequencies above
150 Hz are not recommended for this laser tool, we used 150 Hz for our detailed experi-
ments as an upper limit.
50 Hz
100 Hz
150 Hz
200 Hz
(a) (b)
Figure 5. (a) SEM top view and (b) optical microscopy (×100) bottom view of four different mi-
cropores ablated with the same laser parameters except for different pulse repetition rates: 50, 100,
150, and 200 Hz, respectively.
A parameter of the laser equipment that influences the geometry of the 3D micropore
significantly is the laser beam focus. If the laser is well-focused onto the ablation region
inside the pyramidal shape, the maximum energy will be given to the surface; hence, ab-
lation will be performed effectively at a minimal number of pulses. On the other hand, if
it is focused poorly, the energy will be spread in a larger area, consequently reducing the
energy delivered to the part. Since the laser light is beyond the visible wavelength, the
LightDeck camera system facilitates the focusing of the laser onto the ablation region. To
explore the effect of an off-set in adjusting the focal plane, first the mask patterned was
imaged in focus onto the surface of the NOA81 foil using the off-axis light source, and the
position was set to Z = 0 as the reference plane. Thereafter, the laser was moved in the Z-
direction for a range of distances, and the ablation results were compared to each other.
This test was done from Z = 60 µm (meaning the distance of the lens relative to the foil
surface was moved down compared to the reference plane) to Z = +140 µm. Figure 6 pro-
vides an overview of the results achieved by changing the height of the focal plane in
reference to the foil surface. The resulting apertures for each of the settings were observed
and imaged by an optical microscope with a ×100 objective. Moreover, Figure 6 presents
the aperture diameter size dependent on different positions of the laser, relative to the
reference plane and based on a quality definition of the irregular shape. One expects the
best result with an outer and inner diameter ratio close to one and an inner diameter be-
tween 3 and 4 μm (the design of the through-hole diameter in our application). The results
indicate that, for the distance Z between +40 and +50 µm (not all result shown), the inner
aperture diameter was in its maximum size. Therefore, +45 μm was chosen for all subse-
quent laser experiments performing an analysis of the number of pulses against aperture
shape.
Figure 5.
(
a
) SEM top view and (
b
) optical microscopy (
×
100) bottom view of four different microp-
ores ablated with the same laser parameters except for different pulse repetition rates: 50, 100, 150,
and 200 Hz, respectively.
A parameter of the laser equipment that influences the geometry of the 3D micropore
significantly is the laser beam focus. If the laser is well-focused onto the ablation region
inside the pyramidal shape, the maximum energy will be given to the surface; hence,
ablation will be performed effectively at a minimal number of pulses. On the other hand, if
it is focused poorly, the energy will be spread in a larger area, consequently reducing the
energy delivered to the part. Since the laser light is beyond the visible wavelength, the
LightDeck camera system facilitates the focusing of the laser onto the ablation region. To
explore the effect of an off-set in adjusting the focal plane, first the mask patterned was
imaged in focus onto the surface of the NOA81 foil using the off-axis light source, and
the position was set to Z = 0 as the reference plane. Thereafter, the laser was moved in
the Z-direction for a range of distances, and the ablation results were compared to each
other. This test was done from Z =
60
µ
m (meaning the distance of the lens relative
to the foil surface was moved down compared to the reference plane) to Z = +140
µ
m.
Figure 6provides an overview of the results achieved by changing the height of the focal
plane in reference to the foil surface. The resulting apertures for each of the settings were
observed and imaged by an optical microscope with a
×
100 objective. Moreover, Figure 6
presents the aperture diameter size dependent on different positions of the laser, relative to
the reference plane and based on a quality definition of the irregular shape. One expects
the best result with an outer and inner diameter ratio close to one and an inner diameter
between 3 and 4
µ
m (the design of the through-hole diameter in our application). The
results indicate that, for the distance Z between +40 and +50
µ
m (not all result shown),
the inner aperture diameter was in its maximum size. Therefore, +45
µ
m was chosen for
all subsequent laser experiments performing an analysis of the number of pulses against
aperture shape.
Micromachines 2021,12, 21 8 of 13
Micromachines 2021, 12, x 8 of 13
Figure 6. Micropores were ablated with the same laser parameters, except for the vertical distance
of the beam focus. This height difference ranged from 60 to +140 µm with 0 as a reference plane.
Within steps of 5 μm around a +45 μm off-set, the aperture size maintained an inner diameter be-
tween 3 and 4 micrometers (not all results are shown), so a +45 μm off-set from the reference plane
was chosen for subsequent experiments.
An important parameter in laser micromachining is the number of pulses applied to
the ablation region. This value should be high enough to produce through-holes but
should not make the micropore aperture too wide because cells are very flexible, and with
the cells’ diameter being around 10 µm, there is a risk that the cells slip through. Even in
holes with a diameter smaller than that of the cell size, cells might deform and squeeze
through such micropores in due course of the culture process [40]. Therefore, the fabrica-
tion should guarantee a laser exit hole, i.e., an aperture or bottom opening with a size
around 3 to 4 μm. Figure 7 shows the sequence of laser treatment where an intact micro-
cavity (Figure 7a) is under laser treatment. If the number of pulses is too low, it may not
make a through-hole at all (Figure 7b) or may make through-holes where the diameter of
the bottom openings (D
b
) is too small, yielding an overly high flow resistance, which is
not sufficient for cell capturing (Figure 7c). If the number of laser pulses is optimal, the
achieved D
b
is around 3 to 4 µm (Figure 7d). A higher number of pulses would then widen
the size of the bottom opening, which may ultimately result in a poor capturing rate (Fig-
ure 7e).
Figure 7. Schematic cross-sectional view of pyramidal-shaped micropores consisting of a combined
pyramidal and conical shape to form a 3D micropore. D
t
represents the diameter of the through-
hole top opening made by the laser ablation step in the process, where D
b
represents the bottom
diameter; (a) an intact microcavity prior to ablation; (b) a low number of pulses may not make a
through-hole at all or (c) may make a through-hole with a D
b
that is too small, which is not sufficient
for cell capturing; (d) for an optimal number of laser pulses, the achieved D
b
is around 3 to 4 µm;
(e) a higher number of pulses would widen the size D
b
, which may ultimately result in a poor cap-
turing rate.
Exploring the influence of the number of laser pulses on the geometry of the ablated
region and how the selection of this number affects microsieves with different thicknesses,
a set of laser experiments was performed with patterned NOA81 foils that were spin-
coated at 600, 800, 1000, and 1200 rpm. On each chip (foil assembled with a PMMA ring),
14 micropores were ablated, each with a different number of pulses but all with the same
repetition rate of 150 Hz and with the Z position set to +45 µm. The number of pulses
ranged between 100 and 2000. Since, in this experiment, the maximum number of pulses
Figure 6.
Micropores were ablated with the same laser parameters, except for the vertical distance
of the beam focus. This height difference ranged from
60 to +140
µ
m with 0 as a reference plane.
Within steps of 5
µ
m around a +45
µ
m off-set, the aperture size maintained an inner diameter between
3 and 4 micrometers (not all results are shown), so a +45
µ
m off-set from the reference plane was
chosen for subsequent experiments.
An important parameter in laser micromachining is the number of pulses applied to
the ablation region. This value should be high enough to produce through-holes but should
not make the micropore aperture too wide because cells are very flexible, and with the
cells’ diameter being around 10
µ
m, there is a risk that the cells slip through. Even in holes
with a diameter smaller than that of the cell size, cells might deform and squeeze through
such micropores in due course of the culture process [
40
]. Therefore, the fabrication should
guarantee a laser exit hole, i.e., an aperture or bottom opening with a size around 3 to 4
µ
m.
Figure 7shows the sequence of laser treatment where an intact microcavity (Figure 7a) is
under laser treatment. If the number of pulses is too low, it may not make a through-hole
at all (Figure 7b) or may make through-holes where the diameter of the bottom openings
(D
b
) is too small, yielding an overly high flow resistance, which is not sufficient for cell
capturing (Figure 7c). If the number of laser pulses is optimal, the achieved D
b
is around 3
to 4
µ
m (Figure 7d). A higher number of pulses would then widen the size of the bottom
opening, which may ultimately result in a poor capturing rate (Figure 7e).
Figure 7.
Schematic cross-sectional view of pyramidal-shaped micropores consisting of a combined
pyramidal and conical shape to form a 3D micropore. D
t
represents the diameter of the through-hole
top opening made by the laser ablation step in the process, where D
b
represents the bottom diameter;
(
a
) an intact microcavity prior to ablation; (
b
) a low number of pulses may not make a through-hole
at all or (
c
) may make a through-hole with a D
b
that is too small, which is not sufficient for cell
capturing; (
d
) for an optimal number of laser pulses, the achieved D
b
is around 3 to 4
µ
m; (
e
) a higher
number of pulses would widen the size Db, which may ultimately result in a poor capturing rate.
Exploring the influence of the number of laser pulses on the geometry of the ablated
region and how the selection of this number affects microsieves with different thicknesses,
a set of laser experiments was performed with patterned NOA81 foils that were spin-
coated at 600, 800, 1000, and 1200 rpm. On each chip (foil assembled with a PMMA ring),
14 micropores were ablated, each with a different number of pulses but all with the same
repetition rate of 150 Hz and with the Z position set to +45
µ
m. The number of pulses
ranged between 100 and 2000. Since, in this experiment, the maximum number of pulses
we had applied was 2000 pulses, with the pulse repetition rate of 150 Hz, the required time
for any though-hole ablation was 13.3 s. For the chosen range of pulses, we have observed
Micromachines 2021,12, 21 9 of 13
that 100 pulses did not result in any breaching of the foils regardless of their thickness.
Figure 8a depicts an overview of the results. As expected, laser ablation with a number
of pulses below a certain threshold does not produce a through-hole, which is depicted
schematically in Figure 7b. There was an increasing number of pulses above that threshold
(for the thinnest NOA81 foils, 200 pulses suffice to start breaching it); however, an increase
in the aperture size was observed. In addition, it can be seen from these figures that the
aperture area enlarges when thinner foils (i.e., those made with higher spin-coat speeds)
are ablated with the same number of pulses as thicker ones. This is a critical point because
it shows how important it is to maintain the thickness of the NOA81 foils from device to
device within narrow margins, which can be successfully accomplished by replacing the
previously applied NOA81 drop-casting step with a spin-coating step in the process. The
diameter of each aperture was estimated by projecting an outer and an inner circle onto
the resulting shape, as previously depicted in Figure 6. The aperture increased when the
number of pulses was increased, as anticipated, but it is important to realize that, if the
microsieves are thinner, the previously demonstrated funnel shape, resulting from the laser
process, will be lost. The shape of these 3D micropores made by the combination of replica
molding and laser ablation will then be more similar to the original geometry realized
by silicon micromachining, albeit with a circular aperture rather than a square aperture.
This is also shown in the next section via 3D micropore inspection using focused ion beam
(FIB)-cut and SEM analysis. These results are similar to our previous laser ablation results
in NOA81, as published by Moonen et al. [
31
]; however, here micropattern fidelity is gained
by controlling the NOA81 foil thickness.
Micromachines 2021, 12, x 9 of 13
we had applied was 2000 pulses, with the pulse repetition rate of 150 Hz, the required
time for any though-hole ablation was 13.3 s. For the chosen range of pulses, we have
observed that 100 pulses did not result in any breaching of the foils regardless of their
thickness. Figure 8a depicts an overview of the results. As expected, laser ablation with a
number of pulses below a certain threshold does not produce a through-hole, which is
depicted schematically in Figure 7b. There was an increasing number of pulses above that
threshold (for the thinnest NOA81 foils, 200 pulses suffice to start breaching it); however,
an increase in the aperture size was observed. In addition, it can be seen from these figures
that the aperture area enlarges when thinner foils (i.e., those made with higher spin-coat
speeds) are ablated with the same number of pulses as thicker ones. This is a critical point
because it shows how important it is to maintain the thickness of the NOA81 foils from
device to device within narrow margins, which can be successfully accomplished by re-
placing the previously applied NOA81 drop-casting step with a spin-coating step in the
process. The diameter of each aperture was estimated by projecting an outer and an inner
circle onto the resulting shape, as previously depicted in Figure 6. The aperture increased
when the number of pulses was increased, as anticipated, but it is important to realize
that, if the microsieves are thinner, the previously demonstrated funnel shape, resulting
from the laser process, will be lost. The shape of these 3D micropores made by the combi-
nation of replica molding and laser ablation will then be more similar to the original ge-
ometry realized by silicon micromachining, albeit with a circular aperture rather than a
square aperture. This is also shown in the next section via 3D micropore inspection using
focused ion beam (FIB)-cut and SEM analysis. These results are similar to our previous
laser ablation results in NOA81, as published by Moonen et al. [31]; however, here micro-
pattern fidelity is gained by controlling the NOA81 foil thickness.
(a)
(b)
Figure 8. (a) The view of the openings on the bottom side of four different NOA81 microsieves. Each
has been made with a specific spin-coat speed, which are 600, 800, 1000, and 1200 rpm. On each foil,
14 micropores were realized (selected ones are presented here) with an increasing number of pulses
but all with 150 Hz. The scale bar located in the lower right image is 5 µm for all images. (b) Spread
of the inner and outer diameter of the aperture plotted for each microsieve for an increasing number
Figure 8.
(
a
) The view of the openings on the bottom side of four different NOA81 microsieves. Each
has been made with a specific spin-coat speed, which are 600, 800, 1000, and 1200 rpm. On each foil,
14 micropores were realized (selected ones are presented here) with an increasing number of pulses
but all with 150 Hz. The scale bar located in the lower right image is 5
µ
m for all images. (
b
) Spread
of the inner and outer diameter of the aperture plotted for each microsieve for an increasing number
of laser pulses (the representation of the inner and the outer diameter of a projected circle onto the
resulting shape defines the irregularity).
Micromachines 2021,12, 21 10 of 13
3.3. Microsieves Characterization by Replica and FIB-Cut Sectioning
To examine whether the through-hole was realized with the expected geometry, a cross
section view is obtained. This work was done with an SEM/FIB workstation in Nanolab at
TU/e with an FEI Nova600i NanoLab (FEI-Thermofisher, Hillsboro, OR, USA). Figure 9
shows a single micropore before and after FIB-cut sectioning. The details of the parameters
used in SEM/FIB setup are presented in the Supplementary document. The cross-sectional
view of the 3D pore validates not only the control over position but also the geometry of
the pore. As expected, the bottom opening was narrower than the top opening, which
resembled a funnel-shape through-hole. Although there was no control over reducing the
size of the top opening, as this is determined by the laser beam diameter, which was 10
µ
m,
altering laser parameters, most importantly the number of pulses delivered to the ablation
region, results in different bottom opening areas. The cross-sectional view analysis made
by the FIB-cut tool helped to observe the surface topography on the pore and inside the
through-holes, as this is a potential feature whereby neurite outgrowth can be influenced.
The inner wall surface of the hole ablated by our KrF excimer laser system appeared to be
smooth. Since it is possible that the FIB-cut procedure influences these aspects, an inverted
PDMS copy was made to characterize the micropore surface and visualize the topography
and geometry of the through-holes. For this purpose, a small amount of PDMS was poured
onto a microsieve and peeled off after curing. This inverted PDMS copy of the structure
was also inspected by SEM, as demonstrated in Figure 10. Thanks to the high replication
capability of PDMS at the nanometer scale (as also confirmed in Figure 4), we expected
to transfer the surface relief of the micropore into the copy as well. Figure 10a shows a
wide view of the resulting shapes, including pyramids alone and pyramids with pillars.
Figure 10b provides a closer look at these shapes, where traces of the original 220-nm-thick
electrodes are visible (yellow arrows). Figure 10c provides a close-up of the PDMS copy and
shows the inverted shape of a single 3D micropore geometry with the conical through-hole,
also visualized by FIB-cut sectioning. The smooth interior surface can be noticed, especially
in comparison with the surface roughness on the pyramidal slopes of the 3D micropores,
which results from debris produced in the laser ablation step. This might not be the most
reliable means of verifying the height of a through-hole because the molded PDMS pillar
can be ruptured for any reason during peeling off and thus altering of the height can
occur. Still, this procedure provides for a genuine understanding of the interior surface
roughness of through-holes as well as the surface topography of the 3D micropores and
can be applied for a detailed shape analysis during process optimization. In fact, sectioned
views and inverted PDMS copy analysis together provide the means for further analysis
of the geometry and surface properties of 3D micropores in the quality control process of
manufacturing a larger number of chips. These factors are crucial during cell culturing and
neuronal growth, as it is expected that the cells will respond to mechanical cues during the
development stage.
Micromachines 2021,12, 21 11 of 13
Micromachines 2021, 12, x 11 of 13
(a)
(b)
Figure 9. Focused ion beam (FIB) cross-sectional view of a single micropore (a) before and (b) after
FIB-cut sectioning.
(a)
(b)
(c)
Figure 10. PDMS was cast on a microsieve with an assortment of laser ablated and intact microcav-
ities. The PDMS was then peeled off to demonstrate an inverted copy of the structure; (a) presents
a wide view of the resulting shapes. (b) A closer look at the pyramids where traces of the original
220-nm-thick electrodes are still visible (yellow arrows). (c) Close-up of a single PDMS pillar, which
delivers a genuine reproduction of the interior surface roughness as well as the surface topography
of the 3D micropore.
4. Conclusions
A double replica molding process was employed to fabricate an array of 900 micro-
cavities. In this matrix, individual cavities can be opened in a laser micromachining step
to result in a microsieve. Previously, we demonstrated that such a microsieve can be used
for pairing single cells to electrodes; here, we wanted to demonstrate that replica molding
and the laser ablation process together can be used to fabricate devices within defined
error margins. Image analysis of laser ablation tests studying film thickness, the number
of pulses, and focus plan variations revealed promising results within a sufficiently wide
process window that will also allow for the fabrication of microsieves of a quality fit for a
more extensive biological study. This microsieve scaffold-assisted culture platform can
now be made with enhanced micropattern fidelity thanks to the use of spin-coating to
define the NOA81 film thickness during soft-lithography. This control of NOA81 foil
thickness also improves the control of 3D micropore geometry during the through-hole
ablation process. Although the laser’s minimal beam diameter is 10 µm, it is shown that,
by selecting optimal laser parameters and confining the NOA81 foil thickness, repetitive
apertures with a small enough size for capturing cells can be achieved over a relatively
Figure 9.
Focused ion beam (FIB) cross-sectional view of a single micropore (
a
) before and (
b
) after
FIB-cut sectioning.
Micromachines 2021, 12, x 11 of 13
(a)
(b)
Figure 9. Focused ion beam (FIB) cross-sectional view of a single micropore (a) before and (b) after
FIB-cut sectioning.
(a)
(b)
(c)
Figure 10. PDMS was cast on a microsieve with an assortment of laser ablated and intact microcav-
ities. The PDMS was then peeled off to demonstrate an inverted copy of the structure; (a) presents
a wide view of the resulting shapes. (b) A closer look at the pyramids where traces of the original
220-nm-thick electrodes are still visible (yellow arrows). (c) Close-up of a single PDMS pillar, which
delivers a genuine reproduction of the interior surface roughness as well as the surface topography
of the 3D micropore.
4. Conclusions
A double replica molding process was employed to fabricate an array of 900 micro-
cavities. In this matrix, individual cavities can be opened in a laser micromachining step
to result in a microsieve. Previously, we demonstrated that such a microsieve can be used
for pairing single cells to electrodes; here, we wanted to demonstrate that replica molding
and the laser ablation process together can be used to fabricate devices within defined
error margins. Image analysis of laser ablation tests studying film thickness, the number
of pulses, and focus plan variations revealed promising results within a sufficiently wide
process window that will also allow for the fabrication of microsieves of a quality fit for a
more extensive biological study. This microsieve scaffold-assisted culture platform can
now be made with enhanced micropattern fidelity thanks to the use of spin-coating to
define the NOA81 film thickness during soft-lithography. This control of NOA81 foil
thickness also improves the control of 3D micropore geometry during the through-hole
ablation process. Although the laser’s minimal beam diameter is 10 µm, it is shown that,
by selecting optimal laser parameters and confining the NOA81 foil thickness, repetitive
apertures with a small enough size for capturing cells can be achieved over a relatively
Figure 10.
PDMS was cast on a microsieve with an assortment of laser ablated and intact microcavities.
The PDMS was then peeled off to demonstrate an inverted copy of the structure; (
a
) presents a wide
view of the resulting shapes. (
b
) A closer look at the pyramids where traces of the original 220-nm-
thick electrodes are still visible (yellow arrows). (
c
) Close-up of a single PDMS pillar, which delivers
a genuine reproduction of the interior surface roughness as well as the surface topography of the 3D
micropore.
4. Conclusions
A double replica molding process was employed to fabricate an array of 900 micro-
cavities. In this matrix, individual cavities can be opened in a laser micromachining step to
result in a microsieve. Previously, we demonstrated that such a microsieve can be used
for pairing single cells to electrodes; here, we wanted to demonstrate that replica molding
and the laser ablation process together can be used to fabricate devices within defined
error margins. Image analysis of laser ablation tests studying film thickness, the number
of pulses, and focus plan variations revealed promising results within a sufficiently wide
process window that will also allow for the fabrication of microsieves of a quality fit for a
more extensive biological study. This microsieve scaffold-assisted culture platform can now
be made with enhanced micropattern fidelity thanks to the use of spin-coating to define the
NOA81 film thickness during soft-lithography. This control of NOA81 foil thickness also
improves the control of 3D micropore geometry during the through-hole ablation process.
Although the laser’s minimal beam diameter is 10
µ
m, it is shown that, by selecting optimal
laser parameters and confining the NOA81 foil thickness, repetitive apertures with a small
enough size for capturing cells can be achieved over a relatively large process window.
FIB-cut cross-sectional views and inverted PDMS-copy SEM analysis confirmed the desired
geometry. The results also demonstrate a reproducible surface roughness and a minimal
Micromachines 2021,12, 21 12 of 13
debris formation on the surfaces of the 3D micropores’ pyramidal slopes and on the side
wall of the conical through-hole.
Supplementary Materials:
The following are available online at https://www.mdpi.com/2072-666
X/12/1/21/s1: the details of the parameters used in SEM/FIB setup.
Author Contributions:
R.L. acquired the funding. R.S.-K. and R.L. conceptualized and designed the
manuscript. R.S.-K. acquired the data. R.S.-K. and R.L. analyzed the data. R.S.-K. and R.L. interpreted
the data. R.S.-K. and R.L. prepared the manuscript. R.S.-K. and R.L. contributed equally to the final
approved version of the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding:
This project received funding from the European Union’s Horizon 2020 research and
innovation program H2020-FETPROACT-2018-01 under grant agreement No 824070.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing is not applicable to this article.
Acknowledgments:
The authors thank the members of the Microfab/lab at the Eindhoven University
of Technology for their experimental support. We also acknowledge Multiscale lab and Nanolab at
the Eindhoven University of Technology for SEM and FIB-cut tool support.
Conflicts of Interest:
The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict of interest.
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... Planar MEAs are well-known in the art of neuro-electrophysiology to perform dynamic studies of neural networks and add value as a simple-to-automate read-out in microfluidic brain-on-chips culture concepts, such as published by us recently [23]. Here, we also contributed by demonstrating improvements on the polymer microfabrication of microsieves [30]. These devices serve the idea to transform classical planar MEA systems into 3D read-outs but with the same ease as 2D cultures [31]. ...
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