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Microfabrication of Micropore Array for Cell Separation and Cell Assay

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Micromachines
DOI:
10.3390/mi9120620
Date:
November, 2018
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2018
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english
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PDF, 3.80 MB
micromachines
Article

Microfabrication of Micropore Array for Cell
Separation and Cell Assay
Yaoping Liu 1 , Han Xu 1 , Lingqian Zhang 1,2 and Wei Wang 1,3, *
1
2
3

*

Institute of Microelectronics, Peking University, Beijing 100871, China; yaopingliu@pku.edu.cn (Y.L.);
h.xu@pku.edu.cn (H.X.); zlqpku@gmail.com (L.Z.)
R&D Center of Healthcare Electronics, Institute of Microelectronics, Chinese Academy of Sciences,
Beijing 100029, China
National Key Laboratory of Science and Technology on Micro/Nano Fabrication, Beijing 100871, China
Correspondence: w.wang@pku.edu.cn; Tel.: +86-010-6275-2536

Received: 2 November 2018; Accepted: 21 November 2018; Published: 24 November 2018




Abstract: Micropore arrays have attracted a substantial amount of attention due to their strong
capability to separate specific cell types, such as rare tumor cells, from a heterogeneous sample
and to perform cell assays on a single cell level. Micropore array filtration has been widely used
in rare cell type separation because of its potential for a high sample throughput, which is a key
parameter for practical clinical applications. However, most of the present micropore arrays suffer
from a low throughput, resulting from a low porosity. Therefore, a robust microfabrication process for
high-porosity micropore arrays is urgently demanded. This study investigated four microfabrication
processes for micropore array preparation in parallel. The results revealed that the Parylene-C
molding technique with a silicon micropillar array as the template is the optimized strategy for
the robust preparation of a large-area and high-porosity micropore array, along with a high size
controllability. The Parylene-C molding technique is compatible with the traditional micromechanical
system (MEMS) process and ready for scale-up manufacture. The prepared Parylene-C micropore
array is promising for various applications, such as rare tumor cell separation and cell assays in
liquid biopsy for cancer precision medicine.
Keywords: mic; ropore array; Parylene-C; molding; cell separation; cell assay

1. Introduction
Micropore arrays have attracted lots of attention due to their capability in cell operations at the
single cell level, especially rare tumor cell separation and cell assays from a large volume of clinical
samples in the liquid biopsy. The competitiveness of micropore array-based filtration among the
developed techniques for liquid biopsy is its promising potential to realize a high throughput at
mL/min, along with a high recovery rate [1–4]. To fulfill the above challenges, the micropore array
needs to possess the following properties: (1) a uniform size, geometry, and density of pores to ensure
a high separation precision and a high recovery rate; (2) a large area and a high porosity, i.e., a small
sized supporting structure (edge-to-edge space) between the adjacent pores to realize a high filtration
throughput for the efficient operation of large-volume clinical samples; and (3) a small edge-to-edge
space (high porosity) to achieve a high purity of target cells via eliminating the non-specific adhesion
of non-target cells to facilitate the downstream analysis, such as gene sequencing and drug screening.
A symmetric review of the reported micropore arrays is summarized in Table 1. The earliest
reported micropore array is the polymer filtration membranes prepared via a track-etched method,
which can be traced back to the 1960s [5,6], and has been widely utilized in biological studies and
clinical practice for cell enrichment [7,8]. For the track-etched micropore array, it is easy to realize
Micromachines 2018, 9, 620; doi:10.3390/mi9120620

www.mdpi.com/journal/micromachines

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a large area; however, the size and geometry are uncontrollable (with fusion of two or more pores),
and the porosity is very low (less than 1%), resulting from the random placement of pores with a
relatively low density. During the last decade, several strategies via microfabrication techniques for
micropore arrays have been developed, with a precisely controlled size, geometry, and density of
pores [9–23]. Si micropore arrays with an area of 0.64 cm2 and porosity of 10% were produced via deep
reactive iron etching (DRIE) and KOH etching approaches by Wit et al. [9]. SU-8 micropore arrays with
an area of a 9-mm circle and a porosity of <12.5% were fabricated by Adams et al. [10–12]. A tapered
slit array of SU-8 with a porosity <11% was realized by Kang et al. [13]. A poly(ethylene terephthalate)
(PET) microcavity array [14] and nickel (Ni) [15] micropore array were prepared via laser drilling and
photolithography-based electroforming, respectively, by Hosokawa et al., with a porosity of <2.25%,
although the area was not difficult to extend to >1 cm2 . A mechanically strong polyethylene (glycol)
diacrylate (PEGDA) filter containing conical hole arrays via ultraviolet (UV)-assisted molding with an
area of a 6−9 mm circle and a porosity of <5.88% was fabricated by Tang et al. [16]. Fan et al. utilized a
sandwich molding technique (modified soft lithography) for the preparation of a polydimethylsiloxane
(PDMS) micropore-arrayed membrane from a microfabricated silicon micropillar-arrayed master.
This approach was cost-effective and could potentially be used for large-area fabrication (2.25 cm2 ).
However, the space between the adjacent pores in the produced membrane exceeded 14 µm, and its
porosity was still less than 20% [17]. Several types of Parylene C filtration membrane of microspring
structures [18], rectangular-pore arrays (porosity of 18%) [19], and circular-pore arrays (area of 1 cm2
and porosity of <5.6%) [20] were obtained via photolithography-based micropatterning and oxygen
plasma etching. Three-dimensional (3D) double-layered Parylene C micropore arrays (area of 1 cm2
and porosity of <6.96%) [21] and 3D palladium micropocket arrays [22] (area of 1 cm2 and porosity of
<5.02%) were also reported.
Table 1. Summary and comparison of the reported micropore arrays.
Ref. No.

Material

Fabrication Strategy

Area

Porosity 1

Edge-to-Edge Space

8

Polycarbonate

Track-etching technique

1 cm2

N/A

N/A (Randomly
distributed)

9

Silicon

Photolithography-based micropatterning,
and DRIE and KOH etching

0.64 cm2

10%

9 µm

cm2

10−12

SU-8

Photolithography-based micropatterning

<12.5%

13 µm

13

SU-8

Photolithography-based micropatterning

1 cm2

6.2%/11%

N/A

14

PET

Photolithography-based micropatterning
and laser drilling

4 cm2

0.008%

58 µm

15

Ni

Photolithography-based micropatterning
and electroforming

1 cm2

0.64%

51 µm

16

PEGDA

Micropatterning and UV-assisted
molding

0.81 cm2

3.25−5.88%

22−24.5 µm

17

PDMS

Modified soft lithography

2.25 cm2

20%

14.2−18.1 µm

18

Parylene-C

0.5 cm2

N/A

N/A

19

Parylene-C

0.36 cm2

18%

N/A

20

Parylene-C

1 cm2

<5.6%

10/12 µm

21

Parylene-C

1 cm2

<6.96%

11/12 µm

5.02%

4/26 µm

22

Palladium

Photolithography-based micropatterning
and oxygen plasma etching

Photolithography-based micropatterning
and electroforming

0.64

1

cm2

1

The definition of porosity is the ratio of the opening area (i.e., the total area of micropores) to the whole area of
the filtration membrane (i.e., the area of micropores plus the area of supporting structures named edge-to-edge
space). Some calculations were performed according to the provided parameters in references to extract the values
of porosity for comparison.

From the above, the uniformity in size and geometry of the previously reported micropore
arrays have already presented a good controllability, benefiting from the microfabrication
techniques. Additionally, a relatively large area was achievable for some of the mentioned approaches
[8,13–15,17,20–22]. However, the low porosity, resulting from a relatively large edge-to-edge space

In this study, four different approaches for preparation of the demanding large-scale and
high-porosity micropore arrays were investigated in parallel for comparison. They include the
micropatterning and etching of silicon (Figure 1a), micropatterning and etching of Parylene-C
(Figure 1b), Parylene-C molding with a PDMS micropillar array as the template (Figure 2a), and the
Parylene-C molding technique with a silicon micropillar array as the template (Figure 2b). The
Micromachines 2018, 9, 620
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fabricated micropore arrays via the above four processes were displayed in Figures 3, 4, 5, 6,
respectively. Furthermore, three different layout designs of micropore-arrayed membranes in the
Parylene-C
with
a silicon
template
werefor
tried,
in order
to acquire
the optimal
between
themolding
adjacenttechnique
pores, was
still
a serious
challenge
all the
reported
micropore
arrays;
version
7).for a high filtration throughput, a critical index in the operation of large-volume
while
it (Figure
is critical

clinical samples. The elevation of porosity was restricted by the mechanical strength and the process
2. Materials
and Methods
difficulty,
considering
that the high porosity of micropore arrays may cause a large deformation
at a high filtration throughput, resulting in a decrease of the size separation accuracy. In short,
2.1. Micropatterning and Etching of Silicon for Micropore Array Preparation
a microfabrication technique for uniformly packed micropore arrays of a large area and a high porosity
is urgently
required.
The micropatterning
and etching process for silicon micropore arrays is schematically shown in
In 1a.
this
study,
four different approaches
forand
preparation
the depth
demanding
Figure
First,
the lithography-based
patterning
DRIE of a of
20 μm
on the large-scale
top surface and
of a
high-porosity
micropore
arrayswere
wereperformed,
investigated
in parallel
for sequential
comparison.
They include
the2
double-polished
silicon wafer
followed
by the
depositions
of SiO
micropatterning
etching
silicon (Figure
etching
of Parylene-C
(thickness at 1000and
Å ) and
Si3Nof
4 (thickness
at 10001a),
Å ) micropatterning
via low pressure and
chemical
vapor
deposition
(Figure
1b),
Parylene-C
molding
with aphotolithography-based
PDMS micropillar array
as the template
(LPCVD),
as shown
in Figure
1a1. Then,
micropatterning
and (Figure
reactive 2a),
ion
and
the (RIE)
Parylene-C
with a silicon
micropillar
array
the template
(Figure1a2).
2b).
etching
of Si3Nmolding
4 and SiOtechnique
2 were performed
on the bottom
surface
of aassilicon
wafer (Figure
The
micropore
arrays
via the
processes
weresurface
displayed
Figure
Figure
4,
Next,fabricated
a KOH bath
was used
to etch
theabove
siliconfour
from
the bottom
untilinthe
SiO 23,layer
was
Figure
5, Figure
6, Si
respectively.
three
different
layout
designs of
micropore-arrayed
removed,
and the
3N4 (on the Furthermore,
other side) layer
was
exposed,
sequentially
(Figure
1a3). Finally,
membranes
in the Parylene-C
molding
technique
with
a siliconafter
template
were tried,
in order
silicon micropore
arrays of a 20
μm thickness
were
obtained
the removal
of SiO
2 and to
Si3acquire
N4 in a
the
optimal
version (Figure
7).
buffered
hydrofluoric
acid (BHF)
bath (Figure 1a4).

Figure 1.
Schematic of
of microfabrication
microfabrication process
process via
via micropatterning
micropatterning and
and etching
etching of
of Si
Si (a) and
Figure
1. Schematic
Parylene-C
(b)
for
micropore
arrays
preparation.
Parylene-C (b) for micropore arrays preparation.

2. Materials and Methods
2.2. Micropatterning and Etching of Parylene-C for Micropore Array Preparation
2.1. Micropatterning
and Etching
Silicon process
for Micropore
Array Preparation
The micropatterning
and of
etching
of Parylene-C
for micropore array preparation is
schematically
shown
in
Figure
1b.
First,
Parylene-C
of
a
10
μm
thickness
titanium (Ti)
of a
The micropatterning and etching process for silicon micropore
arrays isand
schematically
shown
3000
Å thickness
sequentially deposited
on a single-polished
1b1).surface
Then,
in
Figure
1a. First,were
the lithography-based
patterning
and DRIE of asilicon
20 µmwafer
depth(Figure
on the top
photolithography
and
etching
(with
RIE
and
BHF,
in
parallel
for
comparison)
were
performed
of a double-polished silicon wafer were performed, followed by the sequential depositions of SiOto2
prepare theatTi
mask
subsequent
etching of Parylene-C (Figure 1b2). Next, RIE of oxygen
(thickness
1000
Å) for
andthe
Si3 N
4 (thickness at 1000 Å) via low pressure chemical vapor deposition
plasma was
to etch
Parylene-C
untilphotolithography-based
the surface of silicon wafer
was exposed, and
followed
by ion
the
(LPCVD),
asused
shown
in Figure
1a1. Then,
micropatterning
reactive
etching (RIE) of Si3 N4 and SiO2 were performed on the bottom surface of a silicon wafer (Figure 1a2).
Next, a KOH bath was used to etch the silicon from the bottom surface until the SiO2 layer was
removed, and the Si3 N4 (on the other side) layer was exposed, sequentially (Figure 1a3). Finally, silicon
micropore arrays of a 20 µm thickness were obtained after the removal of SiO2 and Si3 N4 in a buffered
hydrofluoric acid (BHF) bath (Figure 1a4).
2.2. Micropatterning and Etching of Parylene-C for Micropore Array Preparation
The micropatterning and etching process of Parylene-C for micropore array preparation is
schematically shown in Figure 1b. First, Parylene-C of a 10 µm thickness and titanium (Ti) of a
3000 Å thickness were sequentially deposited on a single-polished silicon wafer (Figure 1b1). Then,
photolithography and etching (with RIE and BHF, in parallel for comparison) were performed to

The schematic of the Parylene-C molding process with a PDMS micropillar array as the template
is schematically shown in Figure 2a. The PDMS micropillar arrays were prepared via the widely used
soft lithography technique. First, silicon microwell arrays (depth at 10 μm, and space at 4 μm) were
prepared via
micropatterning and DRIE on a single-polished wafer,
Micromachines
2018,photolithography-based
9, 620
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followed by soft lithography to prepare the PDMS micropillar array (Figure 2a1). Then, Parylene-C
of a 3 μm thickness was deposited onto the PDMS substrate with a micropillar array with a
prepare
the Tideposition
mask for the
subsequent
of Parylene-C
(Figure
RIE2a2).
of oxygen
commercial
machine
(PDS etching
2010, SCS,
Indianapolis,
IN, 1b2).
USA)Next,
(Figure
Next, plasma
oxygen
was
used
to
etch
Parylene-C
until
the
surface
of
silicon
wafer
was
exposed,
followed
by
the
use of
plasma etching was used to remove Parylene-C until the top of the silicon micropillars was exposed
a(Figure
BHF bath
the removal
of residual
Ti (Figure
1b3).
Finally, the
releasethe
of Parylene-C
2a3).for
Finally,
sonication
in a water
bath was
performed
to release
Parylene-C micropore
micropore
arrays
from
the
silicon
wafer
was
realized
via
sonication
in
the
water
bath
(Figure
1b4).
arrays from the PDMS template (Figure 2a4).

Figure
a polydimethylsiloxane
(PDMS)
(a) (a)
andand
Si
Figure2.
2. Schematic
Schematicof
ofthe
theParylene-C
Parylene-Cmolding
moldingprocess
processwith
with
a polydimethylsiloxane
(PDMS)
(b)
micropillar
array
as
templates
for
micropore
array
preparation.
Si (b) micropillar array as templates for micropore array preparation.

2.3. Parylene-C Molding with PDMS Micropillar Array as the Template for Micropore Array Preparation
2.4. Parylene-C Molding with Silicon Micropillar Array as the Template for Micropore Array Preparation
The schematic of the Parylene-C molding process with a PDMS micropillar array as the template
The schematic of the Parylene-C molding process with a silicon micropillar array as the template
is schematically shown in Figure 2a. The PDMS micropillar arrays were prepared via the widely
is shown in Figure 2b, and this process was also discussed in our previous work [23,24]. First,
used soft lithography technique. First, silicon microwell arrays (depth at 10 µm, and space at 4 µm)
photolithography-based micropatterning and DRIE were used to fabricate the micropillar array
were prepared via photolithography-based micropatterning and DRIE on a single-polished wafer,
(height at 10 μm, and space at 4 μm) on a single-polished silicon wafer (Figure 2b1). Then,
followed by soft lithography to prepare the PDMS micropillar array (Figure 2a1). Then, Parylene-C of
Parylene-C of a 3 μm thickness was deposited onto the silicon template with micropillar arrays
a 3 µm thickness was deposited onto the PDMS substrate with a micropillar array with a commercial
(Figure 2b2). Next, oxygen plasma etching was used to etch Parylene-C off until the top of the silicon
deposition machine (PDS 2010, SCS, Indianapolis, IN, USA) (Figure 2a2). Next, oxygen plasma etching
micropillars was exposed (Figure 2b3). Finally, the release of Parylene-C micropore arrays from the
was used to remove Parylene-C until the top of the silicon micropillars was exposed (Figure 2a3).
silicon template was realized in the HNA (HF:HNO3:HAc = 5:7:11, v/v) bath (Figure 2b4).
Finally, sonication in a water bath was performed to release the Parylene-C micropore arrays from the
For the Parylene-C molding process with a silicon template, three different layout designs of
PDMS template (Figure 2a4).
micropore-arrayed membranes were tried, in order to acquire the optimal version for the ease of
operation
and Molding
application
maximization.
2.4.
Parylene-C
withperformance
Silicon Micropillar
Array as the Template for Micropore Array Preparation
The schematic
of the Parylene-C molding process with a silicon micropillar array as the template
3. Results
and Discussion
is shown in Figure 2b, and this process was also discussed in our previous work [23,24]. First,
photolithography-based
micropatterning
and DRIE were
used tooffabricate
3.1. Micropore Arrays Obtained
from Micropatterning
and Etching
Silicon the micropillar array (height
at 10 µm, and space at 4 µm) on a single-polished silicon wafer (Figure 2b1). Then, Parylene-C of a
The fabricated micropore arrays via the micropatterning and etching of silicon are displayed in
3 µm thickness was deposited onto the silicon template with micropillar arrays (Figure 2b2). Next,
Figure 3. The microfabrication process of silicon in a microelectromechanical system (MEMS) is
oxygen plasma etching was used to etch Parylene-C off until the top of the silicon micropillars was
well-developed, and a high size controllability of microstructures could be maturely realized. The
exposed (Figure 2b3). Finally, the release of Parylene-C micropore arrays from the silicon template was
uniformly close-packed silicon micropore array with edge-to-edge space <4 μm (porosity > 40%) was
realized in the HNA (HF:HNO3 :HAc = 5:7:11, v/v) bath (Figure 2b4).
fabricated, as shown in Figure 3a,b. However, the fragility of silicon led to the generation of cracks,
For the Parylene-C molding process with a silicon template, three different layout designs of
micropore-arrayed membranes were tried, in order to acquire the optimal version for the ease of
operation and application performance maximization.
3. Results and Discussion
3.1. Micropore Arrays Obtained from Micropatterning and Etching of Silicon
The fabricated micropore arrays via the micropatterning and etching of silicon are displayed
in Figure 3. The microfabrication process of silicon in a microelectromechanical system (MEMS)

Micromachines 2018, 9, 620

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is well-developed, and a high size controllability of microstructures could be maturely realized.
The uniformly close-packed silicon micropore array with edge-to-edge space <4 µm (porosity > 40%)
Micromachines 2018, 9, x
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was fabricated, as shown in Figure 3a,b. However, the fragility of silicon led to the generation of
cracks, and the unavoidable defects in the Si N4 layer caused the formation of flaws (Figure 3c,d
and the unavoidable defects in the Si3N4 layer 3caused
the formation of flaws (Figure 3c,d with three
with three cracks and five flaws in an area of
5.24 mm2 ), thus resulting in a low yield for large-area
2
cracks and five flaws in an area of 5.24 mm ), thus resulting in a low yield for large-area micropore
micropore array microfabrication. The frequently appearing defects result in a questionable mechanical
array microfabrication. The frequently appearing defects result in a questionable mechanical strength
strength of large-area and high-porosity silicon micropore arrays, which will degrade operation
of large-area and high-porosity silicon micropore arrays, which will degrade operation ease and cell
ease and cell separation efficiency in practical applications. Besides, the cost of a large-area silicon
separation efficiency in practical applications. Besides, the cost of a large-area silicon micropore array
micropore array is too high, limited by the expensive DRIE, and is still not ready for manufacturing
is too high, limited by the expensive DRIE, and is still not ready for manufacturing for wide
for wide applications. Therefore, a cost-effective alternative microfabrication process for large-area
applications. Therefore, a cost-effective alternative microfabrication process for large-area and
and high-porosity micropore array preparation needs further improvement and development.
high-porosity micropore array preparation needs further improvement and development.

Figure
The
prepared
silicon
micropore
arrays
via micropatterning
and
etching.
(a) The
Figure 3.3.The
prepared
silicon
micropore
arrays via
micropatterning
and etching.
(a) The
well-prepared
well-prepared
silicon
micropore
array;
(b)
the
amplification
view
of
the
squared
area
in
(a);
(c)
defects
silicon micropore array; (b) the amplification view of the squared area in (a); (c) defects (cracks
and
(cracks
flaws)
in silicon
micropillar
due to thestrength;
poor mechanical
strength; (d)
flaws) inand
silicon
micropillar
arrays
due to thearrays
poor mechanical
(d) the amplification
viewthe
of
amplification
view
squared
areaare
in (c).
The(a,b,d)
scale bars
and 100 μm in (c).
the squared area
inof
(c).the
The
scale bars
10 µm
and are
100 10
µmμm
in (a,b,d)
(c).

3.2. Micropore
Micropore Arrays
Arrays Obtained
Obtained from
from Micropatterning
Micropatterning and
and Etching
Etching of
3.2.
of Parylene-C
Parylene-C
Parylene-C is
is aa popular
popular polymer
polymer material
material for
for MEMS
MEMS devices
devices owing
owing to
to its
its good
good biocompatibility
biocompatibility
Parylene-C
and
compatibility
with
the
conventional
microfabrication
processes.
Previously,
Parylene-C
micropore
and compatibility with the conventional microfabrication processes. Previously, Parylene-C
arrays have
beenhave
reported
cell separation.
The reported
Parylene-C
micropore
arrays
micropore
arrays
been [18–21]
reportedfor
[18−21]
for cell separation.
The reported
Parylene-C
micropore
with
a
large
edge-to-edge
space
(10/12
µm
[20],
11/12
µm
[21],
low
porosity)
were
fabricated
arrays with a large edge-to-edge space (10/12 μm [20], 11/12 μm [21], low porosity) were fabricated
via micropatterning
micropatterningand
andetching
etchingwith
withthe
thephotoresist
photoresist
metal
(aluminum
[25,26]
or titanium
[27])
via
oror
metal
(aluminum
[25,26]
or titanium
[27])
as
as
the
etching
mask.
Our
previous
study
investigated
SF
optimized
oxygen
plasma
etching
of
6
the etching mask. Our previous study investigated SF6 optimized oxygen plasma etching of ParyleneParylene-C
[27]
with
micropatterned
Ti
acting
as
the
etching
mask,
and
this
process
was
further
utilized
C [27] with micropatterned Ti acting as the etching mask, and this process was further utilized to try
to try
the fabrication
of high-porosity
Parylene-C
micropore
arrays
instudy.
this study.
The fabrication
results
the
fabrication
of high-porosity
Parylene-C
micropore
arrays
in this
The fabrication
results
are
are
shown
in
Figure
4.
As
shown
in
Figure
4a,b,
the
Parylene-C
micropore
array
of
a
large
edge-to-edge
shown in Figure 4. As shown in Figure 4a,b, the Parylene-C micropore array of a large edge-to-edge
space (>15
(>15μm)
µm)could
couldbe
beobtained.
obtained.However,
However,the
thefabrication
fabricationof
of aahigh-porosity
high-porosity Parylene-C
Parylene-C micropore
micropore
space
array
with
a
small
(4
µm)
edge-to-edge
space
failed
due
to
the
undercutting
during
the
etching
array with a small (4 μm) edge-to-edge space failed due to the undercutting during the etching
process
(Figure
4c
−
f),
resulting
from
the
limited
anisotropic
etching
capability
of
Parylene-C
and
process (Figure 4c−f), resulting from the limited anisotropic etching capability of Parylene-C and thus
thusdifficulty
the difficulty
in obtaining
a high-aspect-ratio
microstructure.
The undercutting
of Parylene-C
the
in obtaining
a high-aspect-ratio
microstructure.
The undercutting
of Parylene-C
in a
in
a
small
sized
area
was
inevitable,
even
though
the
quality
of
the
Ti
mask
was
improved
via
RIE
small sized area was inevitable, even though the quality of the Ti mask was improved via RIE
(Figure
4e,f),
compared
to
that
prepared
via
BHF
etching
(Figure
4c,d).
From
the
above,
a
process
(Figure 4e,f), compared to that prepared via BHF etching (Figure 4c,d). From the above, a process for
for the
microfabrication
of structures
with
a highaspect-ratio
aspect-ratioisisrequired
requiredtoto obtain
obtain high-porosity
high-porosity
the
microfabrication
of structures
with
a high
Parylene-C
micropore
arrays.
Parylene-C micropore arrays.

process (Figure 4c−f), resulting from the limited anisotropic etching capability of Parylene-C and thus
the difficulty in obtaining a high-aspect-ratio microstructure. The undercutting of Parylene-C in a
small sized area was inevitable, even though the quality of the Ti mask was improved via RIE
(Figure 4e,f), compared to that prepared via BHF etching (Figure 4c,d). From the above, a process for
the
microfabrication
Micromachines
2018, 9, 620 of structures with a high aspect-ratio is required to obtain high-porosity
6 of 10
Parylene-C micropore arrays.

Figure 4. The prepared Parylene-C micropore arrays via micropatterning and etching. (a) The
well-prepared Parylene-C micropore array with a large space; (b) the oblique view of a single
micropore in (a); (c) the SEM images of Parylene-C and Ti (prepared via BHF etching) after RIE;
(d) the amplification view of the squared area in (c); (e) the SEM images of Parylene-C and Ti (prepared
via RIE) after RIE; (f) the amplification view of the squared area in (e). The scale bars are 10 µm.

3.3. Micropore Arrays Obtained from Parylene-C Molding with PDMS Template
To fabricate the high-aspect-ratio Parylene-C microstructures, a Parylene C molding process
was developed by Suzuki et al. [28] and Kuo et al. [29] to prepare the suspended microsprings and
microbeams with a size of >10 µm. Our previous study used the molding process for a large-area
and high-porosity micropore array [23,24]. The reported Parylene-C molding fabrications used the
expensive silicon microstructure as the template. Considering the fabrication cost, the Parylene-C
molding process with an economic PDMS microstructure as the template was investigated in this
study. The fabrication results are shown in Figure 5. A large-area (20 × 20 mm) PDMS micropillar
array with space at 4 µm could be well-prepared (Figure 5b) via the widely used soft lithography
process with the silicon microwell array (Figure 5a) as the master. The cost of the PDMS micropillar
arrayed template is very low owing to the repeatability uses of the silicon microwell arrayed master.
After the deposition (Figure 5c) and RIE removal (Figure 5d) of Parylene-C, the micropore array was
expected to be released via a sonication performance in the water bath. However, in fact, the release
failed, even after 24 h continuous sonication, which may be attributed to the fact that the Parylene-C
molecules were imbedded in the porous molecular network of the PDMS matrix during deposition and
thus displayed a very tight adhesion to the PDMS micropillars. Besides, a serious heating effect existed
in long-term oxygen plasma etching, which caused the deformation (Figure 5e) and even adhesion
(Figure 5f) of PDMS micropillars under the vacuum pumping. Finally, the Parylene-C molding process
with the PDMS micropillar array as the template failed to produce the required micropore arrays,
and improvement of the process is still in demand.

molecules were imbedded in the porous molecular network of the PDMS matrix during deposition
and thus displayed a very tight adhesion to the PDMS micropillars. Besides, a serious heating effect
existed in long-term oxygen plasma etching, which caused the deformation (Figure 5e) and even
adhesion (Figure 5f) of PDMS micropillars under the vacuum pumping. Finally, the Parylene-C
molding process
with the PDMS micropillar array as the template failed to produce the required
Micromachines
2018, 9, 620
7 of 10
micropore arrays, and improvement of the process is still in demand.

Figure 5.
5. Fabrication
Fabrication results
results of
of the
the Parylene-C
Parylene-C molding
molding process
process with
with the
thePDMS
PDMStemplate.
template. (a)
(a) Silicon
Silicon
Figure
microwell array
array prepared
prepared via
via photolithography-based
photolithography-based micropatterning
micropatterning and
and DRIE;
DRIE; (b)
(b) micropillar
micropillar
microwell
array of
of PDMS
PDMS prepared
prepared via
via soft
soft lithography
lithography with
with aa silicon
silicon microwell
microwell array
array as
as the
the master;
master; (c)
(c) PDMS
PDMS
array
micropillar
array
after
Parylene-C
deposition
on
the
top
surface;
(d)
PDMS
micropillar
array
with
micropillar array after Parylene-C deposition on the top surface; (d) PDMS micropillar array with
Parylene-C left
left in
in the
the edge-to-edge
edge-to-edge spacing
spacing areas
areas after
after RIE
RIE removal
removal of
of Parylene-C
Parylene-C on
on the top surfaces;
Parylene-C
(e)
(e) deformed
deformed PDMS
PDMS pillars
pillars after
after RIE
RIE of
of Parylene-C;
Parylene-C; (f)
(f) adhered PDMS pillars after RIE of Parylene-C.
The
The scale
scalebars
barsare
are10
10µm.
μm.

3.4.
3.4. Micropore
Micropore Arrays
Arrays Obtained
Obtained from
from Parylene-C
Parylene-CMolding
Moldingwith
withSilicon
SiliconTemplate
Template
After
After the
the poor
poor capabilities
capabilities or
or even
even failures
failures in
in the
the preparation
preparation of
of large-area
large-area and
and high-porosity
high-porosity
micropore
arrays,
the
Parylene
C
molding
process
with
a
silicon
micropillar
array
as
the
was
micropore arrays, the Parylene C molding process with a silicon micropillar array astemplate
the template
used
as
the
final
ability
to
fabricate
the
required
micropore
arrays.
The
fabrication
results
are
shown
was used as2018,
the9,final
ability to fabricate the required micropore arrays. The fabrication results
Micromachines
x
7 ofare
10
in
Figure
A large-area
(>17 × 17(>17
mm)
array with
edge-to-edge
space <4 µm
(porosity
shown
in6.Figure
6. A large-area
× micropore
17 mm) micropore
array
with edge-to-edge
space
<4 μm
>40%)
was>40%)
successfully
achieved. achieved.
The smallest
tried size
could
go down
± 0.07
µm±based
on
(porosity
was successfully
Thesize
smallest
tried
could to
go1.39
down
to 1.39
0.07 μm
the
conventional
UV
lithography.
A
steep
profile
of
the
sidewall
was
obtained
(the
insert
of
Figure
6b),
based on the conventional UV lithography. A steep profile of the sidewall was obtained (the insert of
which
a good
uniformity
pore size,ofand
thus
a high
precision/efficiency
in cell separation
Figure fulfills
6b), which
fulfills
a good of
uniformity
pore
size,
and thus
a high precision/efficiency
in cell
and
cell
assays
in
practical
applications.
The
Parylene-C
micropore-arrayed
membrane
presents
separation and cell assays in practical applications. The Parylene-C micropore-arrayed membranea
high
mechanical
strength and
easy operation,
with either
tweezers
ortweezers
manuallyor(Figure
7). Besides
presents
a high mechanical
strength
and easy either
operation,
with
manually
(Figurethe
7).
preliminary
version of theversion
Parylene-C
membranes inmembranes
our previousinreports
[24,25],
Besides the preliminary
of themicropore-arrayed
Parylene-C micropore-arrayed
our previous
different
layout designs
investigated
and compared
from
thecompared
concerns offrom
mechanical
strength,
reports [24,25],
differentwere
layout
designs were
investigated
and
the concerns
of
easy
operation,
and
filtration
performance,
as
shown
in
Figure
7.
The
3rd
third
generation
of a
mechanical strength, easy operation, and filtration performance, as shown in Figure 7. The 3rd third
totally
freestanding
membrane
with amembrane
100% effective
is thefiltration
optimal design,
could
generation
of a totally
freestanding
withfiltration
a 100% area
effective
area is which
the optimal
process
10
mL
(the
largest
volume
we
ever
tried)
undiluted
whole
blood
without
clogging,
and
more
design, which could process 10 mL (the largest volume we ever tried) undiluted whole blood without
investigations
of various
clinical samples
are ongoing.
clogging, and more
investigations
of various
clinical samples are ongoing.

Figure 6.
6. Fabrication
with aa silicon
silicon template.
template. (a) Si
Fabrication results
results of
of the
the Parylene C molding process with
micropillars prepared via photolithography micropatterning and DRIE; (b) the prepared Parylene-C
micropore array.
array. The
The scale
scale bars
bars are
are 10
10 µm.
μm.

Figure 6. Fabrication results of the Parylene C molding process with a silicon template. (a) Si
Micromachines
2018,prepared
9, 620
micropillars
via photolithography micropatterning and DRIE; (b) the prepared Parylene-C 8 of 10

micropore array. The scale bars are 10 μm.

Figure
Figure7.7.Photos
Photosofofmicropore-arrayed
micropore-arrayedmembranes
membranesprepared
preparedvia
viathe
theParylene-C
Parylene-Cmolding
moldingprocess
processwith
with
an
Si
micropillar
array
as
the
template.
(a,b)
1st
generation:
with
4
mm
wide
flat
film
as
an Si micropillar array as the template. (a,b) 1st generation: with 4 mm wide flat film asaasupport
support
frame
surrounding area
areaand
andeffective
effectivefiltration
filtration
(high-porosity
micropore
array)
in central
the central
frame in
in the
the surrounding
(high-porosity
micropore
array)
in the
area;
area;
(c,d)generation:
2nd generation:
a low-porosity
micropore
array
(4diameter
μm poreand
diameter
and 4 μm
(c,d) 2nd
with a with
low-porosity
micropore
array (4 µm
pore
4 µm edge-to-edge
edge-to-edge
space) as
a support
in the surrounding
area and
effective(high-porosity
filtration (high-porosity
space) as a support
frame
in theframe
surrounding
area and effective
filtration
micropore
micropore
array)
in
the
central
area;
(e,f)
3rd
generation:
totally
freestanding
membrane
with a
array) in the central area; (e,f) 3rd generation: totally freestanding membrane with
a high-porosity
high-porosity
micropore
arrayarea.
in the whole area.
micropore array
in the whole

Thanks to the
high
yield
of this
process
(Table(Table
2), it is2),ready
a scale-up
Thanks
the strong
strongrobustness
robustnessand
and
high
yield
of this
process
it is for
ready
for a
manufacture
in a foundry,
which would
fabrication
cost. The
yield
thisofprocess
could
scale-up
manufacture
in a foundry,
which lower
wouldthe
lower
the fabrication
cost.
The of
yield
this process
be as be
high
85%,
the ratio
of theofnumber
of membranes
without
defects
to the
could
as as
high
as representing
85%, representing
the ratio
the number
of membranes
without
defects
tototal
the
number
of membranes
(both without
and with
defects)
after verification
under a microscope.
Therefore,
total
number
of membranes
(both without
and
with defects)
after verification
under a microscope.
we consider
with the
improved
optimized
fabrication,
this technology
can be very can
cost
Therefore,
wethat
consider
that
with the and
improved
and batch
optimized
batch fabrication,
this technology
effective, which makes it suitable to develop, or to be integrated into, a device for cell separation and
cell assays in the clinics.
Table 2. Comparison of the four microfabrication processes investigated for micropore arrays.
Process

Size
Precision/Controllability

Realization of
High Porosity

Process
Robustness

Realization of
Large Area

Yield

Micropatterning and
etching of silicon

High

Yes

Poor

Difficult

Low

Micropatterning and
etching of Parylene-C

Low

No

Poor

Achievable

Low

Parylene-C molding
with PDMS template

Low

No

Poor

N/A

Low

Parylene-C molding
with silicon template

High

Yes

Strong

Easily
achievable

High

4. Conclusions
In this study, four different microfabrications of micropore arrays for cell separation and
cell assays were investigated and compared in terms of the performances in size precision
and controllability, robustness, and yield (Table 2). The Parylene-C molding process with a
microfabricated silicon micropillar array as the template is the optimized one to prepare the widely
required large-area (>17 × 17 mm) and high-porosity (>40%) micropore arrays, along with a high
size/geometry controllability (good micropore size uniformity and small edge-to-edge space <4 µm).
These advantages make our Parylene-C micropore array attractive in high-efficiency cell separation
and cell assays in the liquid biopsy for potential clinical diagnosis and therapy in Precision Medicine.

Micromachines 2018, 9, 620

9 of 10

Furthermore, the high yield and strong robustness make this process ready for scale-up manufacture
in a foundry, which will be very cost effective and facilitates the broad applications in the fields of
basic biomedical study and practical clinics.
Author Contributions: Y.L. conceived all aspects of this study, performed all the experiments, and prepared the
manuscript. H.X. and L.Z. participated in the Parylene C etching process. W.W. designed the work and revised
the manuscript. All the authors reviewed the manuscript.
Funding: This work was financially supported by the National Natural Science Foundation of China (Grant No.
81611540352, 81471750 and U1613215), the Beijing Natural Science Foundation (Grant No. 4172028 and L172005),
the Advanced Research Program of the Ministry of Education (Grant No. 6141A02033604), the Postdoctoral
Science Foundation of China (Grant No. 2018M631261), and the Seeding Grant for Medicine and Information
Sciences (2018-MI-03) awarded by Peking University.
Acknowledgments: The authors also want to thank the staffs from the National Key Laboratory of Science and
Technology on Micro/Nano Fabrication for their help with the fabrication process.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.

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