direct rapid prototyping of pdms from a photomask ﬁlm for...

TECHNICAL NOTE www.rsc.org/loc | Lab on a Chip

Direct rapid prototyping of PDMS from a photomask film formicropatterning of biomolecules and cells†

Hyundoo Hwang,‡a Gyumin Kang,‡a Ju Hun Yeon,a Yoonkey Nam*a and Je-Kyun Park*ab

Received 18th June 2008, Accepted 19th August 2008

First published as an Advance Article on the web 20th October 2008

DOI: 10.1039/b810341k

The soft lithographic technique is a collection of simple and cost-effective patterning techniques which

applies an elastomeric stamp to transfer a nano/micro-scale pattern. Patterning biological materials

using soft lithography provides procedurally simple control of the surface chemistry and the cell

environments. However, conventional methods for generating microstructures on a substrate require

expensive clean room facilities and skillful training. Here we report a simple and inexpensive clean-

room free process using a conventional photomask film as a master to fabricate elastomeric stamps or

microfluidic channels. This ultra rapid prototyping technique was applied to print FITC labeled poly-

L-lysine with a 10 mm feature size on a glass substrate using soft lithographic processes, such as micro-

contact printing and micromolding in capillaries, for patterning human hepatocellular carcinoma cells,

human skin fibroblasts and hippocampal neurons from E-18 Sprague-Dawley rat. This novel technique

using a photomask film as a master would be very useful ‘hands-on’ tool for the generation of micro-

patterned chemical or biological assays using cells and proteins.

Introduction

Micropatterning of biological materials such as proteins and cells

is a powerful technique for several applications in biology and

chemistry. The ability to pattern proteins at desired locations on

a substrate is essential in a number of emerging technologies,

including biosensors and biochips for biochemical analysis and

diagnosis.1 In addition, the patterning of cells is also necessary,

especially in cell biology, not only for studying intercellular

interactions,2 cell shape changes,3 apoptosis4 and differentiation,5

but also for creating cell-based biosensors6 and microarrays.7

Although several patterning techniques such as photochemical

methods,1,8 electrokinetic methods9 and direct spraying10 or

spotting11,12 have been reported, soft lithographic methods,

including micro-contact printing (mCP) and micromolding in

capillaries (MIMIC), have attracted much attentions as one of

the most simple, cost-effective, and convenient methods for

patterning biological materials on a substrate.13 Soft lithographic

techniques use an elastomeric stamp or mold to transfer a nano-/

micro-scale pattern.14 Patterning proteins and cells using soft

lithography provides procedurally simple control of the surface

chemistry and the cell environments.13,15 However, conventional

methods such as photolithography and electron beam lithog-

raphy for generating microstructures on a silicon wafer require

expensive clean-room facilities and skillful technicians, which are

aDepartment of Bio and Brain Engineering, KAIST, 335 Gwahangno,Yuseong-gu, Daejeon, 305-701, Korea. E-mail: [email protected];[email protected]; Fax: +82-42-350-4310; Tel: +82-42-350-4315;+82-42-350-4322bDepartment of Biological Sciences, KAIST, 335 Gwahangno, Yuseong-gu,Daejeon, 305-701, Korea

† Electronic supplementary information (ESI) available: Cell culturemethod. See DOI: 10.1039/b810341k

‡ These authors contributed equally to this work.

This journal is ª The Royal Society of Chemistry 2009

usually not available for the real potential user group in biology

and chemistry community.

For the simple rapid fabrication of microstructures, direct

printing methods using an office laser printer have been repor-

ted.16–19 Bao et al. have directly utilized the laser-printed film as

a master for a poly(dimethylsiloxane) (PDMS) microchannel

which has applied for capillary electrophoresis.17 However, the

laser-printed master, which was constructed with laser-printed

toners on a polyester film, had too rough surface and low reso-

lution (the minimum line width $ 100 mm) to apply for other

biological and chemical applications. Very recently, a toner was

directly printed onto the copper sheet as a mask for the simple

fabrication of master by selective wet-etching.19 However, the

minimum feature size by this technique was still in the order

of hundreds of micrometers as well as the etching processes

are certainly required.

In this report, we demonstrate a simple, inexpensive, and

clean-room free process to fabricate elastomeric stamps and

molds, which have both smooth surface and high resolution, for

soft lithographic micropatterning of biomaterials. A commer-

cially available high-resolution photomask film was directly used

as a master to produce PDMS stamps or microfluidic channels,

and micrometer-scale cell patterns, including human hepatocel-

lular carcinoma cells, human skin fibroblasts and hippocampal

neurons from E-18 Sprague-Dawley rat, were created by

patterning a cell-adhesive biomolecule, FITC-labeled poly-L-

lysine (PLL-FITC) on a glass substrate with mCP and MIMIC.

Experimental

The desired micropatterns–spots, lines, and grids–were generated

using a conventional computer-aided design (CAD) program

and printed on a photomask film (0.25 mm-thickness; Kodak

Co., Ltd., NY) using a conventional laser film printer (Laser

Lab Chip, 2009, 9, 167–170 | 167

Fig. 1 Schematic diagram of the overall processes for patterning

proteins using the rapid prototyped PDMS stamp and mold.

Fig. 2 SEM images of the printed photomask film as a master (left) and

the molded PDMS microstructures (right). (a) 10 mm-wide grid and (b)

spot array patterns.

Plotter RG 8500; Dainippon Screen Mfg. Co., Ltd., Japan).

According to the data sheet, the minimum laser beam size was

4 mm and the minimum pitch was 1 mm at the highest resolution

(25400 dpi). The printing processes were ordered by a company

for a conventional photomask film printing service (Han & All

Technology, Korea). There is no problem to use an expensive

laser film printer because many customer services for

manufacturing and delivering photomask films are available

elsewhere. A 5:1 mixture of PDMS (Sylgard 184; Dow Corning

Co., MI) and curing agent was poured on the printed photomask

film and cured in a convection oven. After about 1 � 2 h at 65 �C,

the PDMS stamps and molds for the soft lithographic patterning

of PLL-FITC (Sigma-Aldrich, St. Louis, MO) were obtained by

peeling off the cured PDMS from the printed photomask film.

The surface profiles of printed photomask film master and

molded PDMS structures were obtained by using a scanning

electron microscope (SEM 535M; Philips, the Netherlands).

The topographic images were obtained and the thickness was

measured using a white-light scanning interferometry (PWM-

T300; Pemtron Co. Ltd., Korea).

On the basis of the molded PDMS structures, mCP and

MIMIC was used to obtain PLL-FITC micropatterns on a glass

substrate (22 mm � 22 mm, Corning, NY). Glass substrates and

the PDMS stamps (or molds) were rinsed by ultrasonicator with

acetone, isopropyl alcohol, and de-ionized (DI) water sequen-

tially each for 5 min and then blow-dried by air-stream. To coat

PLL-FITC on the surface of the PDMS stamps using mCP, they

were soaked in 0.1 mg/ml PLL-FITC solution for 30 min and

again dried. The PDMS stamp was gently placed on the glass

substrate to transfer PLL-FITC. After 20 s, that is an optimal

time to prevent sagging of the PDMS stamp, the PLL-FITC

patterned substrate was sterilized with 70% ethanol solution.

For MIMIC, we first attached the PDMS mold to a dummy

glass slide and treated with the oxygen plasma for 10 min to

selectively switch PDMS surfaces inside the channel to hydro-

philic while retaining the hydrophobicity of the PDMS surfaces

that make direct contact with the sample surface. Then, the

PDMS mold was transferred to the actual glass surface and

performed the MIMIC process. In this way, we were able to

avoid any irreversible bonding between the PDMS mold and

glass surfaces. The larger capillary force by the hydrophilic

channel surfaces makes the spontaneous injection of 0.1 mg/ml

PLL-FITC solution into the PDMS-glass channel easy. After

overnight incubating in room temperature, the PDMS mold was

detached from the glass substrate and the residual PLL-FITC

was washed out using phosphate-buffered saline (PBS). After the

patterning processes, the substrate was sterilized with 70%

ethanol solution before plating cells.

Fig. 1 shows the overall process for patterning PLL-FITC with

the soft lithographic patterning process such as mCP and MIMIC

using the ultra-rapidly prototyped elastomers. The whole

processes could be performed in office and chemical wet room

without the expensive and huge clean-room facilities. The fluo-

rescence images of PLL-FITC patterns were obtained by using

a fluorescence microscope (Axiovert 200MAT; Carl Zeiss Inc.,

Switzerland) and the intensity profiles were analyzed by using

a conventional image analysis program (i-solution; IMT i-Solu-

tion Inc., Korea). To investigate viability of neurons on the

patterned substrate, LIVE/DEAD Viability/Cytotoxicity Kit

168 | Lab Chip, 2009, 9, 167–170

(L-3224; Invitrogen, CA) was used and the fluorescence image of

calcein-AM and ethidium homodimer-1 stained neurons was

obtained from a confocal microscope (LSM510; Carl Zeiss Inc.).

Results and discussion

Fig. 2a and 2b show the SEM images of the photomask film as

a master and the PDMS mold which has 10 mm-wide grid or spot

array pattern. The topographic images and cross-section profiles

could also be obtained using a white-light scanning interferom-

etry (Fig. 3a and 3b). The photomask film is composed of

a transparent polyester film and a dark silver halide emulsion

layer which typically functions as a masking layer in photoli-

thography but functions as a microstructure in our method. The

measured height of emulsion structures from the polyester

surface of the photomask film was 1.03 � 0.11 mm (mean � S.D.,

n ¼ 15). Although the measured thickness was relatively low, it

was thick enough to make a stamp or a microchannel for


Fig. 3 Topographic images and cross-section profiles of the printed

photomask film as a master (left) and the molded PDMS microstructures

(right). (a) 10 mm-wide grid and (b) spot array patterns.

Fig. 4 Fluorescence images (left) and intensity profiles (right) of (a)

10 mm-wide grid and (b) spot array patterns of PLL-FITC, which was

patterned with micromolding in capillaries (MIMIC) and micro-contact

printing (mCP) processes, respectively (Scale bar ¼ 100 mm).

patterning biomolecules and cells using soft lithographic

methods. In addition, the wide applicable size of the film, the

maximum is 28 � 32 in, allowed us to obtain much larger

patterning area as well as much more stamping or molding units

at a time than the conventional photolithographic method which

uses generally 4 in silicon wafer as a master substrate. After

submitting the mask design, it was delivered in a day. Once the

photomask film arrived, the PDMS mold could be casted

immediately. By eliminating the clean-room access for the master

fabrication, the overall prototyping process from the mask

design to PDMS molding could be done in 24 hours. Despite

repeated molding processes, the photomask film, which acts as

a master in this method, was reusable without any degradation.

In addition, the manufacturing costs would also be reduced since

the expensive conventional lithographic equipments are not

required. This simple rapid prototyping method costs less than

$40, which includes manufacture and a fast delivery service from

companies for photomask film printing.

To determine the minimum feature size of the photomask film,

we designed a test pattern with lines of various sizes of width and

spacing (gap)–1 mm to 10 mm. The minimum line width and the

gap obtained were 7 mm and 2 mm, respectively. The obtained

resolution of the line width was 1 mm. The minimum line width

limited the minimum channel width of the PDMS mold for

MIMC and the minimum gap size limited the minimum contact

printing sizes for mCP. The height of the structures was also

affected by the gap among the master patterns–about 0.45, 0.65,

0.75, and 1 mm height for 2, 3, 4, and $ 5 mm gap spacings. These

height changes affect the minimum feature size of stamped

molecule patterns in consequence.

The grid (Fig. 2a and 3a) and spot array (Fig. 2b and 3b)

patterns were used to demonstrate MIMIC andmCP, respectively.

The line pattern was used for both MIMIC and mCP of the

target biomolecule, PLL-FITC. The relatively low height of

microstructure, which is about 1 mm, often resulted in sagging of


the elastomeric structures. To avoid the problem, we adjusted

the ratio of base agent to curing agent to 5:1 for increasing the

elastic modulus of PDMS.20 Especially in the case of mCP process,

we needed to shorten the stamping time below 20 s. After

patterning PLL-FITC on a glass substrate with MIMIC and mCP,

the fluorescence intensity profile was investigated. The measure-

ments indicate that the fluorescence biomolecule was patterned

well on the substrate with a feature size of 10 mm (Fig. 4a and 4b).

To investigate the minimum feature size of molecule patterns,

we used the test pattern mentioned above and printed the PLL-

FITC by mCP. The minimum printable pattern using the PDMS

mold, which is casted from the test pattern mask, was 4 mm-wide

lines with 7 mm spacings. Patterns with smaller line width and

wider spacing did not produce well-defined PLL-FITC patterns.

At the available ranges of pattern size, we could successfully

control the pattern dimensions with a micrometer scale. However,

at the pattern smaller than 4 mm, the edge of the patterns was

rugged and uncontrollable. For this research, however, wherein

the biological cells were patterned, the order of tens of micrometer

was reasonable resolution. Under these results, we can conclude

this rapid prototyping technique based on the photomask film

can be applicable for micropatterning of proteins.

Three different cell types–hepatocytes, fibroblasts and primary

hippocampal neurons–were cultured on the PLL-FITC

patterned substrate as shown in Fig.5. Poly-L-lysine has been

widely used for promoting cell attachment.21 When the target

cells were plated on the PLL-FITC patterned substrates, they

showed preferential adhesion on the patterned areas, which

resulted in patterned cell cultures on 10 � 30 mm wide patterns.

Consequently, after the washing process which was performed

after 1 h since we seeded the HepG2/C3A cells and CCD-986sk

fibroblasts, only the cells attached onto the PLL-coated region

remained. After the cultivation for 1 to 3 days, they grew along

the PLL-FITC patterns which are square and line shapes

(Fig. 5a-5c). The orientation of the cells on 20 mm-wide lines

was more uniformly aligned along the line pattern than them on

30 mm-side squares which are relatively wide and undirectional

Lab Chip, 2009, 9, 167–170 | 169

Fig. 5 Microscopic pictures of the cells cultured on the PLL-FITC

patterned substrates. (a) HepG2/C3A cells on the 30 mm-side square

array after 1 day and (b) on the 20 mm-wide line pattern after 3 days. (c)

CCD-986sk fibroblasts on the 30 mm-wide lines after 2 days. (d) Live cell

staining (calcein-AM) of neurons patterned on the 10 mm-wide grid

pattern at 3 days in vitro. All scale bars are 150 mm.

pattern. In the case of the cells on the bare glass substrate, the

attachment and the differentiation were not well appeared (data

not shown). The HepG2/C3A cells and CCD-986sk fibroblasts

on the PLL-FITC coated–but not patterned–area grew with

random distribution, orientation and shapes. These results

provided us possibilities to control the shape, size, proliferation

and differentiation of biological cells using various biomolecule

patterns created by the proposed technique.

Fig. 5d shows dissociated rat hippocampal neurons cultured

on the PLL-FITC grid pattern in Fig. 4a, which was created by

utilizing MIMIC with the PDMS mold in Fig. 2a. Neurite

outgrowth as well as somata adhesion was well confined by the

10 mm patterns and resulted in the ordered neuronal networks in

vitro. Calcein-AM staining indicated that most of the neurons

were live on the patterns at 3 days after the culture. The selective

cell adhesion and the effect of guided neurite growth were

comparable to previously reported similar reports using

conventional lithographic techniques.22,23 Judging from the

obtained minimum feature size of biomolecule patterns and

neuronal growth, we believe that the presented rapid prototyping

method can be applied to design various neurobiological assays

such as axon guidance and structured neuronal networks.24

The high degree of freedom of photomask film design is very

valuable to make the complicate micro-patterns. Also the high

resolution printing technology using a laser film printer is in still

development to reduce the minimum pattern width. Now anyone

can easily access to a photomask film with one’s own design from

widely distributed photomask printing service companies

without buying an expensive laser film printer. We expect this

direct rapid prototyping of PDMS from a photomask film can be

widely applicable in soft lithography.

Conclusions

In conclusion, we have demonstrated a simple and inexpensive

clean-room free process to fabricate elastomeric stamps for soft

lithography. A conventional high-resolution photomask film was

170 | Lab Chip, 2009, 9, 167–170

directly used as a master to produce PDMS stamps or molds, and

micrometer-scale cell patterns were created by mCP and MIMIC.

By utilizing this technique, we could print a cell adhesive

biomolecule, PLL-FITC, with a minimum feature size of 10 mm

on glass substrates reliably, and pattern several types of cells such

as human hepatocytes, human fibroblasts and hippocampal

neurons. This ultra rapid prototyping method using a photo-

mask film as a master would be very useful technique for

patterning cells and proteins with simple, rapid and inexpensive

processes as well as with high resolution using soft lithography.

Acknowledgements

This work was supported by the Korea Science and Engineering

Foundation (KOSEF) NRL Program grant funded by the Korea

government (MEST) (R0A-2008-000-20109-0), by the Nano/

BioScience and Technology Program (2005-01291) of the MEST,

and by the System IC 2010 program (10030554) of the Ministry

of Knowledge and Economy (MKE). The authors also thank

the Chung Moon Soul Center for BioInformation and Bio-

Electronics, KAIST.

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direct rapid prototyping of pdms from a photomask ﬁlm for...

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