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A thermoresponsive hydrogel poly(N-isopropylacrylamide) micropatterning method using

microfluidic techniques

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2009 J. Micromech. Microeng. 19 127001

(http://iopscience.iop.org/0960-1317/19/12/127001)

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IOP PUBLISHING JOURNAL OF MICROMECHANICS AND MICROENGINEERING

J. Micromech. Microeng. 19 (2009) 127001 (6pp) doi:10.1088/0960-1317/19/12/127001

TECHNICAL NOTE

A thermoresponsive hydrogelpoly(N-isopropylacrylamide)micropatterning method usingmicrofluidic techniquesHuijie Hou1, Woosik Kim2, Melissa Grunlan2 and Arum Han1,2,3

1 Department of Electrical and Computer Engineering, Texas A&M University, College Station, TX77843-3128, USA2 Department of Biomedical Engineering, Texas A&M University, College Station, TX 77843-3128, USA

E-mail: arum.han@ece.tamu.edu

Received 30 May 2009, in final form 4 August 2009Published 20 October 2009Online at stacks.iop.org/JMM/19/127001

AbstractPoly(N-isopropylacrylamide) (PNIPAAm) is a thermoresponsive hydrogel that has beenwidely used in various biomedical applications, including tissue engineering. MakingPNIPAAm into a microscale structure is an effective method of increasing itsthermoresponsiveness and modulating surface properties compared to bulk PNIPAAm. Thecommonly used method of direct photolithography combined with a photomask is challengingin creating pure PNIPAAm patterns smaller than 10 μm. Also, each time when there is a needto change the sizes of resulting PNIPAAm patterns, a new photomask is required. Here, amicrofluidically controlled micropatterning method utilizing hydrophilic spots on ahydrophobic substrate was developed to create pure PNIPAAm microstructures. This methodenabled the fabrication of pure PNIPAAm microstructures with a wide range of sizes from70 μm to sub-10 μm out of the same substrate preparation by simply varying the flow speed ofthe hydrogel precursor solution through a microfluidic channel. Hydrogel microstructureswith diameters of 12 to 59% of the hydrophilic pattern diameters were successfully fabricatedusing this fabrication scheme. The smallest hydrogel pattern fabricated using this method was2.3 μm in diameter using a 5 μm diameter hydrophilic pattern at a flow speed of 100 mm s−1.This simple and versatile fabrication scheme could be an ideal method for creating large arraysof hydrogel micropatterns with varying sizes.

(Some figures in this article are in colour only in the electronic version)

1. Introduction

Poly(N-isopropylacrylamide) (PNIPAAm) is athermoresponsive hydrogel that has a volume phasetransition temperature (VPTT) of ∼33 ◦C [1] and canreversibly swell/deswell in response to temperature changes.This material has been widely used in biomedical applications

3 Author to whom any correspondence should be addressed.

[2, 3] such as cell release [4–6], drug delivery [7] andtissue engineering [8]. PNIPAAm structures have alsobeen integrated into microfluidic devices such as actuatorsand filters utilizing the thermoresponsive property [9, 10].Thermosensitivity and surface properties are critical factorsin their applications. Current technologies to modulate itsthermosensitivity and surface properties have been focusedon co-polymerization [11], addition of materials that will

0960-1317/09/127001+06$30.00 1 © 2009 IOP Publishing Ltd Printed in the UK

J. Micromech. Microeng. 19 (2009) 127001 Technical Note

generate a porous network after removal [12], surfactants[13], embedded particles that will increase the mechanicalproperties [4, 14] and micropatterning to reduce the geometryof the structures [15].

Size-based modulation of thermoresponsiveness andsurface properties has the advantage of being a simpler andmore versatile method compared to other means such asco-polymerization. For example, thin films of PNIPAAmcopolymers showed improved thermoresponsiveness in cellrelease applications compared to bulk PNIPAAm hydrogels[16]. Influence of gradient microstructures on cell releasingbehavior has also been investigated [17]. PatternedPNIPAAm microstructures have also been widely used inapplications such as microlens arrays [18] and in sensorsfor solvents, pH, thermal and electrical fields [19]. Inthese applications, the size of the micropatterns becomesa critical factor in influencing their functionalities, suchas drastically increased responsiveness of the PNIPAAmmicrostructures with decreasing microstructure sizes. Themost commonly used techniques for hydrogel micropatterningare UV polymerization combined with a photomask [4],microcontact printing (μCP) [20] and reactive ion etching(RIE) [21]. For example, using the RIE micropatterningtechnique, poly(N-isopropylacrylamide-co-methacrylic acid)structures as small as 2.5 μm were successfully fabricated[22]. However, most PNIPAAm microstructures reported sofar are based on co-polymerization of PNIPAAm, and many ofthe developed technologies are material specific and cannot beutilized as a universal microfabrication scheme for variouschoices of material. Weak mechanical properties of purePNIPAAm, light scattering effect, free-radical diffusion andpoor adhesion to substrates are some of the main factorsthat make it challenging to produce sub-10 μm PNIPAAmmicrostructures [19].

‘Dip-coating’ methods have been recently introducedutilizing the differences in surface hydrophilicity/

hydrophobicity to create microchannels with ‘3D’ profiles[23, 24]. In this method, a substrate that had a hydrophilic/hydrophobic profile was dipped into a coating material andslowly pulled out, thus forming microstructures only onthe hydrophilic sites. Utilizing this idea, in this note, wepresent a novel method to micropattern pure PNIPAAm ofvarious sizes on a hydrophobic substrate having hydrophilicmicropatterns. In this method, a microfluidic channel isplaced on top of a substrate having hydrophilic micropatterns,then is filled with PNIPAAm solution, followed by pushingair through the microfluidic channel at a set flow speed.This results in hydrogel precursor solution remaining onlyon the hydrophilic part but not on the hydrophobic part ofthe substrate due to the difference in the surface wettingcharacteristics. The main advantage of this method is thatby varying the air flow speed, the amount of PNIPAAmsolution remaining on the hydrophilic substrate can bevaried, resulting in microfluidically controllable PNIPAAmmicropattern sizes. Another advantage of this method is thatsince the NIPAAm precursor solution forms a defined sizedroplet before photopolymerization, free-radical diffusion,which is the main reason for the limited resolution in most

PNIPAAm micropatterning methods, can be eliminated.Finally, only a small amount of solution is needed comparedto the ‘dip-coating’ method where the whole substrate has tobe immersed into a container filled with NIPAAm precursorsolution. Arrays of microfluidic channels can be used to eitherpattern a large area or easily create PNIPAAm micropatternswith different sizes on a single substrate. We expect thatthis method can also be used to pattern other photocurablepolymers that can be delivered through microfluidic channels.

2. Materials and methods

2.1. Preparation of hydrogel aqueous precursor solutions

Aqueous precursor solutions (20 wt%) containing N-isopropylacrylamide (NIPAAm, 97%) monomer (Aldrich,St Louis, MO), N,N′-methylenebisacrylamide (BIS, 99%)crosslinker (Acros Organics, Morris Plane, NJ) and 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure R© 2959) (Ciba Chemical, Tarrytown, NY)photoinitiator were prepared as follows: in a 50 mL roundbottom flask equipped with a TeflonTM-covered stir bar,NIPAAm (5 g, 8.84 mmol), BIS (0.1 g, 0.13 mmol) andIrgacure-2959 (0.4 g, 0.36 mmol) were dissolved in DI water(25 g) and the solution was stirred under N2 for 15 min.

2.2. Substrate preparation and contact angle measurement

Three types of glass substrates were prepared for contactangle characterization. The first type of glass substrates(50 × 75 mm) were cleaned using ‘piranha’ solution(H2SO4:H2O2 = 3:1 v/v) for 30 min, and then thoroughlywashed with deionized (DI) water. This served asa reference hydrophilic substrate. Hydrophobic glasssubstrates were obtained by exposing these piranha-cleanedsubstrates to a fluorinated trichlorosilane ((tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane, UCT SpecialtiesLLC, Bristol, PA) vapor under vacuum for 15 min. Thethird substrate type was hydrophobic glass substrates rinsedwith acetone for 10 min followed by thorough rinsing withisopropanol and DI water.

Static contact angles (θ static) of sessile DI water droplets(5 μL) and NIPAAm precursor solution (20 wt%, 5 μL)were measured with a CAM-200 contact angle measurementsystem (KSV Instruments, Monroe, CT). Air-dried substrateswere mounted on the stage of the contact angle measurementsystem. θ static was measured at room temperature and valueswere recorded 2 min after the droplet was placed on the surface.Three substrates of each type were measured with three trialson each substrate, resulting in a total of nine measurements foreach type of substrate. The contact angle average and standarddeviation were calculated for each substrate type.

2.3. Hydrophilic pattern fabrication

Piranha-cleaned glass substrates (50 × 75 mm) were placed ina 150 ◦C oven for 15 min for substrate dehydration. Patterningof the substrate was carried out in the following sequence(figure 1(a)). First, MICROPOSITTM S1818TM photoresist

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J. Micromech. Microeng. 19 (2009) 127001 Technical Note

(a) (b)

Figure 1. Fabrication steps. (a) Substrate preparation steps to create hydrophilic patterns on a glass substrate. (b) Illustration of thePNIPAAm micropatterning method controlled with microfluidics.

Table 1. Contact angle measurement of glass substrates before andafter trichlorosilane coating.

Contact anglesSolution/substrate (ave. ± s.d.) (◦)

NIPAAm/‘piranha’ cleaned glass 21 ± 3.24NIPAAm/trichlorosilane treated glass 75 ± 5.23Water/‘piranha’ cleaned glass 19 ± 4.00Water/trichlorosilane treated glass 114 ± 2.13Water/trichlorosilane treated glass (after washa) 113 ± 2.40

a Substrate was washed with acetone, isopropanol and DI water.

(Rohm and Haas Electronic Material LLC, Marlborough,MA) was spin coated on the prepared substrates at4000 rpm for 30 s with an acceleration time of 10 s. Next,the substrates were soft baked at 100 ◦C for 10 min. Thesesubstrates were then exposed using a mask aligner (MJB3,SUSS MicroTec Inc., Waterbury Center, VT) at 12 mW cm−2

(wavelength: 320 nm) for 14 s with a photomask havingarrays of circular patterns with diameters ranging from 5 μmto 200 μm. The substrates were baked at 130 ◦C for10 min, and then developed (MF-319, Rohm and HaasElectronic Material LLC, Marlborough, MA) for 20–40 s.Finally, the substrates were rinsed in DI water and driedwith N2 gas. Photoresist was chosen as a sacrificial patternmaterial for the hydrophilic surface patterning since it is widelyused in conventional photolithography, easy to fabricate high-resolution microstructures and can be easily removed.

Next, these substrates were vapor coated withtrichlorosilane for 15 min. The photoresist was then removedwith acetone, and the substrates were washed thoroughlywith isopropanol and DI water. This resulted in theareas not covered with photoresist micropatterns to becomehydrophobic, while the areas covered by photoresist remainedhydrophilic (table 1). The final substrate had hydrophiliccircular micropatterns with diameters ranging from 5 μm to200 μm, with the surrounding area remaining hydrophobic.

2.4. Microfluidic channel fabrication and PNIPAAmpatterning

The mold for microfluidic channels was fabricated withstandard photolithography in the following procedure.Piranha-cleaned glass substrates were placed in a 150 ◦Coven for 15 min for substrate dehydration. SU-8 2075TM

(MicroChem Corp., Newton, MA) was spin coated on thesubstrates first at a speed of 500 rpm for 10 s with anacceleration time of 10 s, then at a speed of 1500 rpm for10 s with an acceleration time of 5 s, and finally at 2500 rpmfor 30 s with an acceleration time of 5 s. Next, the substrateswere soft baked at 65 ◦C for 40 min and then at 95 ◦Cfor 40 min. These substrates were exposed using a maskaligner (MJB3, SUSS MicroTec Inc., Waterbury Center, VT) at228.2 mJ cm−2 (wavelength: 320 nm) with a photomaskwith 1 mm width channel arrays. Post-exposure baking wasconducted at 65 ◦C for 40 min and then at 95 ◦C for 40 min.The SU-8 structure was then developed in an SU-8 developer(thinner: type P, MicroChem Corp., Newton, MA) for5–10 min and rinsed with isopropanol and DI water.

Microchannels were replicated out of this SU-8 moldusing poly(dimethylsiloxane) (PDMS, Sylgard 184, DowCorning Corporation, Midland, MI) by mixing the base andthe curing agent (10:1 weight ratio) and pouring the mixtureon the mold. After 30 min of degassing, the mixture was curedat 65 ◦C for 30 min. The PDMS replica was then peeled offand fluidic inlet holes were punched for tubing connections.Outlets were made by cutting the channel at the end, whichproduced open-ended channels. The resulting microchannelswere 100 μm high, 1 mm wide and 5 cm long.

To create hydrogel microstructures, the PDMS layercontaining microfluidic channel arrays was placed on theprepared substrate. The PDMS bonded with the substrate withno leakage. The channel was filled with a NIPAAm precursorsolution through the inlet and then air was pushed into thechannel at a controlled speed by a syringe pump (HarvardApparatus, Holliston, MA) (figure 1(b)). This resulted in

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J. Micromech. Microeng. 19 (2009) 127001 Technical Note

(a) (b)

Figure 2. Microfabricated hydrogel arrays. (a) Microscopy images of hydrogel microstructures fabricated at different flow speeds with 50,100 and 200 μm mask patterns. Scale bars: 50 μm. (b) Flow speed controls the size of PNIPAAm micropatterns using the samehydrophilically patterned substrates. Hydrophilic pattern sizes used were 50 μm, 100 μm and 200 μm in diameter. Flow speeds of 7, 42and 150 mm s−1 were used for each hydrophilic pattern size: 50 μm (n = 3), 100 μm (n = 3), 200 μm (n = 3).

droplets of NIPAAm precursor remaining on the hydrophilicspots of the substrate due to the surface tension difference.Flow speeds of 7, 42 and 150 mm s−1 were used on substrateshaving 200, 100 and 50 μm diameter hydrophilic patterns. Thewhole substrate was then placed under a UV light (OmnicureSeries 1000, 15 mW cm−2, 365 nm, Plano, TX) for 10 s. ThePDMS layer was removed after hydrogel polymerization.

2.5. Micropattern characterization

PNIPAAm micropatterns were characterized by opticalmicroscopes (Eclipse LV100 and Eclipse TS100, NikonInstruments Inc., Melville, NY) with a digital camera (DigitalSight DS-2 Mv, Nikon Instruments Inc., Melville, NY) and anoptical surface profilometer (NT9100, Veeco Instruments Inc.,Plainview, NY). Patterns smaller than 5 μm were primarilycharacterized using the optical surface profilometer. Crosssections of the dehydrated micropatterns were characterizedwith an atomic force microscope (AFM, Veeco CP-II, VeecoInstruments Inc., Plainview, NY) in a dry state. Tapping modeAFM was used in ambient air at driving frequencies rangingfrom 70 to 90 kHz. Cantilevers were antimony-doped siliconwith a spring constant of 15 N m−1 and nominal tip radiusof 8 nm (FESP model from Veeco Probes). Only dehydratedmicropatterns were characterized with AFM.

3. Results and discussions

This micropatterning method utilizes the differences insubstrate wetting characteristics of the PNIPAAm solution.Surface properties of substrates used were first characterizedby comparing the contact angles between the piranha-cleanedglass substrate and the hydrophobically coated glass substrate.The NIPAAm precursor solution and DI water used forthe contact angle measurement revealed that the surfacescoated with fluorinated trichlorosilane became less wettable(table 1). Contact angles increased by 95◦ and 54◦,

respectively, for water and NIPAAm after the coating. Sinceacetone, isopropanol and DI water were used to remove thePR patterns after trichlorosilane coating, the effect of thisrinsing procedure on surface property was also characterizedby measuring contact angles of trichlorosilane-coated glasssubstrates with and without solvent cleaning. Contact anglesof 114 ± 0.52◦ (n = 9) and 113 ± 2.40◦ (n = 9) before andafter the rinsing procedure showed that the solvent cleaningprocedure did not affect the surface wettability of the coatedsubstrate.

Figure 2(a) shows microscopy images of PNIPAAmmicropatterns created using the developed method. Sizesof the hydrogel microstructures patterned at three differentflow speeds (7, 42 and 150 mm s−1) on three different sizesof hydrophilic patterns (50, 100, 150 μm in diameter) aresummarized in figure 2(b). When using 50 μm diameterhydrophilic patterns, 5.9, 17.4 and 25.5 μm diameter hydrogelmicrostructures were obtained at flow speeds of 7, 42and 150 mm s−1, respectively. The results show thatthe hydrogel microstructure sizes decrease with decreasingflow speed, and that hydrogel microstructures much smallerthan the hydrophilic pattern size on the substrate couldbe fabricated using this method. Similar trends wereobserved when using substrates having 100 and 200 μmdiameter hydrophilic patterns. When using 100 μm diameterhydrophilic patterns, 16.6, 37.7 and 38.7 μm diameterhydrogel microstructures were obtained at flow speeds of7, 42 and 150 mm s−1, respectively. When using 200 μmdiameter hydrophilic patterns, 25.6, 53.5 and 59.5 μmdiameter hydrogel microstructures were obtained at flowspeeds of 7, 42 and 150 mm s−1, respectively. The resultingPNIPAAm micropatterns were located in the center of thehydrophilic patterns.

Figure 3 shows the cross-sectional profiles of typicalPNIPAAm microstructures in a dry state measured using anAFM. The PNIPAAm microstructure created using a 20 μmdiameter hydrophilic pattern at a flow speed of 150 mm s−1

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J. Micromech. Microeng. 19 (2009) 127001 Technical Note

(a)

(b)

Figure 3. Atomic force microscopy (AFM) images showingcross-sectional profiles of the PNIPAAm microstructures. (a) APNIPAAm microstructure made out of a 20 μm diameterhydrophilic pattern at a flow rate of 150 mm s−1. (b) A PNIPAAmmicrostructure made out of a 50 μm diameter hydrophilic pattern ata flow rate of 42 mm s−1.

Figure 4. Optical surface profilometer image of PNIPAAmmicrostructures made out of 5 μm mask patterns with averagediameter of 2.3 μm (n = 11). Flow speed: 100 mm s−1.

showed a diameter of 7.6 μm and height of 82 nm, with a roundtop profile. The PNIPAAm microstructure created using a50 μm diameter hydrophilic pattern at a flow speed of42 mm s−1 showed a diameter of 15.1 μm and height of 45 nm,with a flat profile. These results show that the resultingPNIPAAm microstructures have thickness below 100 nm indry state; however, considering the swelling capability ofPNIPAAm, the actual height of the PNIPAAm micropatternsin use would be at least in the several hundred nm range.The differences in shapes (flats versus round top) are notexpected to affect the performances of the micropatternedhydrogels.

Hydrogel micropatterns with an average diameter of2.3 μm ± 0.6 μm and height of 7.0 nm ± 0.8 nm(n = 11) could be obtained when using 5 μm diameterhydrophilic patterns at a flow speed of 100 mm s−1

(figure 4). Such small hydrogel structures are extremely

Figure 5. PNIPAAm microstructure sizes controlled by flow speed.Flow speeds from 1.7 to 300 mm s−1 were used on substrates having50, 100 and 200 μm diameter hydrophilic patterns. Averages andstandard deviations were calculated for mask sizes of 50 μm (n =27–39), 100 μm (n = 10–12) and 200 μm (n = 4).

hard to obtain using a direct photopolymerization methodutilizing a photolithography mask, hence demonstrating thehigh-resolution micropatterning capability of the developedhydrogel microfabrication method.

We further investigated and analyzed how flow speed canbe used to control the size of the hydrogel microstructures(figure 5). Using a substrate that has 200 μm diameterhydrophilic patterns, PNIPAAm microstructures ranging from24.8 to 118.4 μm in diameter (12.4 to 59.2% of hydrophilicpattern diameters) could be obtained when varying the flowspeed from 1.7 to 200 mm s−1. With decreasing flowspeed, smaller patterns could be obtained. In the case of100 μm diameter hydrophilic patterns, the average diameterof hydrogel microstructures varied from 16.7 to 46.1 μm(16.7 to 46.1% of hydrophilic pattern diameters) when flowspeeds were varied from 1.7 to 200 mm s−1. For 50 μmdiameter hydrophilic patterns, hydrogel microstructures withdiameters ranging from 5.9 to 25.5 μm (11.8 to 51% ofhydrophilic pattern diameters) were obtained when flow speedvaried from 6.9 to 200 mm s−1. For all three hydrophilicpattern sizes, slower flow speed resulted in smaller hydrogelmicrostructure sizes. An interesting phenomenon observedis that at low flow speed regime, hydrogel microstructuresizes changed more dramatically with varying flow speed. Atflow speed higher than approximately 50 mm s−1, hydrogelmicrostructure sizes increased only gradually and eventuallyreached a saturated value where no significant differencescould be observed with varying flow speed. Typical standarddeviations were less than 15%, showing that this method cancreate uniform hydrogel micropatterns. The capability tocreate hydrogel microstructures having different diameters bysimply varying the flow speed using the same substrate couldbe a versatile hydrogel microfabrication technique where aphotolithography mask does not have to be created every timewhen there is a design change.

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4. Conclusion

We have demonstrated a high-resolution hydrogelmicropatterning method based on wettability differences ofhydrogel precursor solution on hydrophilically patternedhydrophobic substrate, where the hydrogel pattern sizescould be easily controlled by varying the flow speed ofprecursor solutions. By placing a microfluidic channel ontop of a hydrophobic substrate having hydrophilic spots andflowing PNIPAAm precursor solutions through the channelat a controlled speed, uniform PNIPAAm microstructurescould be obtained. With this simple method, PNIPAAmmicrostructures having diameters as small as 2.3 μm weresuccessfully fabricated. PNIPAAm microstructures withdifferent diameters (12 to 54% of the hydrophilic patterndiameters) could be obtained by simply varying the flow speedof precursor solutions through the microfluidic channels.This simple and versatile hydrogel micropatterning methodshows promise for high-resolution patterning of variousother hydrogels or polymer materials on large substrate areaswithout having to modify the properties of the materials to bepatterned.

Acknowledgments

The authors would like to thank John Noel and Dr WonmukHwang for assistance with AFM imaging. This work wasfunded by the National Science Foundation (NSF) under grant#0854462 and the Korean Ministry of Knowledge Economyand Seoul Technopark under grant #1002970790.

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