Protein Pattern Assembly by ActiveControl of a Triblock CopolymerMonolayerChao Yung Fan, † Katsuo Kurabayashi,* ,†,‡ and Edgar Meyh 2ofer* ,‡,§

Department of Electrical Engineering and Computer Science, Department ofMechanical Engineering, and Department of Biomedical Engineering, UniVersity ofMichigan, Ann Arbor, Michigan 48109

Received July 30, 2006; Revised Manuscript Received October 11, 2006

ABSTRACT

We present an electrically programmable technique that can rapidly assemble proteins into high-resolution, arbitrarily shaped patterns. Control ledprotein adsorption is achieved by switching the nanoscale surface arrangement of a triblock copolymer monolayer on microelectrodes in anengineered microfluidic device via an electric potential. Our technique is the first capable of configuring differential protein densities intopatterns by varying the applied control voltage, providing the possibility to tune signals for complex analytes, test conditions, or quantificatio n.

The ability to precisely immobilize proteins on surfaces inwell-defined patterns plays a critical role in the advancementof bioelectronics, proteomic research,1,2 tissue engineering,3

and clinical diagnostics.4,5 Previous research has madesignificant efforts to develop a wide variety of proteinpatterning methods, such as nanoimprint lithography,6 e-beamlithography,7 microstamping,8 ink-jet printing,9 dip-penlithography,10 and protein laser printing.11 However, severaldrawbacks, including time-consuming fabrication or surfacetreatments, tedious serial patterning, and the need forcomplex, expensive instrumentation during the patterningprocess, limit the use of these techniques for various futureapplications we envision for these techniques. In addition,it is frequently not possible to rapidly change protein patternsor configure devices during testing. Initial work on thermallyactivated polymer films12,13 demonstrated the feasibility ofprogrammable protein patterning, but the resulting proteinpatterns were nonuniform and of limited resolution (∼200µm). Consequently, it is desirable to develop new proteinpatterning techniques capable of varying the surface densityof immobilized protein in a spatially programmable manner.We believe that such advances will make it possible to pre-cisely isolate, detect, quantify, or manipulate biomolecules.

To address the above challenges, we developed a micro-fluidic system that incorporates a programmable patterningtechnique. Our technique generates high-resolution proteinpatterns in minutes and has the ability to modulate the density

of immobilized protein molecules. The technique combinesthe electrowetting-on-dielectric (EWOD) phenomenon14 andsteric repulsion of proteins by a monolayer of Pluronic F108(BASF) triblock copolymers. The proposed voltage-con-trolled patterning employing EWOD does not subject proteinmolecules to direct current or electrolysis and avoids expo-sure of proteins to heat, radiation, physical stresses, solvents,or dehydration as do some of the previous methods.8-13,15

EWOD, which has been used for microscale manipulationsof liquids16,17 can locally control, via a voltage bias acrossthe dielectric layer, the wettability or hydrophobicity of thedielectric surface. On the other hand, Pluronic copolymershave been widely studied owing to their biocompatibilityand antifouling properties.18-21 Pluronic copolymers aretriblock copolymers composed of a center hydrophobic poly-(propylene oxide) (PPO) domain and two flexible hydrophilicpoly(ethylene oxide) (PEO) chains, which are attached ateach end of the center PPO domain (Figure 1c).20 DifferentPPO and PEO chain lengths of copolymers in the Pluroniccopolymer family give some variations in Pluronic’s anti-fouling properties.20,21

Antifouling properties of Pluronic copolymers come fromthe fact that the Pluronic copolymer’s hydrophobic PPOdomain binds strongly to hydrophobic surfaces via hydro-phobic-hydrophobic interactions, leaving the flexible hy-drophilic PEO chains free in solution to repel proteins andother adsorbents from the surface by steric repulsion.19

Therefore, we hypothesize that by switching a hydrophobicsurface, which has been coated with a monolayer of Pluroniccopolymers, to a hydrophilic state via EWOD (1) somePluronic copolymers would detach from the surface due to

* Corresponding authors. E-mail: katsuo@umich.edu, meyhofer@umich.edu.

† Department of Electrical Engineering and Computer Science.‡ Department of Mechanical Engineering.§ Department of Biomedical Engineering.

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reduced hydrophobic-hydrophobic interactions and (2) hy-drophilic PEO chains could collapse onto the surface bycomparatively weaker hydrophilic-hydrophilic binding.22

Both conditions suppress Pluronic’s steric repulsion, thusallowing proteins to be adsorbed to the surface. On the basisof these properties, we expect that a hydrophobic surfacewith a monolayer of Pluronic copolymers can be readilyswitched from a protein-repelling to a protein-adsorbing stateby controlling the wettability of these surfaces.

To verify our hypothesis, we designed and fabricated asimple microfluidic system that incorporates the requiredpatterning structures (Figure 1). All electrodes consist of 200nm thick indium-tin oxide (ITO) and down to 3µm inwidth. An approximately 800 nm dielectric film of ParyleneC is deposited over the device surface, which contains thepatterning structures. This film is hydrophobic as indicatedby the contact angle of about 90° and has been tested to beelectrically stable up to the breakdown voltage in excess of100 V, which is beyond the 60 V maximum operating voltageused here. The Pluronic copolymer used for forming the

monolayer on top of the hydrophobic Parylene C layer isPluronic F108, which has a PEO chain length of 128monomer units and PPO chain length of 54 monomer units.21

Since each monomer length is∼0.35 nm,23 a fully extendedhydrophilic PEO chain length in solution would be∼45 nm.The solution in the microfluidic device is referenced with a“solution biasing electrode” (Figure 1), and regions not tobe patterned are inactivated by grounding them to the“solution biasing electrode”. As outlined above, we expectthat the localized voltage drop associated with an activatedelectrode leads to a hydrophobic-to-hydrophilic transition ofthe Parylene C dielectric layer on top of the electrode,causing the Pluronic copolymer monolayer in this region tocollapse or be repelled from the surface, which leads toprotein binding in this region.

The protein patterning is carried out as follows: Initially(step 1, Figure 1c), a monolayer of Pluronic copolymers isformed on the device surface by immersing the surface intoa 2.5 mg/mL Pluronic F108 solution for 10 min. To patternproteins, a bias voltage is applied to the selected patterning

Figure 1. Conceptual drawings showing the patterning device design and its operating principle. (a) A three-dimensional view of themicrofluidic patterning device. The actual device has approximate chamber size of 2 cm× 2 cm× 200µm and is assembled by attachinga substrate containing the patterning structures to a microscope slide using∼200 µm thick double-sided mounting tape. (b) Top viewshowing key electrode arrangements used for protein patterning. The addition of a “grounding electrode” around the patterning electrodesincreases the protein pattern resolution by eliminating floating voltages. For visual clarity, the “grounding electrode” is omitted from otherfigures. (c) Principal device operation in a cross-sectional view. STEP 1: Pluronic copolymers form a monolayer on the device surface.STEP 2: Activation of selected patterning electrodes suppresses the Pluronic’s steric repulsion and allows proteins to be adsorbed. STEP3: Unbound proteins are removed from the microfluidic chamber, leaving the desired protein patterns. STEP 4: Additional protein patternsmay be created on the device structure by repeating STEPS 2 and 3.

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electrodes, and then 300µL of ∼0.4 mg/mL fluorescentlylabeled protein in 1 mM K-PIPES buffer (pH 6.8) isintroduced into the chamber (step 2, Figure 1c). For initialdevice characterizations we used rhodamine-labeled bovineserum albumen (TMR-BSA). Protein molecules are allowedto bind to the surface for 3 min before unbound protein mole-cules are washed away (step 3, Figure 1c). Protein patternsestablished in this manner were quantitatively characterizedby fluorescence microscopy and digital CCD video micros-copy. In subsequent steps (step 4, Figure 1c), other electrodescan be activated and exposed to different proteins to establishcomplex patterns by repeating steps 2 and 3.

The programmability and resolution of the proposedpatterning method were demonstrated by selectively activat-ing electrodes (60 V bias voltage) of various shapes andpatterning fluorescently labeled BSA as outlined above(Figure 2). Fluorescence microscopy showed that proteinswere consistently confined with high resolution (better than3 µm) on the activated electrodes, while inactivated elec-trodes (0 V, Figure 2a) did not attract proteins above thebackground level. Intensity profiles (graphs in Figure 2,which correspond to yellow dashed lines in the images)quantitatively confirmed the selectivity and uniformity of ourprotein patterning technique. The absolute density of im-mobilized molecules in patterned regions was determinedby calibrating the fluorescence intensity from the quantizedbleaching behavior of fluorophores and the known labelingstoichiometry of our BSA preparations (see the SupportingInformation, Method). We observed that under our experi-mental conditions a 3 min patterning interval is sufficient tobind ∼500 BSA molecules/µm2 (∼160 arbitrary intensityunits (AU) of our 12 bit digital CCD camera) to an activatedregion. These adsorbed protein densities are consistent withour working hypothesis and experimentally observed mono-layer densities of BSA on Parylene C of about 11 000

molecules/µm2. Also, the intensity profiles clearly show thatthe binding of protein to inactivated electrodes is low andindistinguishable from unpatterned areas. To demonstrate thatthe described patterning method is not limited to BSA, butrepresents a technique that is generally applicable to differentprotein molecules, we repeated our patterning experimentswith casein. The data in Figure S3 (Supporting Information)show that casein behaves similar to BSA: casein’s adsorptionto the substrate surface was confined with high contrast toelectrodes activated by bias voltages of 60 V while thebackground adsorption remained low. These data suggest thatour method holds significant potential for programmablepatterning a large variety of protein molecules other thanthose examined here.

Furthermore, we characterized how protein patterns canbe sequentially configured with our technique (Figure 3).When three patterning electrodes were consecutively acti-vated with a 60 V bias voltage and exposed to protein probes,they were indeed successively patterned. A fourth inactivatedelectrode served as a reference and remained unpatternedduring the entire procedure. The images in Figure 3 reveala progressive increase in the fluorescence intensity ofpatterned areas (by∼19.4( 4.5%) following each proteinpatterning. This progressive increase in pattern intensitieswas expected as the voltage bias remained on the previouslyactivated electrodes throughout the sequential patterning;however, this behavior is prevented by switching off the biasvoltage to previously activated electrodes when a new setof electrodes are being patterned (see the SupportingInformation, Figure S1). In addition, the experiments re-ported in Figure 3 show that, even 1 h after the removalof the electrode bias, protein patterns remain intact with∼78.0% ( 11.7% of the original signal retained (see theSupporting Information, Figure S2), which suggests thatthe majority of protein molecules are bound irreversibly on

Figure 2. Experimental verification of the programmable protein patterning. The distribution of fluorescently labeled BSA has beenquantitatively characterized with high-resolution light microscopy. (a) Fluorescence micrograph and intensity of two adjacent patterningelectrodes. The pattern on the right was activated by applying a 60 V bias, while the left pattern was inactivated with a 0 V bias. Theinactivated electrode (left) is outlined by a narrow gap in the ITO. (b) Fluorescence micrograph of a 3µm wide swirl protein pattern andits intensity profile with estimated protein density. (c) Arbitrarily shaped protein patterns like the letters “UM” and a finger pattern. Thetransmission differences between ITO-coated and non-ITO-coated regions were corrected in the intensity profiles (see the SupportingInformation, Method).

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the hydrophobic surface24 and that protein patterns couldpossibly be used even after the electrical connections areremoved.

Finally, we present evidence that our technique allows usto quantitatively control protein surface densities. When weapplied different bias voltages to individual patterningelectrodes, the observed protein surface densities variedaccordingly (Figure 4a): surfaces that were activated withlower bias voltages adsorbed fewer proteins than thoseactivated by higher voltages, resulting in a nonlineardependency. To examine the mechanistic basis of thisbehavior, we characterized the hydrophobicity of the ParyleneC film via contact angle measurements using a sessile dropmethod as shown in Figure 4b (see Supporting Information,Method). The contact angle declined nonlinearly fromapproximately 90° to 55° for voltages in the range from 0to 60 V as predicted by

whereθ is the contact angle at a given bias voltageV, θ0 isthe initial contact angle,d and εd are the thickness anddielectric constant of the dielectric layer, respectively, andσlv is the surface tension between the liquid droplet andthe ambient gas.14 We selected a maximum operating volt-age for the device of 60 V since bias voltages higher thanthat did not further decrease the contact angle. Possiblemechanisms for this contact angle saturation have beendescribed previously (e.g., charge trapping in the dielectric),but the exact nature of this behavior remains unre-solved.14,25 A regression analysis showed that the changein hydrophobicity, as characterized by the contact anglemeasurements (before reaching saturation), is directly in-versely proportional (R2 > 0.99) to the patterned proteindensities (P < 0.001). The nonlinear voltage dependence ofthe protein surface density is therefore a consequence ofthe nonlinear voltage dependence of the contact angle.This supports the overall hypothesis that our voltage-controlled protein patterning technique is driven by changes

Figure 3. Sequential protein patterning. We initially activated pattern “1” with a 60 V bias (while others are inactivated with a 0 Vbias)and introduced a BSA solution into the chamber. After a 3 min wait, unbound BSA proteins were washed out with a protein-free buffersolution, leaving behind adsorbed proteins on pattern “1”. Subsequently, the same procedure was repeated for pattern “2” and then for “3”.During these sequential patterning steps, the bias on previously activated electrodes was maintained. Pattern “4” remained inactivated toserve as a reference. One hour after the bias to the electrodes had been removed, the protein patterns were still intact, retaining 78.0%(11.7% of the original protein density (compare to the Supporting Information, Figure S2). Electrode patterns that have not been activatedare outlined by the gap in ITO.

Figure 4. Protein surface density (a) and hydrophobicity (b) as a function of applied voltage bias. The insert in (a) shows a representativefluorescence microscopy image of four patterns to which differential bias voltages were applied. Quantitative measurements reveal a nonlinearrise in the protein surface density with increasing voltage bias. The data points (mean( standard deviation,N ) 18) were averaged fromthree independent experiments and a cubic polynomial was fitted to the data. (b) To relate this nonlinear response to the hydrophobicity ofthe surface, we characterized the contact angle variation from an electrowetting experiment using a sessile drop setup (as shown in theinsert). Data from four different experimental devices were averaged, and the mean and standard deviation were plotted. The surfacehydrophobicity declines nonlinearly up to a bias voltage of 60 V; the dashed line represents a least-squares fit of eq 1 to the data. Aregression analysis of the dependence of the protein surface density on the contact angle suggests that these two parameters are linearlyrelated (P < 0.001). Since the contact angle saturates around 60 V, this value presents the practical operating limit for the device.

cosθ ) cosθ0 +ε0εd

2dσlvV2 (1)

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in the hydrophobicity of the Pluronic copolymer-coatedsurfaces.

In conclusion, we successfully implemented a new, volt-age-controlled protein patterning technique that makes itpossible to control protein binding to the surface of micro-fluidic chambers in the presence of monolayers of Pluronictriblock copolymer by modulating the surface wettability.We demonstrated the feasibility of our technique by usingfluorescently labeled BSA and casein molecules as modelsystems and generated complex protein shapes with about 1µm resolution on lithographically patterned electrodes. Thetechnique is fast, portable, and programmable. We demon-strated that the approach is well suited to create arbitrarilyshaped patterns of these proteins with configurable surfacedensities by using lithographically defined electrodes. Wealso showed that proteins can be patterned in sequence, butmore detailed, quantitative work is necessary to determinepossible cross contaminations that result from patterningdifferent protein species in sequence. A number of challengesremain to demonstrate the full potential of this proteinpatterning method. Research is now under way to furtherinvestigate the versatility and adaptability of EWOD-basedprotein patterning by investigating a variety of differentproteins, their activity, and possible applications. We believethat our protein patterning process can support sub-micrometer resolution. If true, this will allow us to develophigh-resolution, multiplexed protein patterning on multipixelelectrode arrays. Moreover, the technique’s programmabilityand flexibility in the shape of the protein patterns could playan important role in tissue engineering, cell studies, nano-molecular systems, and bioelectronics. We note that thepresented technique holds significant potential for futuredevelopments of configurable devices, especially on-siteapplications that can afford little patterning equipment butrequire rapidly arranging biomolecules into complex, high-definition patterns.

Acknowledgment. We thank Y.-W. Lin for assistancein electrode lithography, M.-T. Kao for advices on proteinlabeling, and C.-T. Lin for advices on device fabrication.This work was supported by NSF, Yamatake Co., Japan, andREA Fellowship from the University of Michigan to C.Y.F.

Supporting Information Available: Experimental meth-ods and figures showing an alternative way to sequentially

pattern proteins, relative fluorescence signal vs time afterremoval of voltage biases, and voltage-controlled proteinpatterning of casein. This material is available free of chargevia the Internet at http://pubs.acs.org

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