Photopatterning of polybutadiene substrates byinterferometric ultraviolet lithography: fabricationof phospholipid microarrays

Aschalew Kassu, Jean-Michel Taguenang, and Anup Sharma

Gratings are written holographically with low power �10 mW�cm2� 244 nm UV light on thin films ofpolybutadiene rubber polymer. The increase of hydrophilicity–wettability of polybutadiene films ismeasured over UV-exposed regions. Sequential fabrication of two orthogonal gratings results inhydrophilic microarrays having applications as functionalized substrates for immobilizing biomol-ecules. This is demonstrated by immobilizing a phospholipid in a microarray pattern. © 2007 OpticalSociety of America

OCIS codes: 160.2900, 170.0170.

1. Introduction

Phospholipids have been proposed as promisingplatforms for protein immobilization in biosensingapplications.1 Phospholipid bilayer is the primarycomponent in cell membrane and lipidic films havebeen widely used as models of biological mem-branres.2 Deep UV lithography with a photomaskhas been used to pattern phospholipid bilayers intomicroarrays for sensor-related applications.3 Wedescribe fabrication of phospholipid microarrays byphotopatterning the underlying polybutadiene sub-strate by interferometric UV lithography.

Interferometric photopatterning of polymer thinfilms into surface relief gratings (SRGs) has been wide-ly investigated4–10 using modest powers �mW�cm2�of visible and UV lines of an argon-ion laser. Re-cently, holographic recording of SRGs was demon-strated in polybutadiene thin films conjugated toazobenzene chromophore.11,12 Holographic SRGshave also been fabricated for distributed feedbacklaser (DFB) applications in dye-doped poly(methylmethacrylate) and polycarbonate.13 Recently, apulsed UV laser was used to fabricate SRGs with anablation technique.14 We describe fabrication of sur-

face relief holographic gratings on films ��700 nmthick) of polybutadiene synthetic rubber on a glasssubstrate without any azobenzene�dye moieties.Low-power density �20 mW�cm2� cw laser precludesany ablation effects. A plausible mechanism of grat-ing formation involves photodissociation followed byelastic relaxation of the rubber film over several min-utes after the UV is blocked. Similar to other poly-mers, SRGs in polybutadiene rubber films could beinteresting for their practical application in opticalrecording, diffractive optics,7 and in DFB lasers.14,15

This research, however, was motivated by nanoscalelithography of substrates that could be functionalizedto immobilize biomolecules for sensing applications.In keeping with this goal, we recently demonstratedgrating formation in an azodye-labeled-phospholipidthin film by UV holographic lithography.10 Gratingformation in a phospholipid thin film with an azoben-zene moiety makes this technique restrictive. Theresults reported here demonstrate fabrication of ahydrophilicity grating and�or microarray on a thinfilm of polybutadiene. UV-enhanced hydrophilicity–wettability of the substrate is used to immobilize aphospholipid in a microarray pattern. Polybutadienefilms possess several desirable properties for thelong-term durability of gratings and�or microarraysincluding, resistance to aging, excellent abrasion re-sistance, resistance to dynamic stress, and good low-temperature flexibility. Gratings on polybutadienefilms were stored under ambient conditions for sev-eral months without any noticeable deterioration oftheir diffraction efficiency.

The authors are with the Department of Physics, Alabama A&MUniversity, Normal, Alabama 35762. A. Sharma’s e-mail address isanup.sharma@email.aamu.edu.

Received 30 May 2006; accepted 10 September 2006; posted 22September 2006 (Doc. ID 71460); published 17 January 2007.

0003-6935/07/040489-06$15.00/0© 2007 Optical Society of America

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2. Fabrication and Characterization of PolybutadieneGratings

Thin films were made by dipping glass slides in asolution of polybutadiene (1% by weight) in cyclohex-ane and draining any excess liquid sticking to thesurface. Coated slides are then vacuum dried in adesiccator for several hours to remove the solvent.Films with a measured thickness of �700 nm arecolorless, transparent, and show excellent opticalquality. The grating fabrication setup (Fig. 1) is sim-ilar to that used by us earlier for gratings in azo-dye-labeled phospholipid thin films.10 Two coherent UVbeams from a frequency-doubled argon-ion laser(244 nm, 2 mW�beam) interfere on the thin film for arange of periods between 900 and 2300 nm. Forma-tion of gratings is monitored in real time by diffract-ing a 5 mW He–Ne laser incident normally to thefilm. Diffracted He–Ne light becomes visible in lessthan a minute. A grating is formed over a 3–4 mm2

area on the film. With 2 mW�beam, it takes about2 min for the diffraction efficiency to reach its maxi-mum. Figure 2 shows some typical grating growthcurves. As can be seen in Fig. 2, the grating continuesto grow for several minutes following brief exposureto UV light. Under these conditions, the final diffrac-tion efficiency in general increases with the fluence�mJ�cm2� of UV exposure and most of the growth ofthe gratings takes place after the UV is blocked.Post-UV increase of the grating growth rate can beseen as a discontinuity in slope where the UV isblocked in Fig. 2. After the UV is blocked the addi-tional growth �I�t� of diffraction intensity in Fig. 2can be fitted to a simple exponential function:

�I�t� � �I����1 � e��t�T�� (1)

�I��� is the maximum additional growth as the equi-librium is reached. Phenomenological time T de-creases with the UV fluence. As can be seen in Fig. 2,theoretical growth curves show a good fit to the ex-perimental data. Post-UV growth is also seen inFig. 3 following several brief UV exposures, each for2 s. Figure 4 shows a comparison of growth of grating

when the UV is always on versus growth following aninitial 40 s UV exposure. Clearly, while the final dif-fraction efficiency is about the same, characteristicsof grating growth are very different. Following ashort initial exposure, the grating continues to evolvefor some time after the UV is blocked.

Figure 5 shows measurements with an atomic forcemicroscope on a grating with a period of 2300 nm.The micrograph shows surface relief amplitude of60–80 nm, i.e., �10% of film thickness. Maximumdiffraction efficiency of the grating is measured to be�0.5%. However, coating the grating with a 20 nmthick gold film increases this efficiency to 5% ineach of the two backward diffracted first-orderbeams. Gold films are known to possess a weakhydrophobic behavior.16 Polybutadiene rubber films

Fig. 1. Schematic of the holographic setup with a 244 nm UVlaser to fabricate SRGs in thin films of polybutadiene on a glasssubstrate. UV light from a frequency-doubled argon-ion laser issplit into two coherent beams (2 mW�beam) with a phase mask(PM). The two UV beams are folded with mirrors (M1 and M2) tointerfere at incident angles of 3°–8° on the polybutadiene rubberfilm for a grating period between 900 and 2300 nm. The gratingbecomes visible by diffraction of a 5 mW He–Ne laser. One of thefirst-order diffracted beams is detected with a photodiode and alock-in amplifier.

Fig. 2. Experimental growth of gratings following a holographicUV exposure of 10 �□�, 20 (Œ), 30 (�), and 60 ��� s. Most of thegrating growth takes place after the UV is blocked. Post-UVgrowth is fitted theoretically (solid curve) with an exponentialfunction [Eq. (1)] with relaxation times T of 48, 30, 28, and 18 s,respectively. The point where the UV is blocked can be seen as adiscontinuity in slope of the growth curve.

Fig. 3. Each segment of this curve represents growth over a 5 minduration following a 2 s holographic exposure to UV light. In eachsegment, the growth of the grating shoots up after the UV isblocked.

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are also hydrophobic. Similar to the effect in manyother polymers,17–19 UV exposure appears to increasethe hydrophilicity–wettability of polybutadiene films.Sputtered gold thus preferentially adheres to regionsnot exposed to UV light. Ambient humidity also has aweak effect on the diffraction efficiency of these grat-ings. This is shown in Fig. 6 where the diffractionefficiency increases by 10%–20%, as the relative hu-midity decreases from near 80% to 55%. The effect ofgold coating and ambient humidity on diffraction ef-ficiency are both related to the increase of hydrophi-licity of polybutadiene films by UV exposure andexplained more thoroughly in Section 3.

3. Increased Hydrophilicity of Polybutadiene byUltraviolet Light

Holographic SRGs described here result in increasedhydrophilicity–wettability of the polybutadiene poly-mer due to photodissociation by UV exposure. Todemonstrate UV-enhanced hydrophilicity of poly-butadiene, we have measured the contact angle of awater droplet on polybutadiene film following itsexposure to various doses of UV. An UV laser beam of5 mW power was expanded to a diameter of 5 mm.The polymer surface was exposed for a maximumtime of 16 min in steps of 2 min; the results are shown

in Fig. 7. As polybutadiene thin film is exposed to UVfluence up to 25 J�cm2, its contact angle with waterdecreases from 120° to 77°. Increase of the wettability–hydrophilicity of polymer by UV exposure as measuredby the decrease of contact angle has been widely re-ported in other polymers17–19 and is attributed to pho-todissociation by the UV into smaller more polarcomponents. This observation is widely used for re-moval of organic contaminants from surfaces by thetechnique of UV cleaning.20 The effect of UV lightis further explained by fabricating hydrophilic–hydrophobic patterns on polybutadiene when thinfilms are exposed to UV light through lithographicmasks. Two types of UV-lithographic mask are usedfor this purpose: (a) Polka-dot neutral density filter,fabricated on fused-silica substrate and (b) electro-formed micromesh of nickel. The polka-dot mask [Fig.8(a)] has an array of reflecting squares with a period of0.15 mm. An UV laser of 5 mW power is expanded toa collimated beam of 0.3 cm2 area and incident on themask. A glass slide with the polybutadiene film isplaced behind the mask at a separation of 0.5 mm.The film is exposed to the UV for 10–30 min and anequivalent fluence of 10–30 J�cm2. After UV expo-sure, the film is coated with a 20–50 nm thick layer of

Fig. 4. Comparison of grating growth following 35 s exposure toUV (solid curve) with growth when UV is continuously on. In theformer case, the point where UV is blocked is seen as a disconti-nuity in slope.

Fig. 5. Atomic force micrograph of a grating with a period of2300 nm. Surface relief amplitude of 60–80 nm is �10% of thefilm thickness �700 nm�.

Fig. 6. Effect of ambient humidity on diffraction efficiency ofgrating. Polybutadiene film is exposed holographically to UV for aperiod of 1 min. Growth of grating is monitored for �30 min undera relative humidity of 76%. Humidity is then decreased to 55% byletting in dry nitrogen in the fabrication chamber. This results inan increase of diffraction efficiency by 10%–20%.

Fig. 7. Increase of hydrophilicity of polybutadiene thin film asit is exposed to increasing amounts of UV energy. Increase ofhydrophilicity–wettability is manifested as a decrease of contactangle of a water droplet on polybutadiene film.

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sputtered gold. Figure 8(b) shows an optical micro-graph of the gold-coated polybutadiene film. Clearly,only those areas in polybutadiene are coated withgold, which were masked by the opaque squares onthe polka-dot filter. As explained earlier, UV expo-sure increases hydrophilicity by photodissociatingpolybutadiene into smaller more polar components.Sputtered–evaporated gold thus preferentially ad-heres to regions not exposed to UV light. UV-inducedhydrophilicity is also demonstrated by coating UV-exposed polybutadiene film with a solution of hydro-philic dye (Rhodamine 6G) in ethylene glycol. As thesolvent dries it leaves a grid of dye only on areasexposed to the UV through the polka-dot filter [Fig.8(c)]. By using electroformed micromesh [Fig. 9(a)] ofnickel in place of the polka-dot filter, this technique isused to generate a hydrophilic microarray separatedby a hydrophobic barrier of unexposed polybutadiene.As before, the microarray is made visible by coatingthe film with a hydrophilic dye [Fig. 9(b)].

One plausible mechanism for growth of grating in-volves elastic relaxation following spatially periodicmechanical weakening of rubber film by UV photo-dissociation. The resultant SRG shows alternatinghydrophilic–hydrophobic regions with associated film-thickness variation (Fig. 10). For small relief heights��10%� as in this case, diffraction efficiency in gen-eral increases21 with the growth of the grating relief

although the quantitative diffraction behavior de-pends on the details of relief surface profile.22

Post-UV growth of grating diffraction is a measure ofmass flow due to lateral, in-plane relaxation fromhydrophilic to hydrophobic regions of the film result-ing in an increase of relief height. Observed effects ofambient humidity as well as gold coating on the grat-ing (Section 2) provide additional evidence in supportof grating formation mechanism described in Fig. 10.Since film thickness over the hydrophilic region isless than that over the hydrophobic region (Fig. 10),adsorption of ambient humidity by hydrophilic areasof the film will reduce relief height and diffractionefficiency decreases, as observed experimentally (Fig.6). Likewise, gold coating adheres largely over hydro-phobic areas of film [Fig. 8(b)], which increases dif-fraction.

Fig. 8. Micrographs showing (a) transmission through a polka-dot lithographic mask, which has an array of reflecting squares witha period of 0.15 mm and a transparent grid. (b) UV exposure of polybutadiene film through the polka-dot mask results in an arrayof hydrophobic squares surrounded by a hydrophilic grid. Sputtered gold film adheres only to hydrophobic areas not exposed to UV light.(c) Hydrophilic dye (Rh6G) adheres only to areas exposed to UV light.

Fig. 9. Micrographs showing (a) transmission through an elec-troformed nickel micromesh with a period of 0.125 mm. (b) UVexposure of polybutadiene film through the mesh results in anarray of hydrophilic squares surrounded by a hydrophobic grid. (b)Hydrophilic dye (Rh6G) adheres only to areas exposed to UV light.

Fig. 10. Plausible mechanism for fabrication of a hydrophilicitygrating. Mechanical weakening of rubber film by UV photodisso-ciation followed by elastic relaxation results in the observed sur-face relief pattern. Ambient humidity is absorbed by hydrophilicregions, resulting in a decrease of grating contrast as well asdiffraction efficiency. Sputtered–evaporated gold gets coated overhydrophobic regions, resulting in an increase of diffraction effi-ciency as observed.

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4. Patterning of Phospholipid Thin Film

We foresee several applications of the work describedhere. The polybutadiene film provides a novel plat-form for fabricating high-density biosensing chipsboth with gold-coated microarrays [Fig. 8(b)] as wellas hydrophilic wells [Fig. 9(b)]. Gold provides a bio-inert shield against a substrate that could denatureproteins. Extensive technology has been developed toimmobilize biopolymers on such surfaces and charac-terize their interactions with biophotonic sensingtechniques23,24 including surface plasmon resonanceimaging.25 Gold electrode microarrays have beenused in multianalyte sensors for biomolecules andviruses.26 Likewise, several DNA sensor chips involveimmobilizing oligonucleotides on an array of hydro-philic wells separated by hydrophobic barriers.27 Thework described here was motivated by the potentialof holographic UV lithography to fabricate high-density arrays with nanoscale patterning of biolog-ical substrates. Figure 11(a) shows a section of anUV-fabricated holographic grating on polybutadienethat is coated with a solution of Rhodamine 6G dye inethylene glycol. As the solvent dries, the hydrophilicdye adheres [as in Fig. 9(b)] only to regions exposed toUV light. The hydrophilicity grating has a period of2 �m. Figure 11(b) shows a reflection optical micro-graph of two perpendicular gratings fabricated oneafter another in polybutadiene. To make it visible,the film is coated with a 50 nm thick gold layer. Goldbeing hydrophobic [Fig. 8(b)], the coating is least overthe hydrophilic dark squares where exposure to UVlight is maximum. The period of gratings in this mi-crograph is 900 nm, and the hydrophilic squares are500 nm on each side. The size of these hydrophilicsquares can be decreased up to 200 nm simply byincreasing the angle between interfering UV beams.Commercially available microarrays have a mini-mum spot size of �10 �m. Such hydrophilic wellsare used to immobilize proteins and oligonucleotidesfor parallel processing in biosensing-related appli-cations.27 UV holographic patterning techniqueshave the potential to reduce spot size by 2 orders ofmagnitude.

We have demonstrated the utility of UV-functionalized polybutadiene substrate by immobi-lizing a phospholipid (phosphatidylcholine) in a

microarray pattern. A 1 mg�ml solution of phospho-lipid in chloroform is used. Due to the hydrophobicnature of the solvent, the phospholipid is depositedon hydrophobic regions of polybutadiene film, i.e.,those regions not exposed to UV light. This is shownin Fig. 12 where a polka-dot lithographic mask [Fig.8(a)] is used for UV patterning. Phospholipid from achloroform-based solution adheres to square patternsthat are not exposed to UV and so are hydrophobic.Adherence of phospholipid from a chloroform solutionon holographically patterned gratings in polybuta-diene is shown in Fig. 13. Evaporation of chloroformdroplets leaves phospholipid fragments [Fig. 13(a)]along the hydrophobic rows of the grating. An in-creased amount of chloroform solution and the result-ing capillary action [Fig. 13(b)] on the film createunbroken rows of phospholipid deposits. With twoorthogonal gratings on polybutadiene substrate, phos-pholipid deposits primarily over hydrophobic areas ina microarray pattern (Fig. 14). The period of gratingsis 2.3 �m, and the spot size of deposited phospholipidis less than 1 �m. Phospholipid molecules typicallyhave a hydrophobic tail and a hydrophobic head andcan dissolve in both hydrophobic as well as hydro-philic solvents. By choosing an appropriate solvent,the phospholipids can be deposited over hydrophilicor hydrophobic regions of the substrate.

Fig. 11. (a) Optical micrograph of a holographic grating in poly-butadiene made by UV light. The grating period is 2300 nm. Rh6Gdye preferentially coats the hydrophilic regions and is used toenhance the visual contrast. (b) Fabrication of a microarray by twoperpendicular holographic gratings in polybutadiene (period of900 nm). The array is made visible by coating it with gold whichpreferably adheres to hydrophobic regions, i.e., those areas notexposed to UV light.

Fig. 12. Phospholipid microarray deposited on a polybutadienethin film following UV lithography using the mask of Fig 8(a).From a chloroform-based solution, phospholipid adheres only tohydrophobic regions of polybutadiene.

Fig. 13. (a) Evaporation of chloroform solvent results in phospho-lipid droplets adhering to hydrophobic regions of a holographic po-lybutadiene grating. (b) Increasing amount of phospholipid solutionin chloroform results in a capillary action along the hydrophobicrows of the holographic grating. Grating parameter is 2300 nm.

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5. Conclusions

We have demonstrated fabrication of surface reliefgratings in polybutadiene rubber films using an UVholographic technique. The grating formation mecha-nism involves UV photodissociation resulting in en-hanced hydrophilicity of the exposed substrate. Elasticrelaxation of rubber films is observed for several min-utes after the UV light is blocked. Submicrometer pat-terning of the substrate is demonstrated and usedto fabricate biofunctional phospholipid microarrayshaving potential as a platform for biosensing appli-cations.

This research was funded by the Center for Bio-photonics in Science and Technology (CBST) and by aRISE-grant, both from the National Science Founda-tion. We thank Michael Curley for the atomic forcemicroscope measurements.

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Fig. 14. Phospholipid microarray is deposited from a chloroform-based solution on two orthogonal gratings on a polybutadiene sub-strate. The period of gratings is 2.3 �m, and the spot size ofdeposited phospholipid is less than 1 �m.

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