Microreactors

DOI: 10.1002/ange.200502822

Biologically Driven Assembly of PolyelectrolyteMicrocapsule Patterns To Fabricate MicroreactorArrays**

Bo Wang, Qinghe Zhao, Feng Wang, andChangyou Gao*

Microcapsules with polyelectrolyte multilayer walls assem-bled using the layer-by-layer process have attracted greatattention because of their potential applications in medicine,drug delivery, artificial cells (viruses), and catalysis.[1] Themost notable feature of such hollow microcapsules is theswitchable permeability of the walls in response to environ-mental stimuli such as pH, salt, and temperature.[2] Therefore,it is possible to conveniently manipulate the interior contentof the capsules by various approaches[3] with respect tospecific application requirements. Meanwhile, many applica-tions require confinement of the capsules with fine spatialselectivity. For example, space-confined materials are aprerequisite for high-throughput multipurpose sensors.[4]

Stimulus-responsive microcapsules have potential for target-ing drug carriers, as the intracellular pH value and ionicstrength of tumor tissues differ from those of normal tissues.[5]

Therefore, the targeting and selective immobilization of themicrocapsules is of both scientific and technological interestand a key issue in this context.Actually, patterning of the capsules has been fulfilled with

both passive[6] (electron-beam lithography) and active[7]

(electrostatic coupling) approaches. The active approachesare more promising for biological systems, but the stableimmobilization of the capsules to maintain the as-preparedpatterns is still an unsolved problem.[7b] Herein, a method thatallows isolation of the individual capsules and patternedassembly with satisfactory spatial selectivity is developed onthe basis of the biological affinity of avidin and biotin[8]

(Figure 1). The selective immobilization of capsules is per-formed on receptor patterns fabricated by microcontactprinting,[9] with a flexible, biocompatible polymer film assubstrate. The stable microcapsule arrays are further used asmicroreactors to synthesize quantum dots (QDs) and othernanoparticles. In addition, the release of the spatially

synthesized products can be readily tuned. Herein, a flexiblepolymer was chosen as the substrate as it may be furthermanipulated into various shapes, such as tubes. Moreover, thepolymer is more like tissue than the commonly used siliconand glass substrates.Biotin was covalently immobilized on the capsule walls by

the reaction of biotinamidohexanoic acid 3-sulfo-N-hydrox-ysuccinimide (biotin-NHS) and the primary amine groups ofpoly(allylamine hydrochloride) (PAH; the outermost layer ofthe capsules) at pH 7–9. Infrared spectroscopy (Figure 2a)recorded the characteristic absorbance bands of biotin at 1710and 1480 cm�1, thus confirming the existence of the biotinmolecules on the capsule walls. This is further evidenced by

Figure 1. Schematic illustration showing the strategy and bioaffinityforce for capsule patterning. Avidin molecules are covalently patternedon a PET film containing pentafluorophenyl ester groups by micro-contact printing, and are used to guide the spatial location ofbiotinylated capsules. Polymers with chelating groups (exemplifiedhere by PVA) are loaded in the capsules to facilitate precipitation orreduction reactions to synthesize QDs, nanocrystals, and nanoparticles(illustrated by the production of ZnS QDs). PET=poly(ethyleneterephthalate), PVA=poly(vinyl alcohol).

Figure 2. a) FTIR spectrum of the biotinylated microcapsules aftersubtraction of the spectrum of the unmodified capsules. Inset: CLSMimage of biotinylated microcapsules bound with Rd-avidin. The scalebar is 10 mm. b) CLSM image of the Rd-avidin covalent patterns on thePET film. c) CLSM image of the microcapsule arrays immobilized onthe avidin patterns recorded at the same place as (b). A drop offluorescein solution (0.05 mgmL�1) was applied for visualization.d) SEM image of the microcapsule arrays immobilized on the avidinpatterns. e) Line profiles of the fluorescence intensity depict thecapsule wall positions after treatment under various conditions. Thescale bars in b)–d) are 60 mm. CLSM= confocal laser scanning micro-scopy; Rd-avidin= rhodamine B isothiocyanate-labeled avidin.

[*] B. Wang, Q. Zhao, F. Wang, Prof. Dr. C. GaoDepartment of Polymer Science and EngineeringZhejiang UniversityHangzhou 310027 (China)Fax: (+86)571-8795-1948E-mail: cygao@mail.hz.zj.cn

[**] We thank Prof. Y. Y. Chen and Prof. J. C. Shen for their stimulatingdiscussions and continuous support. Prof. H. MHhwald is greatlyacknowledged for his critical reading and corrections of themanuscript. This study was financially supported by the NaturalScience Foundation of China (Nos. 20434030 and 90206006) andthe National Science Fund for Distinguished Young Scholars ofChina (No. 50425311).

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the fact that the biotinylated capsules adsorbed ten timesmore avidin (driven by the bioconjugation force; Figure 2a,inset) than the unmodified capsules (driven by electrostaticforces). A water-soluble biotinylation reagent with a nega-tively charged headgroup (SO3

2�) was selected to improve thebiotinylation activity. The spacer segment �(CH2)6� canprovide freedom for the ligand[10] to recognize its receptorin an optimal manner.Avidin patterns on a poly(ethylene terephthalate) (PET)

film were fabricated by microstamping onto an activatedpolymer surface (MAPS), which was initially developed byChilkoti and co-workers.[11] The COOH groups that wereintroduced onto the PET film by hydrolysis[12] were convertedinto active pentafluorophenyl esters in a solution of penta-fluorophenol (PFP) and 1-ethyl-3-(dimethylamino)propylcar-bodiimide (EDAC).[13] The hydrolysis time, which was trackedby scanning force microscopy (SFM) and contact anglemeasurement, was set at 3 hours to give a homogeneous,flat surface with sufficient COOH groups, which is veryimportant for the selectivity of the capsule assembly.[7b] Theactivated surface was then brought into conformal contactwith a plasma-pretreated poly(dimethylsiloxane) (PDMS)stamp having periodic pillars inked with avidin solution. Theavidin was stably patterned through formation of amidelinkages with the PET film,[14] as shown by confocal laserscanning microscopy (CLSM; Figure 2b). Post-rinsing withbuffer was performed, and the unreacted pentafluorophenylesters were deactivated with lysine.Then the patterned polymer film was placed in a

suspension of biotinylated capsules (diameter� 15 mm). Cap-sule arrays were formed within 30 minutes, as observed byCLSM (Figure 2c, wet state) and scanning electron micro-scopy (SEM; Figure 2d, dry state). The location of eachcapsule corresponded exactly to the site of the avidin receptor(see Figure 2b and c). Control experiments showed that nocapsule patterns could be formed by incubating the samepatterned film in unmodified capsule or avidin-saturatedbiotinylated capsule suspensions, which demonstrates that thecapsule patterns shown in Figure 2c were mediated by ligand–receptor recognition. Although the process has to be fine-tuned, satisfactory capsule arrays with a dimension of severalmillimeters can be obtained. Actually, reducing the nonspecificadsorption of the capsules is a key issue. The introduction ofpoly(ethylene glycol) (PEG) onto capsules[15] or vesicles[16] hasbeen demonstrated as an effective method. Tween20 detergentwas added to our capsule suspension to avoid any potentialinfluence on the capsule permeability by the grafted PEGnonfouling chains.[15] This detergent covered the capsulesurfaces and showed a very effective blocking ability.[17]

To ensure that only one capsule is placed on a singlepattern, the sizes of the patterns and the capsules should bematched. We used patterns with diameters of 4, 20, and 40 mmand a spacing of 6, 40, and 60 mm, respectively, and capsuleswith diameters of 4, 5, and 15 mm. Isolated individual capsulearrays were obtained only in cases where the ratio betweenthe capsule diameters and the pattern sizes was approxi-mately 1:2 to 3:4. When the capsule diameter was too large,the capsules bridged the spaces between patterns, whereasseveral capsules located on a single pattern if the capsule

diameter was relatively small. However, no precise controlover the number of capsules deposited was achieved byvarying the dimensions, which can probably be attributed tothe softness of the capsule wall.Notably, biotin and avidin have to be covalently coupled

to the capsule or substrate. If the avidin (positively charged atneutral pH)[18] was attached to the substrate through electro-static interaction, the very strong affinity (binding constant ofbiotin and avidin ca. 1015m�1)[19] would detach the avidinmolecules.The strong binding and the stability of the avidin

molecules[20] endow the capsule patterns with enough stabilityto withstand relatively harsh conditions. In fact, the as-prepared capsule arrays could survive treatments by ultra-sonication, concentrated salt, acid, and base, as well aselevated temperature (Figure 2e). The representative fluo-rescence intensity line profiles obtained by CLSM confirmthat the intact periodicity of the capsule patterns waspreserved, except that the annealed capsules shrank to someextent as a result of the rearrangement of polyelectrolyteswithin the multilayers.[21]

In a subsequent step, the interiors of such alignedmicrocontainers were manipulated by the incorporation ofmetal ions and subsequent chemical reactions to synthesizeQDs. Polymers with chelating groups, for example poly(vinylalcohol) (PVA), were incorporated into a CaCO3 template asa crystallizationmanipulator.[22] After layer-by-layer assemblyand dissolution of the cores with ethylenediaminetetraaceticacid disodium salt (EDTA-Na), PVA was simultaneouslyincorporated into the capsules during their formation, whichwas confirmed by the absorbance at 1085 cm�1 in the infraredspectrum. After incubation of the capsule arrays in ZnCl2solution for 2 h, Zn2+ ions were adsorbed onto the PVAmatrix in the capsules by complexation with the hydroxygroups.[23] Successively immersing the capsule arrays intoNa2S solution yielded ZnS QDs

[24] exclusively within thecapsules, with no detected fluorescence in solution. Ramanspectroscopy also confirmed the formation of ZnS nano-particles.[25] Under UV radiation, blue capsule arrays wereobserved by fluorescence microscopy (Figure 3a). The size ofthe ZnS QDs was estimated as ca. 1 nm according to thefluorescence spectrum (Figure 3b). Neither the excitation northe emission maxima were changed after complete desicca-tion of the capsules (Figure 3b), which indicates that noagglomeration of the QDs occurred because of stabilizationby the PVA matrix.When capsule arrays loaded with ZnS QDs were incu-

bated in buffer, the QDs were steadily released into thesolution (Figure 3c, solid line). This effect lasted up to 40 days(Figure 3c, left inset image). We regard this as a refreshingprocess, which can be followed by a new reaction cycle.Actually, three cycles were carried out, and no change inproperties of the produced ZnS was found during these cycles.On the other hand, the leaching of the QDs could be blockedto create stable QD-loaded capsule arrays of long durability.For instance, by assembling six additional polyelectrolytelayers on the capsules to reseal the pores,[26] less than 5% ofthe QDs escaped from the capsules within 40 days (Figure 3c,dashed line and right inset image).

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To illustrate the versatility of this approach, carboxy-methyl cellulose, poly(acrylic acid) (PAA), poly(vinyl pyrro-lidone) (PVP), and chitosan were incorporated into thecapsules as well. Cadmium(ii), copper(ii), silver(i), and gold-(iii) ions were similarly coordinated within the capsulesthrough the ligand groups of these polymers, respectively.Nanocrystals or nanoparticles of cadmium sulfide (CdS),copper sulfide (CuS), silver, and gold were successfullysynthesized by precipitation or reduction reactions. Immobi-lization of the capsules on the surface facilitated thefabrication and refreshing processes, as separation of thecapsules and excess reagents could be accomplished by simplywashing instead of centrifugation, filtration, and/or dialysis.In conclusion, micropatterning of polyelectrolyte micro-

capsules has been realized on a polymer film with high spatialselectivity driven by specific biotin–avidin recognition. Forthis to occur, biotin is covalently immobilized on micro-capsules with PAH as the outermost layer, while avidin iscovalently patterned onto the PET film with pentafluorophe-nol groups on its surface by microcontact printing. Thecreated microcapsule arrays are very stable against harshtreatments, such as ultrasonication, acid, base, and elevatedtemperature. Several kinds of polymers with chelating groups(with PVA as a representative example) are loaded into thecapsules to site-specifically synthesize ZnS QDs, nanocrystals,and nanoparticles through precipitation and reduction reac-tions. The QDs can be released from the aligned capsules forup to 40 days. The synthesis can be performed again and sucha cycle has been repeated several times. The leaching of theQDs can also be blocked to create stable QD-loaded capsule

arrays with long durability by simply coating additional layersonto the capsules. A flexible polymer is chosen as thesubstrate, which facilitates the further shaping of devices.This study also provides a model for active drug targeting.

Experimental SectionCapsule fabrication: Ca(NO3)2 solution (0.025m, 100 mL) was mixedwith 5% PVA solution (Mw= 85–124 kDa, 4 mL), and Na2CO3solution (0.025m, 100 mL) was added rapidly under strong agitation.CaCO3 particles with an average diameter of 15 mm were formedimmediately and collected by filtration. The suspension of CaCO3particles (concentration ca. 5 wt%) was alternately incubated for10 min in solutions of poly(styrene sulfonate) sodium salt (PSS,Aldrich,Mw= 70 kDa, 1 mgmL

�1) and PAH (Aldrich,Mw= 70 kDa),both containing NaCl (0.5m). A centrifugation/washing protocol wasapplied to remove excess polyelectrolytes. After (PSS/PAH)5 wasdeposited, the CaCO3 cores were dissolved by incubation in EDTA-Na solution (0.2m) three times, each for 30 min. Capsules withdiameters of 4 or 5 mm were similarly obtained by using commercialsilicon dioxide colloids as templates.

Biotinylation of the capsules: Biotin-NHS (1.0 mg) was added tothe capsule suspension (� 105 capsulesmL�1, 2 mL) in phosphate-buffered saline (pH 8.0). After incubation for 1 h at room temper-ature, centrifugation and washing were performed to purify theresulting capsules.

Activation of the PET film: A PET film (Melinex, DuPont) wascleaned by sequentially rinsing in distilled water, methanol, andrefluxing hexane, each for 2 h. The cleaned film was incubated inNaOH solution (1m) for 3 h. After hydrolysis, the film was sequen-tially rinsed with HCl (0.1m) and distilled water, then dried underreduced pressure at 50 8C for 24 h. The COOH groups on the filmwere activated by immersion in EDAC (0.1m, Sigma) and PFP (0.2m,Aldrich) ethanolic solution for 15 min. The film was rinsed withanhydrous ethanol, dried by a nitrogen flow, and used immediately.

Fabrication of the avidin patterns: A PDMS (Dow Corning)stamp with periodic pillars[9] was inked with rhodamine B isothiocya-nate-labeled avidin (Rd-avidin, Sigma) solution (0.1 mgmL�1) for30 min, rinsed with water, and dried with a gentle nitrogen stream.The stamp was immediately pressed onto the PET substrate with anormalized force of 2 Ncm�2 for 30 min. The patterned substrate wasthen removed from the stamp, washed with ethanol, cleaned byultrasonication for 2 min, and finally dried by a nitrogen flow. Theunreacted pentafluorophenyl esters in both the avidin patterns andthe continuous regions were decomposed by reaction with lysine(0.1m) for 20 min.

Capsule assembly: Capsules were assembled by incubating anavidin-patterned film in biotinylated capsule suspensions(� 105 capsulesmL�1) for 30 min, followed by rinsing with water.Tween20 (0.02% v/v) was supplemented in the capsule suspensionbefore assembly. After mixing for 20 min, the excess Tween 20 wasremoved by centrifugation. Tween20 was also occasionally usedduring the rinsing step.

In situ synthesis of ZnS QDs within the microcapsules: The PETfilm with capsule arrays was incubated in ZnCl2 aqueous solution(0.01m) for 2 h, followed by ultrasonication and extensive washing.Then it was immersed in Na2S solution (0.05m) for 30 min. Finally, thefilm was washed with distilled water to remove the excess Na2S.

Characterization: The CLSM and SEM images were obtainedwith a Bio-Rad Radiance 2100 confocal laser scanning microscopeand a Stereoscan 260 Cambridge electron microscope, respectively.Fluorescence spectra were recorded on a fluorescence spectropho-tometer (Hitachi F-4500). The infrared spectra were obtained with aBruker Vector22 spectrometer on dry capsules.

Received: August 9, 2005Published online: January 27, 2006

Figure 3. a) Fluorescence image showing the existence of ZnS QDsformed exclusively within the patterned microcapsules. b) Excitationand emission spectra of aligned microcapsules containing ZnS QDs.Black: excitation at dry state, red: excitation in suspensions, green:emission at dry state, blue: emission in suspensions. c) Evolution ofthe fluorescence emission at 470 nm as a function of time. The solidand dashed lines represent the fluorescence intensity from the as-prepared QDs containing capsule arrays without and with additional(PSS/PAH)3 layers, respectively. The insets show the fluorescenceimages of capsule patterns after incubation in buffer for 36 days. Thescale bars in (a) and (c) are 30 and 20 mm, respectively. PSS=poly-(styrene sulfonate) sodium salt; PAH=poly(allylamine hydrochloride).

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.Keywords: microreactors · nanostructures · polyelectrolytes ·polymers · quantum dots

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