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Biologically Driven Assembly of Polyelectrolyte Microcapsule Patterns To Fabricate Microreactor Arrays.

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Communications
Microreactors
DOI: 10.1002/anie.200502822
Biologically Driven Assembly of Polyelectrolyte
Microcapsule Patterns To Fabricate Microreactor
Arrays**
Bo Wang, Qinghe Zhao, Feng Wang, and
Changyou Gao*
Microcapsules with polyelectrolyte multilayer walls assembled using the layer-by-layer process have attracted great
attention because of their potential applications in medicine,
drug delivery, artificial cells (viruses), and catalysis.[1] The
most notable feature of such hollow microcapsules is the
switchable permeability of the walls in response to environmental stimuli such as pH, salt, and temperature.[2] Therefore,
it is possible to conveniently manipulate the interior content
of the capsules by various approaches[3] with respect to
specific application requirements. Meanwhile, many applications require confinement of the capsules with fine spatial
selectivity. For example, space-confined materials are a
prerequisite for high-throughput multipurpose sensors.[4]
Stimulus-responsive microcapsules have potential for targeting drug carriers, as the intracellular pH value and ionic
strength of tumor tissues differ from those of normal tissues.[5]
Therefore, the targeting and selective immobilization of the
microcapsules is of both scientific and technological interest
and 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 approaches
are more promising for biological systems, but the stable
immobilization of the capsules to maintain the as-prepared
patterns is still an unsolved problem.[7b] Herein, a method that
allows isolation of the individual capsules and patterned
assembly with satisfactory spatial selectivity is developed on
the basis of the biological affinity of avidin and biotin[8]
(Figure 1). The selective immobilization of capsules is performed on receptor patterns fabricated by microcontact
printing,[9] with a flexible, biocompatible polymer film as
substrate. The stable microcapsule arrays are further used as
microreactors to synthesize quantum dots (QDs) and other
nanoparticles. In addition, the release of the spatially
[*] B. Wang, Q. Zhao, F. Wang, Prof. Dr. C. Gao
Department of Polymer Science and Engineering
Zhejiang University
Hangzhou 310027 (China)
Fax: (+ 86) 571-8795-1948
E-mail: cygao@mail.hz.zj.cn
[**] We thank Prof. Y. Y. Chen and Prof. J. C. Shen for their stimulating
discussions and continuous support. Prof. H. MHhwald is greatly
acknowledged for his critical reading and corrections of the
manuscript. This study was financially supported by the Natural
Science Foundation of China (Nos. 20434030 and 90206006) and
the National Science Fund for Distinguished Young Scholars of
China (No. 50425311).
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Figure 1. Schematic illustration showing the strategy and bioaffinity
force for capsule patterning. Avidin molecules are covalently patterned
on a PET film containing pentafluorophenyl ester groups by microcontact printing, and are used to guide the spatial location of
biotinylated capsules. Polymers with chelating groups (exemplified
here by PVA) are loaded in the capsules to facilitate precipitation or
reduction reactions to synthesize QDs, nanocrystals, and nanoparticles
(illustrated by the production of ZnS QDs). PET = poly(ethylene
terephthalate), PVA = poly(vinyl alcohol).
synthesized products can be readily tuned. Herein, a flexible
polymer was chosen as the substrate as it may be further
manipulated into various shapes, such as tubes. Moreover, the
polymer is more like tissue than the commonly used silicon
and glass substrates.
Biotin was covalently immobilized on the capsule walls by
the reaction of biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide (biotin-NHS) and the primary amine groups of
poly(allylamine hydrochloride) (PAH; the outermost layer of
the capsules) at pH 7–9. Infrared spectroscopy (Figure 2 a)
recorded the characteristic absorbance bands of biotin at 1710
and 1480 cm 1, thus confirming the existence of the biotin
molecules on the capsule walls. This is further evidenced by
Figure 2. a) FTIR spectrum of the biotinylated microcapsules after
subtraction of the spectrum of the unmodified capsules. Inset: CLSM
image of biotinylated microcapsules bound with Rd-avidin. The scale
bar is 10 mm. b) CLSM image of the Rd-avidin covalent patterns on the
PET film. c) CLSM image of the microcapsule arrays immobilized on
the avidin patterns recorded at the same place as (b). A drop of
fluorescein solution (0.05 mg mL 1) was applied for visualization.
d) SEM image of the microcapsule arrays immobilized on the avidin
patterns. e) Line profiles of the fluorescence intensity depict the
capsule wall positions after treatment under various conditions. The
scale bars in b)–d) are 60 mm. CLSM = confocal laser scanning microscopy; Rd-avidin = rhodamine B isothiocyanate-labeled avidin.
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 1560 –1563
Angewandte
Chemie
the fact that the biotinylated capsules adsorbed ten times
more avidin (driven by the bioconjugation force; Figure 2 a,
inset) than the unmodified capsules (driven by electrostatic
forces). A water-soluble biotinylation reagent with a negatively charged headgroup (SO32 ) was selected to improve the
biotinylation activity. The spacer segment (CH2)6 can
provide freedom for the ligand[10] to recognize its receptor
in an optimal manner.
Avidin patterns on a poly(ethylene terephthalate) (PET)
film were fabricated by microstamping onto an activated
polymer surface (MAPS), which was initially developed by
Chilkoti and co-workers.[11] The COOH groups that were
introduced onto the PET film by hydrolysis[12] were converted
into active pentafluorophenyl esters in a solution of pentafluorophenol (PFP) and 1-ethyl-3-(dimethylamino)propylcarbodiimide (EDAC).[13] The hydrolysis time, which was tracked
by scanning force microscopy (SFM) and contact angle
measurement, was set at 3 hours to give a homogeneous,
flat surface with sufficient COOH groups, which is very
important for the selectivity of the capsule assembly.[7b] The
activated surface was then brought into conformal contact
with a plasma-pretreated poly(dimethylsiloxane) (PDMS)
stamp having periodic pillars inked with avidin solution. The
avidin was stably patterned through formation of amide
linkages with the PET film,[14] as shown by confocal laser
scanning microscopy (CLSM; Figure 2 b). Post-rinsing with
buffer was performed, and the unreacted pentafluorophenyl
esters were deactivated with lysine.
Then the patterned polymer film was placed in a
suspension of biotinylated capsules (diameter 15 mm). Capsule arrays were formed within 30 minutes, as observed by
CLSM (Figure 2 c, wet state) and scanning electron microscopy (SEM; Figure 2 d, dry state). The location of each
capsule corresponded exactly to the site of the avidin receptor
(see Figure 2 b and c). Control experiments showed that no
capsule patterns could be formed by incubating the same
patterned film in unmodified capsule or avidin-saturated
biotinylated capsule suspensions, which demonstrates that the
capsule patterns shown in Figure 2 c were mediated by ligand–
receptor recognition. Although the process has to be finetuned, satisfactory capsule arrays with a dimension of several
millimeters can be obtained. Actually, reducing the nonspecific
adsorption of the capsules is a key issue. The introduction of
poly(ethylene glycol) (PEG) onto capsules[15] or vesicles[16] has
been demonstrated as an effective method. Tween 20 detergent
was added to our capsule suspension to avoid any potential
influence on the capsule permeability by the grafted PEG
nonfouling chains.[15] This detergent covered the capsule
surfaces and showed a very effective blocking ability.[17]
To ensure that only one capsule is placed on a single
pattern, the sizes of the patterns and the capsules should be
matched. We used patterns with diameters of 4, 20, and 40 mm
and a spacing of 6, 40, and 60 mm, respectively, and capsules
with diameters of 4, 5, and 15 mm. Isolated individual capsule
arrays were obtained only in cases where the ratio between
the capsule diameters and the pattern sizes was approximately 1:2 to 3:4. When the capsule diameter was too large,
the capsules bridged the spaces between patterns, whereas
several capsules located on a single pattern if the capsule
Angew. Chem. Int. Ed. 2006, 45, 1560 –1563
diameter was relatively small. However, no precise control
over the number of capsules deposited was achieved by
varying the dimensions, which can probably be attributed to
the softness of the capsule wall.
Notably, biotin and avidin have to be covalently coupled
to the capsule or substrate. If the avidin (positively charged at
neutral pH)[18] was attached to the substrate through electrostatic interaction, the very strong affinity (binding constant of
biotin and avidin ca. 1015 m 1)[19] would detach the avidin
molecules.
The strong binding and the stability of the avidin
molecules[20] endow the capsule patterns with enough stability
to withstand relatively harsh conditions. In fact, the asprepared capsule arrays could survive treatments by ultrasonication, concentrated salt, acid, and base, as well as
elevated temperature (Figure 2 e). The representative fluorescence intensity line profiles obtained by CLSM confirm
that the intact periodicity of the capsule patterns was
preserved, except that the annealed capsules shrank to some
extent as a result of the rearrangement of polyelectrolytes
within the multilayers.[21]
In a subsequent step, the interiors of such aligned
microcontainers were manipulated by the incorporation of
metal ions and subsequent chemical reactions to synthesize
QDs. Polymers with chelating groups, for example poly(vinyl
alcohol) (PVA), were incorporated into a CaCO3 template as
a crystallization manipulator.[22] After layer-by-layer assembly
and dissolution of the cores with ethylenediaminetetraacetic
acid disodium salt (EDTA-Na), PVA was simultaneously
incorporated into the capsules during their formation, which
was confirmed by the absorbance at 1085 cm 1 in the infrared
spectrum. After incubation of the capsule arrays in ZnCl2
solution for 2 h, Zn2+ ions were adsorbed onto the PVA
matrix in the capsules by complexation with the hydroxy
groups.[23] Successively immersing the capsule arrays into
Na2S solution yielded ZnS QDs[24] exclusively within the
capsules, with no detected fluorescence in solution. Raman
spectroscopy also confirmed the formation of ZnS nanoparticles.[25] Under UV radiation, blue capsule arrays were
observed by fluorescence microscopy (Figure 3 a). The size of
the ZnS QDs was estimated as ca. 1 nm according to the
fluorescence spectrum (Figure 3 b). Neither the excitation nor
the emission maxima were changed after complete desiccation of the capsules (Figure 3 b), which indicates that no
agglomeration of the QDs occurred because of stabilization
by the PVA matrix.
When capsule arrays loaded with ZnS QDs were incubated in buffer, the QDs were steadily released into the
solution (Figure 3 c, solid line). This effect lasted up to 40 days
(Figure 3 c, left inset image). We regard this as a refreshing
process, which can be followed by a new reaction cycle.
Actually, three cycles were carried out, and no change in
properties of the produced ZnS was found during these cycles.
On the other hand, the leaching of the QDs could be blocked
to create stable QD-loaded capsule arrays of long durability.
For instance, by assembling six additional polyelectrolyte
layers on the capsules to reseal the pores,[26] less than 5 % of
the QDs escaped from the capsules within 40 days (Figure 3 c,
dashed line and right inset image).
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
1561
Communications
arrays with long durability by simply coating additional layers
onto the capsules. A flexible polymer is chosen as the
substrate, which facilitates the further shaping of devices.
This study also provides a model for active drug targeting.
Experimental Section
Figure 3. a) Fluorescence image showing the existence of ZnS QDs
formed exclusively within the patterned microcapsules. b) Excitation
and 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 of
the fluorescence emission at 470 nm as a function of time. The solid
and dashed lines represent the fluorescence intensity from the asprepared QDs containing capsule arrays without and with additional
(PSS/PAH)3 layers, respectively. The insets show the fluorescence
images of capsule patterns after incubation in buffer for 36 days. The
scale bars in (a) and (c) are 30 and 20 mm, respectively. PSS = poly(styrene sulfonate) sodium salt; PAH = poly(allylamine hydrochloride).
To illustrate the versatility of this approach, carboxymethyl cellulose, poly(acrylic acid) (PAA), poly(vinyl pyrrolidone) (PVP), and chitosan were incorporated into the
capsules as well. Cadmium(ii), copper(ii), silver(i), and gold(iii) ions were similarly coordinated within the capsules
through the ligand groups of these polymers, respectively.
Nanocrystals or nanoparticles of cadmium sulfide (CdS),
copper sulfide (CuS), silver, and gold were successfully
synthesized by precipitation or reduction reactions. Immobilization of the capsules on the surface facilitated the
fabrication and refreshing processes, as separation of the
capsules and excess reagents could be accomplished by simply
washing instead of centrifugation, filtration, and/or dialysis.
In conclusion, micropatterning of polyelectrolyte microcapsules has been realized on a polymer film with high spatial
selectivity driven by specific biotin–avidin recognition. For
this to occur, biotin is covalently immobilized on microcapsules with PAH as the outermost layer, while avidin is
covalently patterned onto the PET film with pentafluorophenol groups on its surface by microcontact printing. The
created microcapsule arrays are very stable against harsh
treatments, such as ultrasonication, acid, base, and elevated
temperature. Several kinds of polymers with chelating groups
(with PVA as a representative example) are loaded into the
capsules to site-specifically synthesize ZnS QDs, nanocrystals,
and nanoparticles through precipitation and reduction reactions. The QDs can be released from the aligned capsules for
up to 40 days. The synthesis can be performed again and such
a cycle has been repeated several times. The leaching of the
QDs can also be blocked to create stable QD-loaded capsule
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Capsule fabrication: Ca(NO3)2 solution (0.025 m, 100 mL) was mixed
with 5 % PVA solution (Mw = 85–124 kDa, 4 mL), and Na2CO3
solution (0.025 m, 100 mL) was added rapidly under strong agitation.
CaCO3 particles with an average diameter of 15 mm were formed
immediately and collected by filtration. The suspension of CaCO3
particles (concentration ca. 5 wt %) was alternately incubated for
10 min in solutions of poly(styrene sulfonate) sodium salt (PSS,
Aldrich, Mw = 70 kDa, 1 mg mL 1) and PAH (Aldrich, Mw = 70 kDa),
both containing NaCl (0.5 m). A centrifugation/washing protocol was
applied to remove excess polyelectrolytes. After (PSS/PAH)5 was
deposited, the CaCO3 cores were dissolved by incubation in EDTANa solution (0.2 m) three times, each for 30 min. Capsules with
diameters of 4 or 5 mm were similarly obtained by using commercial
silicon dioxide colloids as templates.
Biotinylation of the capsules: Biotin-NHS (1.0 mg) was added to
the capsule suspension ( 105 capsules mL 1, 2 mL) in phosphatebuffered saline (pH 8.0). After incubation for 1 h at room temperature, centrifugation and washing were performed to purify the
resulting capsules.
Activation of the PET film: A PET film (Melinex, DuPont) was
cleaned by sequentially rinsing in distilled water, methanol, and
refluxing hexane, each for 2 h. The cleaned film was incubated in
NaOH solution (1m) for 3 h. After hydrolysis, the film was sequentially rinsed with HCl (0.1m) and distilled water, then dried under
reduced pressure at 50 8C for 24 h. The COOH groups on the film
were activated by immersion in EDAC (0.1m, Sigma) and PFP (0.2 m,
Aldrich) ethanolic solution for 15 min. The film was rinsed with
anhydrous 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 isothiocyanate-labeled avidin (Rd-avidin, Sigma) solution (0.1 mg mL 1) for
30 min, rinsed with water, and dried with a gentle nitrogen stream.
The stamp was immediately pressed onto the PET substrate with a
normalized force of 2 N cm 2 for 30 min. The patterned substrate was
then removed from the stamp, washed with ethanol, cleaned by
ultrasonication for 2 min, and finally dried by a nitrogen flow. The
unreacted pentafluorophenyl esters in both the avidin patterns and
the continuous regions were decomposed by reaction with lysine
(0.1m) for 20 min.
Capsule assembly: Capsules were assembled by incubating an
avidin-patterned film in biotinylated capsule suspensions
( 105 capsules mL 1) for 30 min, followed by rinsing with water.
Tween 20 (0.02 % v/v) was supplemented in the capsule suspension
before assembly. After mixing for 20 min, the excess Tween 20 was
removed by centrifugation. Tween 20 was also occasionally used
during the rinsing step.
In situ synthesis of ZnS QDs within the microcapsules: The PET
film 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 (0.05 m) for 30 min. Finally, the
film was washed with distilled water to remove the excess Na2S.
Characterization: The CLSM and SEM images were obtained
with a Bio-Rad Radiance 2100 confocal laser scanning microscope
and a Stereoscan 260 Cambridge electron microscope, respectively.
Fluorescence spectra were recorded on a fluorescence spectrophotometer (Hitachi F-4500). The infrared spectra were obtained with a
Bruker Vector 22 spectrometer on dry capsules.
Received: August 9, 2005
Published online: January 27, 2006
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 1560 –1563
Angewandte
Chemie
.
Keywords: microreactors · nanostructures · polyelectrolytes ·
polymers · quantum dots
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