Shape-Encoded Chitosan–Polyacrylamide Hybrid Hydrogel

Polymeric hydrogel microparticle-based suspension arrays with shape-based encoding offer powerful alternatives to planar and bead-based arrays toward ...
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Shape-Encoded Chitosan−Polyacrylamide Hybrid Hydrogel Microparticles with Controlled Macroporous Structures via Replica Molding for Programmable Biomacromolecular Conjugation Eunae Kang, Sukwon Jung, John H. Abel, Allison Pine, and Hyunmin Yi* Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts 02155 United States S Supporting Information *

ABSTRACT: Polymeric hydrogel microparticle-based suspension arrays with shape-based encoding offer powerful alternatives to planar and bead-based arrays toward high throughput biosensing and medical diagnostics. We report a simple and robust micromolding technique for polyacrylamide(PAAm-) based biopolymeric−synthetic hybrid microparticles with controlled 2D shapes containing a potent aminopolysaccharide chitosan as an efficient conjugation handle uniformly incorporated in PAAm matrix. A postfabrication conjugation approach utilizing amine-reactive chemistries on the chitosan shows stable incorporation and retained chemical reactivity of chitosan, readily tunable macroporous structures via simple addition of low content long-chain PEG porogens for improved conjugation capacity and kinetics, and one-pot biomacromolecular assembly via bioorthogonal click reactions with minimal nonspecific binding. We believe that the integrated fabrication-conjugation approach reported here could offer promising routes to programmable manufacture of hydrogel microparticle-based biomacromolecular conjugation and biofunctionalization platforms for a large range of applications.

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INTRODUCTION Rapid and selective assembly and assay of biomacromolecules is crucial in many application areas including medical diagnostics, biosensing and biological threat detection.1−4 While much progress has been made in the general areas of high throughput bioassays in DNA and protein arrays, printed or synthesized planar and bead arrays have limitations in probe titer, selectivity, and assay time. These limitations arise from the limited conjugation capacity and slow mass transfer from the bulk solution to the surfaces as well as high nonspecific binding. Suspension arrays of polymeric hydrogel microparticles offer powerful alternatives to such surface-based assay platforms. First, the high water content and hydrophilic nature of the hydrogels provide favorable solution kinetics and minimal liquid−solid transition. Second, such hydrophilic environment minimizes distortion of the probe and target molecules unlike surface-based assays, leading to selective binding and low nonspecific adsorption for improved selectivity. Third, 3D polymer network offers high probe titer for a wide dynamic detection ranges. In addition, much advancement in a range of hydrogel microparticle fabrication techniques has allowed various encoding schemes for multiplexed assays, including complex 2D and 3D shape-based encoding.5−8 For example, flow lithographic techniques offer rapid fabrication of complex-shaped microparticles by exploiting the laminar nature of the microfluidic flows. For such techniques, poly(ethylene glycol) (PEG) has been the material of choice © XXXX American Chemical Society

due to rapid photoinduced polymerization from PEG diacrylate (DA) and PEG’s highly nonfouling property. Inert PEG porogens have been successfully utilized to create macroporous structures for large biomacromolecule assays.9−12 Yet, the cross-linked nature of the PEGDA-derived networks still limits simple and programmable creation of macroporous structures for rapid biomacromolecular probe conjugation and target capture. While fabrication of hydrogel materials from longer chain PEGDA up to 20 kDa has been reported,13−15 the resulting pore size is still much limited. In contrast to the inherently cross-linking PEGDA, polyacrylamide (PAAm) hydrogels synthesized from acrylamide monomers offer an attractive alternative due to its inherently linear chain-forming nature. Theoretically, the mesh size could be readily tuned by modifying the cross-linker content (e.g., bis-acrylamide).16−18 While such traits have led to a widespread use of PAAm in gel electrophoresis (PAGE) for protein analysis, its slow polymerization rate19,20 has limited its use in microparticle fabrication through rapid microfluidic fabrication techniques. Our approach to addressing these challenges is to exploit a simple and robust replica molding technique for controlled fabrication of PAAm microparticles with simple 2D shape-based Received: December 20, 2015 Revised: March 26, 2016

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DOI: 10.1021/acs.langmuir.5b04653 Langmuir XXXX, XXX, XXX−XXX

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In this report, we present programmable fabrication of PAAm-based hydrogel microparticles with controlled 2D shapes containing CS as an efficient conjugation. Tunable macroporosity for improved biomacromolecular conjugation capacity and kinetics is readily imparted by low content (≤4 w/ v %) long chain (LC) PEG porogen. Simple fluorescent labeling with an amine-reactive N-hydroxyl succinimidyl (NHS) ester form of fluorescein show chitosan’s retained chemical reactivity, as well as highly uniform and stable incorporation. Protein conjugation studies using a model red fluorescent protein R-phycoerythrin (R-PE) reveal tunable macroporous structures imparted by simple addition of low content LC PEG for enhanced protein conjugation capacity via rapid bioorthogonal tetrazine−trans-cyclooctene (Tz−TCO) ligation reaction with minimal nonspecific binding. Further examination of the protein conjugation kinetics shows much improved, rapid conjugation reaching complete penetration and conjugation within 1 h. Finally, we show that two bioorthogonal click reactions (i.e., Tz−TCO and strainpromoted alkyne−azide cycloaddition (SPAAC) reactions) combined with simple shape-based encoding enable simultaneous one-pot assembly of biomacromolecules (single-stranded DNA and R-PE), all with high selectivity and minimal nonspecific binding or fouling. Combined, these results illustrate for the first time facile fabrication of novel biopolymeric-synthetic hybrid microparticles based on PAAm hydrogel matrix with high uniformity and chemical functionality for efficient and rapid protein conjugation in an integrated fabrication-conjugation approach. We envision that the methods reported here may be readily extended to the manufacture of hydrogel microparticle platforms with tunable mesh size and improved performances for a wide range of biosensing and medical diagnostic applications.

encoding, combined with a postfabrication conjugation approach utilizing chemically reactive conjugation handles within the microparticles. For this purpose, we utilize a potent amino-polysaccharide chitosan that offers abundant primary amine groups with uniquely low pKa value (∼6.5) at nearly every glucosamine monomer unit. As shown in the schematic diagram of Figure 1a, our fabrication scheme involves filling

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EXPERIMENTAL SECTION

Materials. Chitosan oligosaccharide lactate (CS, average Mn 5 kDa, > 90% deacetylation), acrylamide, bis-acrylamide (Bis), poly(ethylene glycol) (PEG, average MW 8 kDa), 2-hydroxy-2-methylpropiophenone (photoinitiator, PI) and saline sodium citrate (SSC) buffer (20× concentrate, molecular biology grade) were purchased from SigmaAldrich (St. Louis, MO). 5- (and 6-) carboxyfluorescein succinimidyl ester (NHS−fluorescein) was purchased from Pierce Biotechnology (Rockford, IL). Tetrazine (Tz)−PEG5−NHS ester, azadibenzocyclooctyne (ADIBO)-sulfo-NHS ester, and trans-cyclooctene (TCO)− PEG4−NHS ester were purchased from Click Chemistry Tools (Scottsdale, AZ). Tween 20 (TW20) and poly(dimethylsiloxane) (PDMS) elastomer kits (Sylgard 184, Dow Corning, Auburn, MI) were purchased from Thermo Fisher Scientific. Single-stranded (ss) DNA used in this study were purchased from Integrated DNA Technologies (Coralville, IA); azide-terminated and fluorescently labeled ssDNA (F−DNA−azide, 5′-/azide/ATGATGATGATGATGATG/FAM/-3′). Red fluorescent protein R-Phycoerythrin (R-PE in sodium phosphate buffer, pH 7.0 with ammonium sulfate) was purchased from AnaSpec (Fremont, CA). All the reagents were analytical grade and used without further purification unless noted otherwise. Fabrication of CS−PAAm Microparticles via Replica Molding. Chitosan−polyacrylamide (CS−PAAm) microparticles were fabricated according to methods in our recent report with minor modifications.22 Briefly, the composition of the preparticle solution was as follows: 0.5% (w/v) chitosan oligomer, 15% (w/v) acrylamide and bis-acrylamide (AAm:Bis =29:1), and 2% (v/v) PI with or without 1−4% PEG porogen (8 kDa). The chitosan oligomers were dissolved in deionized water and mixed directly with the other components in the aqueous prepolymer solution. As shown in Figure 1a, the preparticle solution was placed into a PDMS mold (1600 wells per a

Figure 1. Fabrication of CS−PAAm microparticles via replica molding. (a) Schematic diagram of the microparticle fabrication procedure. (b) Chemical structures of chitosan, acrylamide and bis-acrylamide. (c) Bright-field micrographs of simple shape-encoded CS−PAAm microparticles. Coefficient of variation (CV) was measured from minimum 20 microparticles for each shape. Scale bars represent 200 μm.

2D-shaped polydimethylsiloxane (PDMS) microwell patterns with simple aqueous mixture (prepolymer solution) consisting of chitosan (CS, Figure 1b), acrylamide (AAm), the cross-linker bis-acrylamide (Bis), and photoinitiator (PI). Irradiation of the prepolymer solution with a simple hand-held UV lamp (365 nm) triggers photoinduced radical polymerization, yielding highly uniform (coefficient of variation, CV ∼ 1%) CS−PAAm microparticles with reliable replication of simple 2D shapes, as shown in the bright-field micrographs of Figure 1c. This simple fabrication scheme leads to stable incorporation of chitosan with retained chemical reactivity for further chemical conjugation through amine-reactive chemistries.21 The batch processing-based nature of the replica molding scheme negates the needs for delicate microflow control and costly equipment, and allows for sufficient polymerization time for slowpolymerizing monomers such as acrylamide in a simple and readily scalable manner, expanding the fabrication parameter spaces including monomers that can be enlisted than in microfluidic fabrication techniques. B

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molecules (fluorescein and F-DNA) and the R-PEs respectively, and the fluorescence micrographs were captured with a DP70 digital microscope camera. Fluorescence intensity was evaluated with ImageJ software (http://imagej.nih.gov/ij/). Confocal micrographs were acquired on a Leica DMIRE2 microscope (Wetzlar, Germany). The particles were analyzed with a 20× objective (0.7 NA) at 488 and 543 nm excitation for the green fluorescent molecules and the R-PEs respectively, and the depth scan increment was 1 μm.

mold), which was formed with Sylgard 184 following overnight incubation at 65 °C on a silicon master mold fabricated by standard photolithography.22,23 Upon the addition of the prepolymer solution to the micromold, the air bubbles in the microwells were removed by rubbing the mold with a disposable plastic pipet tip. The excess preparticle solution was simply taken away by pipetting, and then the filled mold was sealed with a PDMS-coated glass slide except for the square region for microwells (roughly 0.7 cm × 0.7 cm) to make a small gap between the glass surface and the top portion of the microwells. To prevent rapid evaporation of the preparticle solution, the procedures above were carried out in a humidity chamber with approximately 92% relative humidity. The sealed mold was then placed on an aluminum mirror (Thorlabs, Newton, NJ) and exposed to 365 nm UV light with an 8 W hand-held UV lamp (Spectronics Corp., Westbury, NY) for 1 h. The polymerized particles were released from the microwells by physically bending the mold, and then water containing 0.5% (v/v) TW20 was placed on the top of the mold to collect the particles by pipetting. The microparticles were then transferred to a microcentrifuge tube and rinsed to remove any unreacted chemicals as follows: mixing the particles in 5× SSC buffer solution containing 0.05% (v/v) TW20 by pipetting, allowing them to settle to the tube bottom, and removing the supernatant. The rinsing procedure was repeated at least five times. Fluorescent Labeling of the Microparticles. For fluorescent labeling, the CS−PAAm microparticles (roughly 1000 particles) were incubated in 5× SSC buffer solution containing 0.05% (v/v) TW20 with 0.5 mM NHS−fluorescein for 1 h on a rotator at room temperature covered with aluminum foil to minimize exposure to light. The unreacted NHS−fluorescein molecules were then removed by rinsing the particles 5 times using the rinsing procedure described above. TCO-activation of R-PEs. In order to activate R-PEs with TCO molecules, we first carried out the buffer exchange of the R-PE solution with borate buffered saline (50 mM borate, 300 mM NaCl, pH 8.5) via centrifugal filtration at 4 °C. The R-PEs (2 mg/mL) were then reacted with 20-fold molar excess of TCO−PEG4−NHS ester or NHS− PEG12−azide for 30 min at room temperature. Unreacted chemicals were separated from the R-PE solution via centrifugal filtration (Amicon Ultra 0.5) with PBS buffer (pH 7.4). Concentrations of the final R-PE solutions were measured by UV−vis spectrophotometry (Evolution 300 UV−vis Spectrophotometer, Thermo scientific, Waltham, MA) with the characteristic absorbance peaks and molar extinction coefficients of the R-PE (1.96 × 106 M−1cm−1 at 565 nm). Tz−TCO Reaction for R-PE Conjugation. We conjugated R-PEs with CS−PAAm particles via Tz−TCO cycloaddition reaction. For this, the CS−PAAm particles were first activated with Tz molecules upon 1 h incubation with 500 μM Tz−PEG5−NHS ester in 5× SSC buffer solution containing 0.05% (v/v) TW20 at room temperature, and rinsed with the washing procedures described above. The Tzactivated CS−PAAm particles were then reacted with 2 μM TCOactivated R-PEs in 5× SSC buffer solution containing 0.05% (v/v) TW20 for 24 h at room temperature. For the protein conjugation kinetic study (Figure 4), the Tz-activated CS−PAAm particles were also reacted with TCO-activated R-PEs under identical conjugation conditions for various periods. The unconjugated R-PEs were separated from the particle solutions via the rinsing procedures as described above. SPAAC Reaction for Conjugation of ssDNA. CS−PAAm microparticles were incubated in 5× SSC buffer solution containing 0.05% (v/v) TW20 with 500 μM ADIBO-sulfo-NHS ester for 1 h on a rotator at room temperature for activation with ADIBO. The unreacted ADIBO-sulfo-NHS ester molecules were rinsed 5 times using the rinsing procedure. The ADIBO-activated microparticles were then reacted with 10 μM of azide-terminated ssDNAs (F−DNA− azide) for 24 h at room temperature. The unconjugated DNAs were then rinsed 5 times using the rinsing procedure. Imaging Analysis. The CS−PAAm microparticles were visualized with an Olympus BX51 epifluorescence microscope using standard green (U−N31001) and red (U−N31002) filter sets (Chroma Technology Corp., Rockingham, VT) for the green fluorescent

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RESULTS AND DISCUSSION Chemically Functional CS−PAAm Microparticles. First to examine the chemical functionality of the chitosanincorporated hydrogel (CS−PAAm) microparticles, we utilized a simple amidation reaction with an NHS-ester form of fluorescein (NHS−fluorescein), as shown in Figure 2. For this, we fabricated hexagon-shaped microparticles via replica molding (shown in Figure 1a) with prepolymer solution composed of 0.5% (w/v) short-chain chitosan (Mn 5 kDa), 15% (w/v) monomers (AAm:Bis = 29:1) and varying contents of long-chain (LC) PEG porogen (MW 8 kDa). These microparticles were then exposed to NHS−fluorescein as shown in the schematic diagram of Figure 2a. Note that, we initially hypothesized that low Bis content or low total monomer content should allow for programmable creation of macroporous network structures for conjugation and assay of large biomacromolecules with improved titer and kinetics. However, AAm−Bis content as low as 3000:1 is needed for meaningful macroporosity,24 and such fabrication parameters led to microparticles with insufficient mechanical integrity (data not shown). We have found that simple addition of small content of LC PEG porogen (MW 8 kDa) in the prepolymer mixture enabled the formation of consistent and highly tunable macroporous network structures (further discussed in Figures 2 and 3). As shown in the bright-field micrographs at the top row of Figure 2b, the CS−PAAm microparticles were fabricated in a consistent manner with all the LC PEG porogen conditions examined, indicating the robust nature of the replica molding technique. The microparticles fabricated with 4% (w/v) PEG porogen showed faint brown color (rightmost image, Figure 2b), suggesting the formation of macropores.25 Next, the fluorescence micrographs at the second row of Figure 2c show that the CS−PAAm microparticles are uniformly labeled with fluorescein resulting from the acyl substitution (i.e., amidation) reaction between the amine’s unshared pair of electrons and the NHS−fluorescein.26,27 Specifically, the average fluorescence intensity measured from at least 5 microparticles for each condition ranged from 38 to 51 at 0.1 s exposure time, with consistently small standard deviations for all the LC PEG porogen conditions enlisted in our study. While fluorescence intensity is a semiquantitative measure for comparison without absolute physical ground, this result clearly indicates the incorporation and retained chemical activity of the chitosan, consistent with CS−PEG microparticle results in our recent study.22 Meanwhile, PAAm microparticles fabricated without chitosan showed minimal fluorescence upon exposure to NHS−fluorescein (Figure S1, Supporting Information), further confirming that the fluorescence observed in Figure 2c results from the specific amidation reaction with minimal nonspecific binding between fluorescein and the PAAm backbone. Furthermore, fluorescein labeling results on the CS−PAAm microparticles stored for 2 weeks showed minimal decrease in the fluorescence, indicating stable incorporation of chitosan in C

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Figure 2. Fluorescent labeling of CS−PAAm microparticles. (a) Schematic diagram of amidation reaction for fluorescent labeling. (b) Bright-field micrographs of the CS−PAAm microparticles fabricated with varying LC PEG porogen contents upon fluorescent labeling, and (c) their corresponding fluorescence micrographs. Inset numbers indicate average fluorescence intensity (FI) with standard deviations measured from at least five microparticles per each condition. (d) Confocal micrographs at the center plane of independent sets of fluorescently labeled microparticles for each PEG porogen content. (e) 3D fluorescence contour plot of the fluorescently labeled CS−PAAm microparticles with 4% PEG 8 kDa. Scale bars represent 200 μm.

PE conjugation (top row, Figure 3b). Second, the fluorescence micrographs in the middle row of Figure 3c show stark contrast in the fluorescence intensity of the R-PE-conjugated microparticles with and without the LC PEG porogens under identical imaging conditions. Specifically, the microparticles prepared without LC PEG porogen show minimal fluorescence (FI = 2.4), while all the other three conditions show bright and uniform red fluorescence resulting from high density conjugation of R-PE. The average fluorescence intensity appeared to increase as the LC PEG content increased, reaching a maximum value of 70 at 4% PEG content under the imaging conditions used (e.g., exposure time 5 ms). Also, uniform fluorescence among the particles for every condition is further indicated by the consistently small standard deviations acquired from at least 5 particles per each condition. Notably, particles prepared with 1% and 2% LC PEG porogen (Figure 3c) show higher fluorescence around the particle edges. This higher fluorescence is further confirmed with the confocal images taken at the center plane of the microparticles in Figure 3d, while the epifluorescence and confocal images in Figure 3c,d respectively show complete penetration of R-PE for the 4% PEG condition. Importantly, these fluorescence results clearly indicate that simple addition of small amount of LC PEG porogens (i.e., ≤4 w/v %) in the prepolymer mixtures leads to substantially larger mesh sizes in direct contrast to the particles prepared without the PEG porogen (leftmost column in Figure 3c,d), allowing the large R-PE proteins (MW 240 kDa, ∼ 11 nm diameter)30 to diffuse in and react with the Tz-activated chitosan in the microparticles. The increasing penetration depths (PD = 2.9 and 13.6 μm for 1% and 2% PEG porogen conditions respectively, Figure 3d) combined with the trend of increasing average fluorescence (Figure 3e) further confirm that the mesh size and protein conjugation capacity can be readily tuned by small amounts of LC PEG porogen in a consistent manner. In the meantime, the CS−PAAm microparticles without Tz-activation showed minimal fluorescence upon exposure to TCO-modified R-PE’s for all the PEG conditions examined (Figure S3, Supporting Information). This result clearly indicates minimal nonspecific binding between R-PE protein and the CS−PAAm backbone, in addition to

the CS−PAAm microparticles (Figure S2, Supporting Information). Notably, the fluorescence across each microparticle’s area appears highly uniform for all the conditions examined, unlike our recent results with CS−PEG microparticles22 where outer edges showed higher fluorescence rising from polymerizationinduced phase separation.9,10,28,29 This uniform fluorescence is further confirmed by the 3D fluorescence contour plot from one of the CS−PAAm microparticles with 4% PEG porogen (Figure 2e), as well as by the confocal microscopy images taken at the center plane of independent sets of labeled microparticles as shown in Figure 2d. These results thus suggest that the chitosan is incorporated throughout the microparticles in a highly uniform manner (i.e., minimal phase separation during polymerization between growing PAAm networks and chitosan) under all the fabrication conditions with varying LC PEG porogen contents examined. Finally, these results also indicate that the CS−PAAm microparticles with 15% total polymer content shown here pose minimal mass transfer limitation for the diffusion and conjugation of small fluorescein markers (MW 473.4 Da) regardless of the LC PEG porogen content. In short summary, the results in Figure 2 clearly show uniform distribution and retained chemical reactivity of chitosan in the CS−PAAm microparticles enabling further conjugation reactions. Protein Conjugation via Rapid Tetrazine−trans-Cyclooctene (Tz−TCO) Ligation Reaction. Next, we examined the effect of LC PEG porogen on the formation of macroporous network structures in CS−PAAm microparticles via conjugation of a red fluorescent protein R-phycoerythrin (R-PE) using rapid Tz−TCO ligation reaction, as shown in Figure 3. For this, we first activated the chitosan’s primary amine moieties in triangle-shaped CS−PAAm microspheres prepared with varying concentrations of LC PEG porogens with an NHS−ester form of Tz, as shown in the schematic diagram of Figure 3a. We then exposed these Tz-activated microparticles to TCO-modified RPE (2 μM) for 24 h at room temperature. First, the bright-field micrographs of R-PE-conjugated microparticles show uniform particle shapes and red color for the particles with LC PEG porogen, suggesting high density RD

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Figure 3. R-PE conjugation with CS−PAAm microparticles via Tz−TCO ligation reaction. (a) Schematic diagram of Tz−TCO ligation reaction for R-PE conjugation with the microparticles. (b) Bright-field micrographs of the CS−PAAm microparticles fabricated with varying PEG contents upon R-PE conjugation, and (c) their corresponding epifluorescence micrographs. (d) Confocal micrographs at the center plane of the microparticles shown in panels b and c. (e) Average fluorescence intensity plot of the R-PE conjugated CS−PAAm microparticles shown in panel c. Scale bars represent 200 μm in panels b and c and 100 μm in panel d.

less fluorescence intensity (70% compared to the 4% PEG condition), also consistent with the results shown in Figure 3. Meanwhile, 1% and no LC PEG conditions took longer time to reach the reaction completion (∼8 h), reaching lower fluorescence compared to the microparticles prepared with higher LC PEG contents. These results clearly illustrate readily tunable and macroporous network structures that permit rapid diffusion and conjugation of large biomacromolecules by simple addition of low content LC PEG porogen. Specifically, our model protein R-PE has M.W. 240 kDa, which corresponds to hydrodynamic diameter of ∼11 nm.30 Complete penetration and conjugation of R-PE within 1 h for the 4% PEG condition indicates the mesh size substantially larger than 11 nm (also shown in Figure 3d); these results are encouraging for protein biosensing and medical diagnostics applications, as the complete conjugation of large proteins (e.g., antibodies, MW ∼ 150 kDa) can be achieved well within a clinically relevant assay time of less than 3 h.1 In comparison to our recent studies, the CS−PAAm microparticles examined here provide substantially improved conjugation kinetics. Specifically, CS−PEG microparticles prepared with 40% poly(ethylene glycol) diacrylate (PEGDA, MW 700 Da) in a similar replica molding-based fabrication method did not reach completion of R-PE conjugation reaction for over 48 h.35 CS−PEG microspheres prepared with 10% PEGDA took over 10 h to reach conjugation completion.27 In contrast, the CS−PAAm microparticles in this study yielded conjugation completion within 8 h even without PEG porogens and completion as short as 1 h under 4% LC PEG content condition. This apparent conjugation rate is similar to the one on surface-assembled nanotube templates, where R-PE proteins were conjugated on to viral nanotemplates (i.e., tobacco mosaic virus) without diffusion through polymer networks.26 This strongly suggests that there exists minimal mass transfer limitation of R-PE diffusion through our CS−PAAm microparticles (4% LC PEG), attesting to the highly macroporous network. In addition, the observed trend of fluorescence intensity (i.e., protein conjugation capacity, 4% > 2% > 1% > no porogen) is also relatively consistent with the results acquired with triangle-shaped particles shown in Figure 3, suggesting reliable fabrication of macroporous microparticles with tunable

fluorescein (shown in Figure S1), further confirming the nonfouling nature of the CS−PAAm hybrid materials,31 and showing promise for selective biosensing and biomacromolecular conjugation. These results on the meso- to macro-pore formation of PAAm gels with LC PEG porogen are consistent with previous studies, where partial miscibility or immiscibility of the two polymers leads to phase separation during polymerization (i.e., PIPS) with the LC PEG forming uniform or heterogeneous phases.10,32 For example, Swamy et al.33 reported complete immiscibility of PAAm/PEG-6000 or -4000, while Silva et al.34 recently reported partial miscibility with PEG2000. In our study, short chain PEG (MW 400 or 600 Da) did not lead to macropore formation even at high PEG content (data not shown), while LC PEG readily formed large pores at low content, also consistent with previous studies.32 In short summary, the results in Figure 3 show that macroporous structures can be readily created and tuned by simple addition of small amount of LC PEG porogen to the CS−PAAm prepolymer mixtures, allowing complete penetration and conjugation of large R-PE proteins via Tz−TCO ligation reaction. Effect of Porogens on Protein Conjugation Kinetics. Next, we carried out an in-depth analysis of R-PE protein conjugation kinetic behavior with the CS−PAAm microparticles with varying LC PEG porogen contents, as shown in Figure 4. For this, we exposed disk-shaped, Tz-activated CS− PAAm particles with 2 μM of TCO-activated R-PE, and examined the average fluorescence intensity as well as the R-PE penetration profiles at various reaction times. First, the normalized fluorescence intensity plot of Figure 4a shows rapid protein conjugation for the CS−PAAm microparticles with the highest LC PEG porogen content (4%, solid squares). Specifically, fluorescence intensity reached 74% of the maximum value (i.e., reaction completion) within the first 30 min, and 91% within the first hour. This result clearly indicates highly macroporous structure of the CS−PAAm microparticles prepared with 4% PEG porogens, while also providing the highest protein conjugation capacity among the conditions examined (consistent with the results in Figure 3). Next, particles prepared with 2% LC PEG (solid circles, Figure 4a) reached maximum fluorescence within the first 4 h and showed E

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Figure 4. Protein conjugation kinetics with CS−PAAm microparticles fabricated with varying PEG contents. (a) Normalized fluorescence intensity plots of the CS−PAAm microparticles upon R-PE conjugation for varying times (0−16 h). (b−e) Confocal micrographs at the center plane of the CS−PAAm microparticles with (b) 4%, (c) 2%, (d) 1%, and (e) 0% PEG 8 kDa upon R-PE conjugation for varying times. All the error bars and standard deviations were acquired from at least five particles per each condition examined. All the scale bars represent 200 μm.

penetration likely occurs through loosely cross-linked networks formed by oxygen inhibition (radical scavenger) near the particle surface exposed to air and porous PDMS molds.7,22,36,37 However, considering much rapid protein conjugation with the CS−PAAm particles compared to that of the CS−PEG ones (8 h vs 48 h conjugation completion), the CS−PAAm microparticles shown here may have more loosely cross-linked networks near the particle surfaces due to inherently linear chain-forming nature of PAAm hydrogels from the acrylamide monomer unlike the PEGDA system. One of the key parameters in the mesh size of polymer networks is the distance between cross-links.24 Unlike the PEGDA that inherently forms cross-linked networks leading to limited control of mesh sizes in our recent studies,22,26,35 acrylamide monomers form linear PAAm chains upon radical polymerization. Ideally, the distance between cross-linking points and mesh size of the PAAm hydrogel formed from the mixtures of AAm and Bis should thus be readily tuned and increased by lowering the Bis content. Yet, previous studies and our own examination of such conditions show that PAAm polymers show meaningful macropores only at extremely low AAm:Bis ratio (i.e., less than 3000:1), where substantial swelling and compromised mechanical integrity limits the reliable particle formation and application.38 In the meantime, our results shown here in Figure 4 presents a drastically simple and controlled route to creating macroporous network structures, presumably due to the defects in network formation by LC PEG.32,39 In short summary, the protein conjugation kinetics results in Figure 4 clearly illustrate substantially improved protein conjugation capacity (∼4-fold vs no porogen) and kinetics (complete penetration and conjugation within 1 h) by simple addition of LC PEG porogens. One-Pot Assembly via Orthogonal Conjugation and Simple Shape-Based Encoding. Finally, as shown in Figure 5, we demonstrate orthogonal one-pot biomacromolecular assembly with simple shape-encoded CS−PAAm microparticles in order to examine utility of our integrated fabrication-

porosity and retained chemical functionality. Finally, relatively small error bars obtained from five particles per each condition for all the R-PE-conjugated microparticles in Figure 4a indicate consistent and uniform fabrication and conjugation, as further illustrated in the original fluorescence images shown in Figure S4 (Supporting Information). In sum, the protein conjugation kinetics results in Figure 4a clearly illustrate significantly improved and rapid protein conjugation by simple tuning of the prepolymer condition with small content of the LC PEG porogen. Next, we examined the cross-sectional fluorescence profiles and penetration depths (PD) of the R-PE-conjugated microparticles at various conjugation times shown in Figure 4a (also in Figure S4) via confocal microscopy at the particles’ center plane as shown in Figure 4b−e. First, the confocal image at the leftmost column of Figure 4b shows near-complete penetration of R-PE at 0.5 h for the particles prepared with 4% LC PEG, clearly illustrating the macroporous nature. The confocal images of the particles after this time point consistently show complete penetration and uniform fluorescence profile throughout the particle cross-section, consistent with the epifluorescence results in Figures 3c and 4a. Next, the PD of the particles prepared with 2% PEG gradually increases from 6.5 to 12.6 μm over 8 h (Figure 4c), also consistent with Figures 3d and 4a. This trend is also observed for the 1% PEG microparticles (Figure 4d), where the PD increases from 3.3 to 6.5 μm. Finally, the particles prepared without any LC PEG porogen (Figure 4e) showed minimal increase in the PD (0 to 3.3 μm) over 8 h reaction period examined here. This minimal increase and the small penetration depth of ∼3 μm are similar to the ones observed for CS−PEG microparticles fabricated with 40% PEGDA in our recent study under equivalent fabrication conditions via replica molding.35 This result suggests that the prepolymer conditions enlisted in the current study (i.e., 15% total polymer content, 29:1 AAm:Bis ratio, no LC PEG porogen) yield mesh size smaller than the R-PE’s size similar to the 40% PEGDA condition, where the observed protein F

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TCO-activated R-PE (red) in one pot. CS−PAAm particles without any activation were used as a negative control. First, the overlay image of Figure 5b shows the three types of particles with reliable replication of the 2D shapes. Next, the fluorescence overlay image of Figure 5, parts d and e (Figure 5c) clearly shows minimal fluorescence of the negative control particles, again indicating the nonfouling nature of the CS− PAAm matrix for multiple biomacromolecular targets (e.g., ssDNA and R-PE) as well as the orthogonal nature of the two reactions. Finally, the green and red fluorescence images of Figure 5d,e clearly show the orthogonal conjugation of two chemically distinct biomacromolecules (ssDNA and R-PE) on each particle type via SPAAC and Tz−TCO reaction, respectively. In other words, the ssDNA conjugation via SPAAC reaction (green) and R-PE conjugation via Tz-TCO ligation reaction (red) yielded minimal cross-reactivity. Combined with the simple shape-based encoding and nonfouling nature of our CS−PAAm hybrid materials, this orthogonality illustrates potential for selective high-throughput conjugation and sensing of various types of biomacromolecular probes and targets.41,42 In short summary, the results in Figure 5 illustrate simultaneous conjugation of two distinct types of biomacromolecules with simple 2D shape-encoded CS−PAAm microparticles in an orthogonal, one-pot procedure.

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CONCLUSIONS In this report, we demonstrated an integrated fabrication− conjugation approach for programmable fabrication of shapeencoded CS−PAAm microparticles and improved biomacromolecular conjugation via robust replica molding and high-yield bioorthogonal click reactions. First, the simple replica molding technique yielded highly uniform microparticles with simple 2D shape-based encoding. Fluorescent labeling results indicated uniform and stable incorporation, as well as the retained chemical activity of chitosan toward amine-reactive NHS ester chemistry. R-PE conjugation results clearly indicated the readily tunable mesh structure by simple addition of low content LC PEG porogen, leading to complete penetration and conjugation at the 4% LC PEG porogen condition. Furthermore, in-depth protein conjugation results illustrated high capacity and rapid penetration and conjugation, achieving near-completion within the first hour for the 4% PEG condition. Finally, the one-pot assembly results suggested significant potential for highthroughput assembly of multiple biomacromolecular probes and targets. Importantly, all the conjugation studies showed high selectivity with minimal nonspecific binding, confirming the highly nonfouling nature of the hybrid CS−PAAm material toward selective biomacromolecular conjugation and assay. The batch processing-based nature of the replica molding technique provides a number of advantages over continuous processes (e.g., microfluidic and jetting).23,43 First, the technique does not rely on exquisite control of rapid microflows, thus eliminates the need for delicate flow control or complex equipment making it a simple, cost-efficient and readily scalable process via parallelization. Second, all the components are confined within each microwell, making it an inherently clean process with minimal need for extensive rinsing or detergents, unlike microfluidic procedures. Third, a wider range of monomers and components can be utilized without the need for process modification, e.g., acrylamide with slower polymerization rate19,20 in our study. Finally, the replica molding can readily achieve 100% monodispersity unlike flow-

Figure 5. Site-specific biomolecular conjugation with shape-encoded CS−PAAm microparticles in a one-pot manner using bioorthogonal click reactions. (a) Schematic diagram of the one-pot assembly of biomolecules (i.e., ssDNA and R-PE) with three types of shapeencoded CS−PAAm microparticles that are assigned to SPAAC, Tz− TCO and no reaction, respectively. (b−e) Microscopy results of the orthogonal one-pot biomolecular assembly with the shape-encoded CS−PAAm microparticles: (b) overlay image of bright-field, (d) green fluorescence and (e) red fluorescence micrographs, and (c) fluorescence overlay image of parts d and e.

conjugation approach toward selective and high-throughput conjugation of various probes. For this, we enlisted two high yield bioorthogonal click reactions to conjugate single-stranded DNA (ssDNA) and R-PE on to the CS−PAAm particles with different 2D shapes simultaneously in a one-pot manner, as shown in the schematic diagram of Figure 5a. Specifically, strain-promoted alkyne−azide cycloaddition (SPAAC) reaction was utilized to anchor azide-modified ssDNA onto CS−PAAm particles activated with (azadibenzocyclooctyne) (ADIBO), and Tz−TCO ligation reaction was used to anchor TCO-activated R-PE onto Tz-activated CS−PAAm particles with a different 2D shape. These two bioorthogonal reactions have recently been reported to be orthogonal to each other,40 thus should allow for programmable functionalization of multiple biomolecular probes simultaneously. For this, CS−PAAm microparticles with three distinct 2D shapes were fabricated by replica molding (Figure 1). Upon activation of the chitosan’s primary amines with ADIBO and Tz respectively, the three types of particles were mixed together and exposed to a mixture of azide-modified ssDNA with fluorescein label (green) and G

DOI: 10.1021/acs.langmuir.5b04653 Langmuir XXXX, XXX, XXX−XXX

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based techniques, where the particles formed during initial and final stages often contain irregularities. Next, our results reported in this study attest to several advantages of the hybrid CS−PAAm material. These include high chemical activity rising from the uniquely low pKa of chitosan, which is incorporated in a uniform and stable manner by simply mixing with polymerizable monomers. Second, all the labeling and conjugation results clearly show highly nonfouling nature against hydrophobic dyes, proteins and nucleic acids. Third, tunable macroporosity and improved mass transfer without compromised conjugation capacity is readily achieved by simple addition of the LC PEG porogen. Finally, the two bioorthogonal click reactions (Tz−TCO and SPAAC reactions) have been reported to possess several traits ideal for programmable biomacromolecular conjugation.26,27,35,44,45 These include high conjugation yield, bioorthogonality, stability of functional groups in complex biological fluids, tunable reaction rate, minimal cross-reactivity, and nontoxicity toward biological materials. Taken together, we believe that the results and the fabrication−conjugation methodologies reported in this study should be readily extended to other polymeric materials and biomolecular targets, and show significant potential for attractive routes to multifunctional microparticle suspension array-based biosensing and medical diagnostic applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b04653. Fluorescence micrographs of PAAm microparticles without chitosan upon fluorescent labeling and CS− PAAm microparticles without Tz-activation upon R-PE conjugation, stable incorporation and remaining chemical reactivity of chitosan within CS−PAAm microparticles, and fluorescence micrographs of CS−PAAm microparticles upon conjugation with R-PE for varying times (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(H.Y.) Telephone: (617) 627-2195. Fax: (617) 627-3991. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Chang Hyung Choi at Harvard University and Professor Chang-Soo Lee at Chungnam National University, Korea, for providing the silicon mastermolds.

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REFERENCES

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DOI: 10.1021/acs.langmuir.5b04653 Langmuir XXXX, XXX, XXX−XXX