Uniform Core–Shell Photonic Crystal Microbeads as Microcarriers for

Sep 18, 2014 - Using PC microbeads as optical encoding microcarriers, one of the most important features is the stability of their reflection peaks (a...
0 downloads 0 Views 6MB Size
Download PDF
Article pubs.acs.org/Langmuir

Uniform Core−Shell Photonic Crystal Microbeads as Microcarriers for Optical Encoding Xiaolu Jia, Yuandu Hu, Ke Wang, Ruijing Liang, Jingyi Li, Jianying Wang,* and Jintao Zhu* Key Laboratory of Large-Format Battery Materials and Systems of the Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, P. R. China S Supporting Information *

ABSTRACT: We demonstrate a rapid and robust method to fabricate uniform core−shell photonic crystal (PC) microbeads by the microfluidic and centrifugation−redispersion technique. Colored crystalline colloidal arrays (CCAs) were first prepared through centrifugation−redispersion approach by self-assembly of polystyrene−poly(N-isopropylacrylamide) (PS−PNIPAm) core/ shell nanoparticles (NPs). Different from the conventional NPs (e.g., charged PS or PNIPAm NPs), PS−PNIPAm NPs could easily self-assemble into well-ordered CCAs by only one purification step without laborious pretreatment (e.g., dialysis or ion exchange) or slow solvent-evaporation process. The CCAs is then encapsulated into a transparent polymer shell with functional groups (e.g., copolymer of ETPTA and butyl acrylate (BA)), triggering the formation of core− shell PC microbeads which can be used as optical encoding microcarriers. Importantly, this technique allows us to produce core−shell PC microbeads in a rapid and robust way, and the optical reflections of the PC microbeads appear highly stable to various external stimuli (e.g., temperature, pH value, and ionic strength) relying on the features of the CCAs core and protection of the polymer shell. Moreover, various probe biomolecules (e.g., proteins, antibodies, and so on) can be easily linked on the surface of the core−shell PC microbeads owing to the hydrophilic modification induced by the hydrolysis of BA on the microbead surface, enabling the multiplex biomolecular detection. angle dependence.20−22 The optical stop band of PC microbeads is independent of the rotation under illumination of the surface at a fixed incident angle of the light due to the spherical symmetry, significantly improving the accuracy and efficiency for multiplex assays. Close-packed colloidal crystal microbeads based on monodispersed silica NPs have first been developed by combing the microfluidic and solvent-evaporation technique, and the probe molecules could further be linked on the surface of silica NPs.21 Stable encoding and decoding events by using these PC microbeads as encoding microcarriers could be realized owing to the inert feature of silica. Yet, the preparation method is timeconsuming induced by the slow solvent evaporation process and post-treatment (e.g., rinsing and calcination).23 Therefore, core− shell PC microbead, consisting of a PC core and a polymer shell, has been developed by the microfluidic and photopolymerization technique.24 Nonresponsive PC core offers stable coding signal, and poly(ethylene glycol) (PEG) shell with carboxyl groups allows for immobilizing various probe molecules, broadening their application range as optical encoding microcarriers. Yet again, these core−shell PC microbeads still suffer from some shortcomings. For example, the preparation of PC core still needs at least tens of hours since the crystalline colloidal arrays

1. INTRODUCTION Multiplex assays,1−3 detection and quantification of a broad variety of analytes in a single and simultaneous fashion, have attracted much attention due to their broad applications in drug discovery and clinical diagnostics.4,5 The encoding elements of spectrum encoding (fluorescent dyes,6,7 quantum dots (QDs),8,9 photonic crystal (PC),10−12 among others) are of great value in multiplex assays for identifying different analytes. By comparison, PC shows more advantages over others, including noninterference, high chemical stability, nonbleaching/quenching, and high sensitivity, which lies in the features of PC (e.g., photonic band gap (PBG)) induced by the periodic dielectric arrangements of colloidal nanoparticles (NPs) on the optical wavelength scale.13,14 To date, PC microparticles (e.g., PC microplates and microbeads) have been developed for the application of optical encoding and multiplex assays due to the high flexibility, fast reaction, and good reproducibility for detecting analytes compared to planar chips.15−17 Although two-dimensional (2D) PC microplates with color barcodes based on optical lithography show enhanced encoding capability, limitations of the PC microplates are also obvious, such as complicated preparation procedure, high cost, viewing angle dependence, and others.18,19 Fortunately, 3D PC microspheres/beads based on colloidal assembly technique have been created, which could effectively address the above issues, especially for the viewing © 2014 American Chemical Society

Received: July 19, 2014 Revised: August 31, 2014 Published: September 18, 2014 11883

dx.doi.org/10.1021/la502858f | Langmuir 2014, 30, 11883−11889

Langmuir

Article

Figure 1. Schematic illustration showing the preparation process of uniform PC microbeads. The outer aqueous phase, middle oil phase, and internal aqueous phase consist of a PVA and glycerol aqueous solution, ETPTA along with an UV-sensitive initiator and BA, and core−shell PS−PNIPAm NPs aqueous suspension, respectively. The PS−PNIPAm NPs suspension in ETPTA was sheared into droplets at the tip of the orifice by the outer flow. When the droplets are collected and exposed under the UV lamp, the ETPTA shell phase is polymerized and PC microbeads are obtained within 5 min.

Figure 2. (a) TEM image of the core−shell PS−PNIPAm NPs with a size of 139 ± 5 nm. Inset in (a) is the high-magnification TEM image showing the core−shell structure. (b) Optical microscopy image of the obtained core−shell PC microbeads. (c) SEM image of the fractured core−shell PC microbead with shell thickness of ∼28 μm. (d) SEM image of PS−PNIPAm NPs assembled on the inner surface of the PC microbead. (e−g) Optical microscopy images of core−shell PC microbeads with different colors under reflection mode using (e) 122 nm PS−PNIPAm NPs, (f) 139 nm PS− PNIPAm NPs, and (g) 178 nm PS−PNIPAm NPs. (h) Reflection spectra of the core−shell PC microbeads in (e−g) measured by a fiber-optic spectrometer.

shell PC microbeads rapidly. In this case, the stimuli-responsive building blocks, such as thermal sensitive poly(N-isopropylacrylamide) (PNIPAm) nanogels or magnetic NPs (Fe3O4 NPs), are generally not suitable for the application as optical encoding microcarriers since their reflection spectra (the stop band position or the reflection intensity) are not stable under external stimuli, even though protected by the polymer shell. Herein, we report a facile and robust strategy for the preparation of core−shell PC microbeads with stable spectral read-out by using a double-emulsion microfluidic device consisting of a coflow and flow-focusing system28 and the centrifugation−redispersion technique. Monodispersed PS− PNIPAm core−shell NPs are used as building blocks for the formation of CCAs. Although the building blocks (e.g., PS− PNIPAm core−shell NPs) are thermal responsive, the resulting

(CCAs) were also obtained from the solvent-evaporation process, and the hydrogel shell layer is easily broken due to the weak mechanical stability, affecting the detection of analytes. Thus, it is highly desired for the preparation of PC microbeads with ease-encoding and stable spectral decoding in a fast and robust way for the development of optical encoding microcarriers. Encapsulating CCAs into a spherical polymer microcapsule could be one of the most effective strategies for addressing the above problems. Yet, preparation of the conventional CCAs based on charged polystyrene (PS) NPs is still time-consuming, which needs a complex pretreatment process, including purification, dialysis, ion exchange, etc.25−27 Recently, we demonstrated a new strategy by microfluidic and centrifugation−redispersion technique,22 which allows us to obtain core− 11884

dx.doi.org/10.1021/la502858f | Langmuir 2014, 30, 11883−11889

Langmuir

Article

Biomolecules can be easily linked on the surface of the PC microbeads owing to the hydrophilic modification induced by the hydrolysis of BA on the microbead surface. Another aqueous solution containing PVA and glycerol is used as the outer aqueous phase. Generally, glycerol is added to increase the viscosity of the outer phase for the stable generation of doubleemulsion droplets. The above three different kinds of fluids are pumped through the double-emulsion microfluidic channel simultaneously. Uniform water/oil/water double emulsion droplets could be generated after the breaking-up of three fluids at the orifice induced by the strong shearing force of external fluids and interfacial tension. PVA in the outer phase acts as surfactant to stabilize the interface of outer aqueous/middle oil phase and prevent the coalescence of double emulsion droplets in the downstream of the collection tube and container effectively. Subsequently, UV light is used to initiate the polymerization of monomers in the shell layer of double-emulsion droplets, resulting in the formation of uniform core−shell PC microbeads (Figure 2b). The internal microstructure of core−shell PC microbeads is confirmed by using scanning electron microscope (SEM), as shown in Figure 2c,d. Clearly, the core−shell structure of the PC microbeads can be observed from the cross section of the fractured microbeads. In general, it is hard to characterize the crystalline structure of CCAs in the aqueous core owing to the water evaporation during characterization; however, the wellordered structure of PS−PNIPAm NPs at the inner surface of the fractured core−shell microbeads can be observed, which could give a clear hint for the crystalline structure of CCAs in the core (Figure 2d). Importantly, the crystalline structure of CCAs can further be confirmed by the colors and reflection peaks of the core−shell PC microbeads since the polymer shell layer is transparent without influencing the display of structural colors of the core region. As shown in Figure 2e−h, core−shell PC microbeads show three typical vivid colors (e.g., blue, green, and red), and their corresponding reflection spectra have a single sharp peak, indicating the formation of well-ordered crystalline structures in the CCAs. The position of the maximum reflection peak (λmax) of the PC microbeads follows Bragg’s law:

CCAs are proven to be able to provide stable reflection spectra under external stimuli (e.g., variation of temperature) when the concentration of NPs in CCAs is lower than a critical value.30 Aqueous suspensions containing CCAs as inner phase can be obtained rapidly by using the centrifugation−redispersion technique without slow solvent evaporation and laborious post-treatment process, which significantly shortens the preparation time. The color of the CCA suspension can be tuned by simply changing diameters of the PS−PNIPAm NPs at fixed NP concentration (∼22 wt %). A mixture of the photocurable monomers, ethoxylated trimethylolpropane triacrylate (ETPTA) and butyl acrylate (BA), is used as the middle oil phase. The outer aqueous solution contains poly(vinyl alcohol) (PVA), which acts as surfactant to stabilize the interface between the middle oil and the outer aqueous phase. When the above three fluids flow through the double-emulsion microfluidic system (Figure 1), the water (aqueous suspension of CCAs)/oil (ETPTA and BA)/water (PVA aqueous solution) doubleemulsion droplets are generated at the orifice under the strong shearing force of outer phase and interfacial tension. Once the double-emulsion droplets are collected in a container, UV irradiation is applied to polymerize the monomers in the middle phase. This strategy not only enables the rapid preparation of core−shell PC microbeads with various colored cores but also ensures the read-out of stable reflection spectra of core−shell PC microbeads which can resist various external stimuli (e.g., temperature, pH value, ionic strength, etc.). Moreover, various probe molecules, such as protein, antibodies, etc., can be easily linked on the hydrophilic surface of PC microbeads by using the simple physical absorption after the hydrolysis of BA, enabling the optical encoding and multiplex bioassay.

2. RESULTS AND DISCUSSION 2.1. Preparation of Uniform Core−Shell PC Microbeads. To realize the rapid preparation of uniform core−shell PC microbeads, a combination of the microfluidic and centrifugation−redispersion technique is created in this work. The doubleemulsion microfluidic devices are composed of coflow and flowfocusing system, which could generate water/oil/water double emulsion droplets and well-defined core−shell PC microbeads (Figure 1). Typically, the aqueous suspensions of CCAs are prepared by using the centrifugation−redispersion method, which are used as the inner phase for the microfluidic. In this case, monodispersed PS−PNIPAm core−shell NPs, acting as building blocks for the formation of CCAs, are synthesized through emulsion copolymerization.29 Figure 2a shows the TEM image of the obtained PS−PNIPAm core−shell NPs. Obviously, the PS−PNIPAm core−shell NPs have a uniform size (diameter: 139 ± 5 nm from TEM measurement). From the inset image in Figure 2a, core−shell structure is clearly seen due to the intense contrast between PS and PNIPAm domain. Interestingly, different from the conventional NPs such as charged PS or PNIPAm NPs, PS−PNIPAm core−shell NPs could easily selfassemble into well-ordered crystalline structures (CCAs) through only one purification step by centrifuging and redispersing process without laborious pretreatment (e.g., dialysis or ion exchange) or slow solvent-evaporation process.25,26 The CCAs can be formed in a few minutes, relying on the electrostatic repulsion among charged PS−PNIPAm core−shell NPs, which significantly shorten the overall synthesis time and facilitate the applications of PC microbeads. Furthermore, a photocurable monomer (e.g., ETPTA), mixed with 1 wt % BA and 1 wt % photoinitiator, is used as the middle oil phase.

λmax = 2neff d

(1)

where neff is the effective refractive index and d is the center-tocenter space of the crystal planes next to each other. Here, d is a key parameter to determine the position of the reflection peaks and structural colors since neff is a constant in a given system. Thus, in this case, colors of core−shell PC microbeads can be tuned by varying diameters of NPs related to d at a fixed concentration (∼22 wt %). For example, when diameters of PS− PNIPAm core−shell NPs decrease from 178 ± 3, 139 ± 5, to 122 ± 4 nm (hydrodynamic diameter: 193.8, 149.9, and 135.1 nm, respectively), the reflection peak positions of PC microbeads blue-shift from red to blue correspondingly, and their colors change from red, green, and then to blue. 2.2. Stability of Reflection Peaks of Core−Shell PC Microbeads. Using PC microbeads as optical encoding microcarriers, one of the most important features is the stability of their reflection peaks (as codes for probe molecules) under various external stimuli. To demonstrate the reflection peak stability, the formed core−shell PC microbeads are subjected to various external environments (e.g., temperature, pH value, ionic strength, etc.). First, the stability of core−shell microbeads under various temperature is investigated, as shown in Figure 3. The 11885

dx.doi.org/10.1021/la502858f | Langmuir 2014, 30, 11883−11889

Langmuir

Article

colors (Figure S2), indicating that the microbeads have good stability of reflection peaks under various temperature. The reason for the thermal stability of the microbeads can be attributed to the property of the building blocks PS−PNIPAm core−shell NPs. Interestingly, PNIPAm is a kind of thermalresponsive polymer, which would change its conformation from coil to globule when the temperature is above the low critical solution temperature (LCST) of ∼32 °C. Correspondingly, the hydrodynamic diameter of PS−PNIPAm core−shell NPs would decrease from 149 to 142 nm by increasing temperature from 10 to 50 °C (Figure S3). However, the positions of reflection peaks almost keep the same with temperature once the PS−PNIPAm NPs form the CCAs at low NP concentration even though the PS−PNIPAm NPs change their diameters slightly. It has been reported that the CCAs show almost no thermosensitivity when the concentration of PS−PNIPAm NPs is lower than 30 wt %, which is attributed to the unchanged interparticle distance maintained by the electrostatic interactions which is not affected largely by the temperature. 30 Based on this, the NP concentrations of ∼22 wt % are used for all of our experiments, which are below the critical NP concentration triggering thermoresponse. Therefore, the reflection peak positions of the obtained core−shell PC microbeads would not be affected by the temperature variation in our experiment. Besides temperature, the effect of other external conditions (e.g., pH value and ionic strength) on the stability of reflection peaks of core−shell PC microbeads should be further examined since the traditional CCAs are highly sensitive to the ions, and the practical applications such as bioassay are usually operated in various buffer solutions. In this case, the as-prepared core−shell PC microbeads are immersed in solutions with different pH value and ionic strength for a period of time (Figure 4). The reflection peaks of core−shell red PC microbeads remain nearly constant

Figure 3. (a−c) Optical microscopy images of core−shell PC microbeads under reflection mode at (a) 5, (b) 35, and (c) 55 °C. (d) Reflection spectra of the core−shell PC microbeads with temperature changing from 5 to 55 °C. Clearly, the obtained core−shell PC microbeads display good stability of reflection peaks when varying external temperature.

obtained core−shell PC microbead appears nearly the same red color, and the reflection peak almost keeps the same position by increasing temperature from 5 to 55 °C. Similar results can be obtained for the core−shell PC microbeads with blue and green

Figure 4. (a, b) Reflection spectra of core−shell PC microbeads immersed in aqueous NaCl solution with different concentrations (a) 0.1 M and (b) 1 M with varied immersed time. (c, d) Reflection spectra of core−shell PC microbeads immersed in aqueous solution with different pH value (c) pH = 1 and (d) pH = 13 with varied immersed time. 11886

dx.doi.org/10.1021/la502858f | Langmuir 2014, 30, 11883−11889

Langmuir

Article

from the beginning to 3 days after they are immersed in aqueous solutions with 0.1 M NaCl (Figure 4a), 1 M NaCl (Figure 4b), pH = 1 (Figure 4c), and pH = 13 (Figure 4d). This result is in consistency with Yang’s report where the charged PS NPs have been incorporated in a polymer shell.31 Also, similar results can be obtained for green and blue PC microbeads (Figures S4 and S5). The reason is that the hydrophobic polymer shell layer can effectively protect the crystalline structure of CCAs in the core from the ions interference outside the shell. Notably, no obvious change was observed for the reflection peaks of core−shell PC microbeads when dispersing the PC microbeads in the above solutions for at least three months, implying that the ETPTA/BA copolymer shows the capability as barrier to block out the ion transfer through the shells. Besides, the core−shell PC microbeads appear good reflection peak stability when subjected to other external stimuli such as vibration and electric/magnetic fields because of the protection of the polymer shell layer. Thus, highly stability of reflection peaks under various external environments (e.g., temperature, pH value, ionic strength, etc.) enables the core−shell PC microbeads to be suitable candidates as microcarriers for optical encoding and multiplex assays. 2.3. Optical Encoding and Multiplexed Assays. Based on the above results, the core−shell PC microbeads, consisted of CCAs core and P(ETPTA-co-BA) polymer shell, can be used as optical encoding carriers for multiplexed assays since the CCAs core offers the highly stable signal (reflection peak position) which can be used as codes for representing various probe molecules. To realize the link between the probe molecules and PC microbeads, BA is introduced into the shell layer of doubleemulsion droplets and copolymerized with ETPTA to form the P(ETPTA-co-BA) polymer shell, which can generate the carboxylic groups after the hydrolysis process under base solution (Figure S6). Notably, hydrophilic monomers (e.g., acrylic acid (AA), 2-hydroxyethyl methacrylate (HEMA), etc.) would affect the crystalline structure of the CCAs in the core although the hydrophilic monomers can be copolymerized with ETPTA in the shell. In contrast, the relative hydrophobic monomers (e.g., BA) would not influence the well-ordered structure and display of structure colors of the CCAs in the core since the hydrophobicity of BA limits the molecule diffusion toward aqueous core before solidification of shell layer. Therefore, biomolecules can be easily linked on the surface of the core−shell PC microbeads due to the hydrophilic modification induced by the hydrolysis of BA on the microbead surface. To demonstrate the multiplexed assay using these core−shell PC microbeads, three kinds of core−shell PC microbeads (e.g., red, green, and blue microbeads) are chosen and immersed in the three different kinds of solutions containing human, pig, and chicken immunoglobulin G (Ig G), respectively, for 12 h at 4 °C. Consequently, three kinds of probe molecules (e.g., human, pig, and chicken IgGs) are physically linked onto the red, green, and blue PC microbeads, respectively. The sites on the PC microbeads surfaces that did not absorb protein are blocked with 1 wt % bull serum albumin (BSA) in phosphate buffer saline (PBS) at room temperature for 2 h and washed with PBS several times. Each PC microbead has a distinguished brilliant color (Figure 5a−c), which can be used as a code for a specific probe molecule on the surface of PC microbead. Subsequently, the modified three kinds of PC microbeads are dispersed into an aqueous solution containing fluorescein isothiocyanate (FITC)labeled goat antihuman and antipig IgG. After incubation for 2 h

Figure 5. Scheme of multiplex analysis based on PC microbeads. (a−c) Optical microscopy images of the PC microbeads under reflection mode. (d−f) and (g−i) are the corresponding fluorescence microscopy images and fluorescence/optical reflection spectra of the PC microbeads showing in (a−c). The red, green, and blue microbeads were immobilized with human, pig, and chicken IgG, respectively. The three microbeads were mixed and put into a solution containing FITCtagged goat antihuman IgG and goat antirabbit IgG. Clearly, the fluorescence signals only display on the red and green microbeads, indicating that fluorescence-labeled protein interacts specifically with its probe molecule on the surface of PC microbeads.

at 37 °C, the above three PC microbeads are washed using PBS and deionized water to remove the unbounded anti-IgGs. The PC microbeads would show intense fluorescence signal under the fluorescence microscope (Figure 5d-f) because fluorescence-labeled protein interacts specifically with its probe molecule on the surface of PC microbeads. Fluorescence and reflection spectra are measured after the rinsing treatment (Figure 5g−i). In this case, the red and green PC microbeads show intense fluorescence signals at 650 nm under the excitation of green laser, while almost no fluorescence signal is detected on the blue PC microbead (Figure S7). The results indicate that FITC-labeled goat antihuman and antipig IgG could be detected simultaneously in one tube owing to the specific binding between IgG and its corresponding anti-IgG. Therefore, the novel core− shell PC microbeads not only solve the problems existed in the conventional PC microbeads (e.g., time-consuming, laborious post-treatments, lack of functional groups, etc.) but also enable multiplexed assays as optical encoding microcarriers. Notably, the relatively broad reflection peak of PC microbeads in this system may affect the encoding capacity, which will certainly be solved by improving the crystalline quality or using quantum dots/other fluorescence lables with narrow emission wavelengths.

3. CONCLUSION In summary, we have demonstrated a rapid and robust strategy for the preparation of uniform core−shell PC microbeads by the microfluidic and centrifugation−redispersion technique. The obtained core−shell PC microbeads are composed of a CCAs core based on self-assembly of PS−PNIPAm core−shell NPs and 11887

dx.doi.org/10.1021/la502858f | Langmuir 2014, 30, 11883−11889

Langmuir

Article

colloidal arrays were used as the inner fluid while mixture of ETPTA with 1 wt % butyl acrylate (BA) and 1 wt % photoinitiator (1173) was used as the middle oil fluid. The outer aqueous phase contained 4 wt % PVA and 30 wt % glycerol. PVA is used as surfactant for stabilizing the oil/water interfaces while glycerol can significantly increase the viscosity of the aqueous solution. The above three different fluids were pumped through the microfluidic device. Once the three fluids met at the orifice, uniform double emulsion droplets would be generated under the strong shearing force of the outer phase and the interfacial tension of oil/water. Typically, to form stable emulsion droplets, the flow rates of inner, middle, and outer phases were controlled at 450, 600, and 3000 μL/h, respectively. The double emulsion droplets were then collected in a glass beaker and solidified under UV irradiation (8 W UV lamp) for 5 min. Finally, the uniform core−shell PC microbeads were obtained, which were rinsed several times using deionized water and stored at the deionized water for further use. Modification of Core−Shell PC Microbeads. The ester groups on the shell of core−shell PC microbeads were partially hydrolyzed by immersing the PC microbeads into a 1 M sodium hydroxide solution containing 10 wt % N,N,N′,N′-tetramethylethylenediamine (TEMED) for 10 min. Subsequently, the treated PC microbeads were washed with deionized water several times. The treatment could render the surface of core−shell PC microbeads hydrophilic due to the presence of the carboxylic groups. Optical Encoding. The above hydrophilic-treated core−shell PC microbeads with blue, green, and red colors were first washed with PBS (pH = 7.4) and then immersed in PBS containing 0.5 mg/mL of human, pig, or chicken IgG, respectively, at 4 °C for 12 h. Subsequently, the above PC microbeads were washed with PBS several times. The sites on the PC microbeads surfaces that did not absorb protein were blocked with 1 wt % BSA in PBS at room temperature for 2 h and washed with PBS several times. Multiplex Assays. Three kinds of the microbeads were mixed together, and FITC tagged goat anti-human IgG and goat anti-pig IgG (30 mg/mL) in PBS was added to the microbeads mixture with continuous shaking at 37 °C for 2 h. The resulting microbeads were washed with PBST (0.1 wt % Tween-20 in PBS solution) and observed using optical and fluorescence microscopes. 4.3. Characterization. Formation of the double emulsion droplets was monitored by an inverted optical microscope (IX71, Olympus) in bright-field or phase-contrast modes. The reflection spectra of the PC microbeads were measured by using a fiber-optic spectrometer (USB4000, Ocean Optics Inc.) equipped with a DM2500P optical microscope. The temperature stability of PC microbeads was measured by using temperature controller (TS-4MP, Physitemp Instruments Inc). The PS−PNIPAm NPs were characterized by SEM (Sirion 200, FEI) and transmission electron microscope (TEM, Tecnai G2 20, FEI). Microstructure of core−shell PC microbeads was characterized by using SEM (Sirion 200, FEI). Fluorescence spectra were performed by the laser confocal Raman spectrometer (LabRAM HR800, HORIBA Jobin Yvon). Water contact angles (CA) were measured on contact-angle system (Shanghai Zhongchen Technique Inc.).

a P(ETPTA-co-BA) transparent shell, which can be applied as microcarriers for optical encoding and multiplexed assays. On the one hand, the CCAs core could offer the stable reflection peak positions (codes for probe molecules) which cannot be affected by the external stimuli (e.g., temperature, pH value, ionic strength, etc.). On the other hand, the hydrophobic shell not only shows good mechanical stability and protection for the CCAs core, but the hydrophilic surface of shell also enables ease links between the probe molecules and PC microbeads. It is expected that this novel generation approach opens up a new avenue for the development of advanced PC encoding microcarriers. For example, the other encoding elements, such as dyes, quantum dots (QDs), etc., can be easily introduced into the inner CCAs core of core−shell PC microbeads, which could significantly enhance the coding capability. Furthermore, this technique will potentially expand the application of core−shell PC microbeads in diverse areas, such as sensing, display, drug screening, clinical diagnostics, and combinatorial chemistry. Although the decoding efficiency of the method is low, the microfluidic detection technique has been developed, which pose great potential in high throughput analysis.32

4. EXPERIMENTAL SECTION 4.1. Materials. Sodium dodecyl sulfate (SDS, purity ≥99%), ethoxylated trimethylolpropane triacrylate (ETPTA, purity ≥99%), butyl acrylate (purity ≥99%), 2-hydroxy-2-methylpropiophenone (commercial name 1173, purity ≥99%), bull serum albumin (BSA, (purity ≥99%), and poly(vinyl alcohol) (PVA, Mw = 13K−23K, 87− 89% hydrolyzed) were purchased from Aldrich. Glycerol (purity ≥99%), potassium peroxydisulfate (KPS, purity ≥98%), and styrene (purity ≥98%) were purchased from Sinopharm Chemical Reagent Co. while N-isopropylacrylamide (NIPAM, purity ≥99%) was obtained from Aladdin. N-Isopropylacrylamide was purified by recrystallization from n-hexane which was purified by neutral alumina column before use. Human immunoglobulin (IgG), rabbit IgG, mouse IgG, fluorescein isothiocyanate (FITC)-tagged goat anti-human IgG, goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Biodee Biotechnology Co. 4.2. Methods. Preparation of PS−PNIPAm Core−Shell NPs. PS− PNIPAm core−shell NPs were synthesized through emulsifier-free copolymerization route.29 Typically, SDS (0.09 g) and NIPAm (0.187 g) were dissolved in deionized water (50 mL) in a round-bottomed flask. Under a N2 atmosphere, styrene (15 mL) was added into the reaction flask. The mixture was heated up to 80 °C, and then KPS (0.035 g) was added. After slowly cooling down to room temperature for 8 h, aqueous suspension of the NPs with uniform size was obtained. Sizes of the NPs were tuned by varying the amount of added SDS. Finally, PS−PNIPAm core−shell NPs with diameter of 178 ± 3, 139 ± 5, and 122 ± 4 nm, respectively, were obtained. We note that the as-prepared PS−PNIPAm NPs were purified only one time by centrifugation and washing with deionized water. Preparation of Crystalline Colloidal Arrays. The above PS− PNIPAm NPs suspensions were diluted with water at desired concentration by ultrasonication for half an hour. The resulting PC suspensions containing PS−PNIPAm NPs with sizes of 178 ± 3 nm (21.8 wt %), 139 ± 5 nm (21.5 wt %), and 122 ± 4 nm (22.7 wt %) show brilliant red, green, and blue color, respectively. Fabrication of Microfluidic Device. The microfluidic device is composed of two coaxial round tapered capillaries in a square glass capillary.28 Round glass capillary tubes (World Precision Instruments) with outer and inner diameters of 1.0 mm and 580 μm, respectively, were tapered to the desired orifice using a micropipet puller (Narishige PC-10) and a microforge (Narishige MF-900). The size of injection and collection orifice is ∼90 and ∼280 μm, respectively. Preparation of Core−Shell PC Microbeads. Core−shell PC microbeads were prepared by combing the microfluidic and photopolymerization techniques. Aqueous colored suspensions of crystalline

■

ASSOCIATED CONTENT

■

AUTHOR INFORMATION

S Supporting Information *

Additional figures and plots, including zeta potential of PS− PNIPAm NPs, size and size distribution of PS−PNIPAm NPs, hydrodynamic diameter versus temperature for PS−PNIPAM NPs, contact angle measurement, optical microscopy images, and reflection spectra of PC microbeads. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Authors

*E-mail [email protected] (J.W.). *E-mail [email protected] (J.Z.). Notes

The authors declare no competing financial interest. 11888

dx.doi.org/10.1021/la502858f | Langmuir 2014, 30, 11883−11889

Langmuir

■

Article

(21) Chun, S.; Ding, L. H.; Zyrianov, Y.; Fenniri, H. Preparation, physical properties, on-bead binding assay and spectroscopic reliability of 25 barcoded polystyrene−poly(ethylene glycol) graft copolymers. J. Am. Chem. Soc. 2003, 125, 10546−10560. (22) Xu, H. Q.; Zhang, A. H.; Auclair, C.; Xi, X. G. Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization. Nucleic Acids Res. 2003, 31, e70. (23) Zhao, Y. J.; Zhao, X. W.; Sun, C.; Li, J.; Zhu, R.; Gu, Z. Z. Encoded silica colloidal crystal beads as supports for potential multiplex immunoassay. Anal. Chem. 2008, 80, 1598−1605. (24) Zhao, Y. J.; Gu, Z. Z. Multifunctional photonic crystal barcodes from microfluidics. NPG Asia Mater. 2012, 4, 1−5. (25) Li, J.; Zhao, X. W.; Zhao, Y. J.; Hu, J.; Xu, M.; Gu, Z. Z. Colloidal crystal beads coated with multicolor CdTe quantum dots:microcarriers for optical encoding and fluorescence enhancement. J. Mater. Chem. 2009, 19, 6492−6497. (26) Sharma, A. C.; Jana, T.; Kesavamoorthy, R.; Shi, L. J.; Virji, M. A.; Finegold, D. N.; Asher, S. A. A general photonic crystal sensing motif: creatinine in bodily fluids. J. Am. Chem. Soc. 2004, 126, 2971−2977. (27) Kanai, T.; Lee, D.; Shum, H. C.; Weitz, D. A. Fabrication of tunable spherical colloidal crystals immobilized in soft hydrogels. Small 2010, 6, 807−810. (28) Wang, J. Y.; Hu, Y. D.; Deng, R. H.; Xu, W. J.; Liu, S. Q.; Liang, R. J.; Nie, Z. H.; Zhu, J. T. Construction of multifunctional photonic crystal microcapsules with tunable shell structures by combining microfluidic and controlled photopolymerization. Lab Chip 2012, 12, 2795−2798. (29) Bazin, G.; Zhu, X. X. Understanding the thermo-sensitivity of crystalline colloidal arrays formed by poly(styrene-co-N-isopropylacrylamide) core−shell microspheres. Soft Matter 2012, 8, 1909−1915. (30) Jiang, Y. N.; Yang, X. D.; Ma, C.; Wang, C. X.; Li, H.; Dong, F. X.; Zhai, X. M.; Yu, K.; Lin, Q.; Yang, B. Photoluminescent smart hydrogels with reversible and linear thermoresponses. Small 2010, 6, 2673−2677. (31) Kim, S. H.; Jeon, S. J.; Yang, S. M. Optofluidic encapsulation of crystalline colloidal arrays into spherical membrane. J. Am. Chem. Soc. 2008, 130, 6040−6046. (32) Zhao, Y. J.; Zhao, X. W.; Hu, J.; Xu, M.; Zhao, W. J.; Sun, L. G.; Zhu, C.; Xu, H.; Gu, Z. Z. Encoded porous beads for label-free multiplex detection of tumor markers. Adv. Mater. 2009, 21, 569−572.

ACKNOWLEDGMENTS We gratefully acknowledge funding for this work provided by National Basic Research Program of China (973 program, 2012CB932500), National Natural Science Foundation of China (51103050), and China Postdoctoral Science Foundation (20110491144 and 2012T50643). We also thank HUST Analytical and Testing Center for allowing us to use its facilities.

■

REFERENCES

(1) Walt, D. R. Bead-based fiber-optic arrays. Science 2000, 287, 451− 452. (2) Schwartz, M. P.; Alvarez, S. D.; Sailor, M. J. Porous SiO2 interferometric biosensor for quantitative determination of protein interactions: binding of protein a to immunoglobulins derived from different species. Anal. Chem. 2007, 79, 327−334. (3) Walt, D. R. Fiber optic array biosensors. Biotechniques 2006, 41, 529−535. (4) Lee, W.; Choi, D.; Kim, J. H.; Koh, W. G. Suspension arrays of hydrogel microparticles prepared by photopatterning for multiplexed protein-based bioassays. Microdevices 2008, 10, 813−822. (5) Zhi, Z. L.; Murakami1, Y.; Morita, Y.; Hasan, Q.; Tamiya, E. Multianalyte immunoassay with self-assembled addressable microparticle array on a chip. Anal. Biochem. 2003, 318, 236−243. (6) Dunbar, S. A. Applications of luminex® xmap technology for rapid, high-throughput multiplexed nucleic acid detection. Clin. Chim. Acta 2006, 363, 71−82. (7) Nolan, J. P.; Sklar, L. A. Suspension array technology: evolution of the flat-array paradigm. Trends Biotechnol. 2002, 20, 9−12. (8) Wang, D. Y.; Rogach, A. L.; Caruso, F. Semiconductor quantum dot-Labeled microsphere bioconjugates prepared by stepwise selfassembly. Nano Lett. 2002, 2, 857−861. (9) Kuang, M.; Wang, D.; Bao, H.; Gao, M.; Möhwald, H.; Jiang, M. Fabrication of multicolor-encoded microspheres by tagging semiconductor nanocrystals to hydrogel spheres. Adv. Mater. 2005, 17, 267−270. (10) Zhao, X. W.; Cao, Y.; Ito, F.; Chen, H. H.; Nagai, K.; Zhao, Y. H.; Gu, Z. Z. Colloidal crystal beads as supports for biomolecular screening. Angew. Chem., Int. Ed. 2006, 45, 6835−6838. (11) Cunin, F.; Schmedake, T. A.; Link, J. R.; Li, Y. Y.; Koh, J.; Bhatia, S. N.; Sailor, M. J. Biomolecular screening with encoded porous-silicon photonic crystals. Nat. Mater. 2002, 1, 39−41. (12) Lee, H.; Kim, J.; Kim, H.; Kim, J. Y.; Kwon, S. Colour-barcoded magnetic microparticles for multiplexed bioassays. Nat. Mater. 2010, 9, 745−749. (13) Kim, J. Y.; Osterloh, F. E. Planar gold nanoparticle clusters as microscale mirrors. J. Am. Chem. Soc. 2006, 128, 3868−3872. (14) Wang, Z. Y.; Zong, S. F.; Li, W.; Wang, C. L.; Xu, S. H.; Chen, H.; Cui, Y. P. SERS-fluorescence joint spectral encoding using organicmetal-QD hybrid nanoparticles with a huge encoding capacity for highthroughput biodetection: putting theory into practice. J. Am. Chem. Soc. 2012, 134, 2993−3000. (15) Hu, J.; Zhao, X. W.; Zhao, Y. J.; Li, J.; Xu, W. Y.; Wen, Z. Y.; Xu, M.; Gu, Z. Z. Photonic crystal hydrogel beads used for multiplex biomolecular detection. J. Mater. Chem. 2009, 19, 5730−5736. (16) Zhou, H.; Roy1, S.; Schulman, H.; Natan, M. J. Solution and chip arrays in protein profiling. Trends Biotechnol. 2001, 19, 34−39. (17) Nolan, J. P.; Sklar, L. A. Suspension array technology: evolution of the flat-array paradigm. Trends Biotechnol. 2002, 20, 9−12. (18) Deubel, M.; Freymann1, G. V.; Wegener, M.; Pereira, S.; Busch, K.; Soukoulis, C. M. Direct laser writing of three dimensional photonic crystal templates for the telecommunications. Nat. Mater. 2004, 3, 444− 447. (19) Oader, O.; Chan, T.; John, S. Photonic band gaparchitectures for holographic lithography. Phys. Rev. Lett. 2004, 92, 1−4. (20) Hu, Y. D.; Wang, J. Y.; Wang, H.; Wang, Q.; Zhu, J. T.; Yang, Y. J. Microfluidic fabrication and thermoreversible response of core/shell photonic crystalline microspheres based on deformable nanogels. Langmuir 2012, 28, 17186−17192. 11889

dx.doi.org/10.1021/la502858f | Langmuir 2014, 30, 11883−11889