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Photonic Crystal Microcapsules for Label-free Multiplex Detection Baofen Ye, Haibo Ding, Yao Cheng, Hongcheng Gu, Yuanjin Zhao,* Zhuoying Xie,* and Zhongze Gu* In recent years, there has been immense interest in multiplex and high-throughput assays, which are very important for diagnosis, gene expression, drug screening, and so on.[1,2] One successful biotechnology developed in this field is suspension arrays, which use self-encoded microcarriers as elements for multiplexing.[3–5] Compared with conventional planar microarrays, suspension array-based assays show greater flexibility in the preparation of new assays, fast reaction because of radial diffusion, good reproducibility and high sensitivity.[6] Many encoding microcarriers have been proposed for suspension arrays, including segmented nanorods,[7,8] photopatterning particles,[9] semiconductor quantum dots (QDs),[10] fluorescent particles[11] and photonic crystal (PhC) particles.[12,13] In particular, PhC particles hold immense promise as the microcarriers of suspension arrays because of their excellent optical properties. These include minimal spectral width, remarkable stability and freedom from fluorescent background.[14,15] A key technique for using suspension arrays, including the photonic crystal particle arrays, is target analysis, which is generally realized by detecting fluorescence or absorbance labels attached to the target molecules.[16] As most labeled technologies involve complex labeling and/or require specific apparatus,[17–19] it is desirable to develop a simple, label-free suspension assay. Responsive photonic crystals, which can offer synchronous optical signals along with a change in the periodic lattice spacing or the refractive index contrast, are a good solution to this challenge.[20–26] They have been used successfully as self-reporting sensors to measure various physicochemical changes.[27–32] Thus, it is conceivable that the combination of responsive PhCs with encoding microcarriers, especially PhC particles, would form an unprecedented self-reporting suspension array technique. In this research, we present the desired suspension array with novel PhC microcapsules as its encoding microcarriers. The PhC capsule microcarriers have close-packed Dr. B. F. Ye, Dr. H. B. Ding, Dr. Y. Cheng, Dr. H. C. Gu, Prof. Y. J. Zhao, Prof. Z. Y. Xie, Prof. Z. Z. Gu State Key Laboratory of Bioelectronics Southeast University Nanjing 210096, China E-mail: [email protected]; [email protected]; [email protected] Dr. B. F. Ye Department of Analytical Chemistry China Pharmaceutical University Nanjing 210009, China Prof. Y. J. Zhao, Prof. Z. Y. Xie, Prof. Z. Z. Gu Laboratory of Environment and Biosafety Research Institute of Southeast University in Suzhou Suzhou 215123, China
DOI: 10.1002/adma.201305035
opal PhC cores as their encoding units and responsive inverseopal PhC hydrogel shells as their sensing units. When used in multiplex targets analyses, the PhC cores of the capsule microcarriers could offer stable diffraction peaks for encoding the cross-linked probes in the capsule shells and distinguishing the targets that correspond to the probes, while the PhC hydrogel shells could specifically recognize and react with the targets, causing the shells to shrink. This could be detected as a corresponding blue shift in the Bragg diffraction peak position of the PhC shell, which can be used for quantitatively estimating the amount of the target (as indicated in Figure 1). This feature of the PhC microcapsules makes them ideal encoding microcarriers of suspension arrays for label-free multiplex detection. The PhC microcapsules were fabricated by the template replication of silica colloidal crystal beads (SCCBs), as indicated in Figure 2a. SCCB templates with high monodispersity and brilliant structural colors were prepared by self-assembling of silica nanoparticles in microfluidic droplets, as shown in Figure 2b. The structural colors of the SCCBs originate from the ordered arrangement of the spherical silica nanoparticles composing them. These packed spherical nanoparticles form connected nanopores throughout the SCCBs. The pre-gel solution entered these nanopores and filled all the void spaces between the silica nanoparticles of the SCCBs by capillary force. The mixture was then exposed to UV light to polymerize the pre-gel solution around and between the SCCBs. Then the polymerized hydrogel was mechanically disrupted whilst immersed in a buffer solution, and the hybrid SCCBs at the split could fall off from the hydrogel body. Removal of the silica nanoparticles of the hybrid SCCBs yielded inverse opaline PhC hydrogel beads (Figure 2c). Because of the different relative refractive indexes of the SCCBs and the PhC hydrogel beads, the reflection peak of the hydrogel beads shifted towards blue from 660 nm to 620 nm (Figure 2e and 2f). The change of the reflection peak positions before and after etching the silica nanoparticles implied that we could obtain PhC beads with two reflection peaks if the selected etching was carried out. We found that a novel PhC microcapsule was formed when the etching conditions were controlled. The different relative refractive indexes of the shell and core created two reflection peaks in the PhC microcapsule (Figure 2d and 2g). We found that the relative intensity of the reflection peaks of the shell and the core was influenced by the thickness of the PhC microcapsule shells (Figure S1b). Thin shell were difficult to detect, while thicker shells prevented recording of the reflection peak of the PhC microcapsule core. The shell thickness could be controlled by the etching time; the relationship is shown in Figure S1a. Therefore, an optimized etching time of about 5 min was used to fabricate the microcapsules for the following experiments. Under this condition, microcapsules
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from the SCCB templates should have a similar highly ordered inverse opal structure. Figures 3c and 3d show the SEM images of the microcapsules. It can be observed that the PhC microcapsules have porous surfaces. As expected, the pores form a mainly hexagonal symmetry (Figure 3c). The pores were also interconnected and extended inside the inverse-opaline hydrogel shell (Figure 3d), which offered easier accessibility for the targets to diffuse into the cross-linked probes. Characteristic reflection peaks are a remarkable property of the PhC microcapsules. Under normal incidence, the peak positions λ of the microcapsules can be estimated by Bragg's equation:
λ = 1.633 dnaverage
(1)
where d is the center-to-center distance between two neighboring nanopores or silica nanoparticles, and naverage is the average refractive index of the system. Therefore, by changing the diameters of the silica nanoparticles and their derived pores, a series of PhC microcapsules that show the same size (Figure S2) with different diffraction-peak positions and colors could be obtained (Figure 4). In these microcapsules, the reflection peaks of the photonic cores originated from the close-packed silica nanoparticles, and thus they were very stable against the environmental variation and could be employed for encoding,
Figure 1. Schematic illustration of the PhC microcapsules for label-free multiplex detection.
with shells of uniform thickness and distinguishable core and shell reflections could be achieved. The microstructures of the PhC microcapsules were characterized by a scanning electron microscope (SEM), as shown in Figure 3. It can be observed that the nanoparticles on the surface of the SCCB templates formed a hexagonal alignment (Figure 3a), and this structure extended to the inside of the beads (Figure 3b). Thus, the PhC microcapsules replicated
Figure 2. (a) Schematic illustration of the fabrication of the microcapsules. (b-g) The reflection image (top) and the reflection spectra (bottom) of the SCCBs (b, e), PhC hydrogel beads (c, f) and PhC microcapsules (d, g). Scale bars are 200 µm.
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while the reflection peaks from the inverse-opaline hydrogel shells of the PhC microcapsules were flexible and changeable when the hydrogel was integrated with stimulus-responsive elements. This shell feature gave the PhC microcapsules the function of self-sensing physicochemical stimuli around them. To demonstrate the self-sensing capability of the PhC microcapsules, an aptamer was selected as the target recognition unit to construct the stimulus-responsive hydrogel.[33,34] The specific binding interaction between the aptamer and its target changes the aptamer’s conformation and so triggers the shrinkage of the hydrogel.[35] Here, an Hg2+-responsive aptamer cross-linked hydrogel was employed as the scaffold polymer to construct the microcapsules. Hence, the reflection peak of the PhC microcapsule shell shifted towards blue when the microcapsules were in an Hg2+ solution. The quantitative behavior of the microcapsules was assessed using different concentrations of Hg2+. In
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Figure 3. (a) SEM image of the template SCCB surface showing the hexagonal alignment of nanoparticles. (b) Image of a split bead showing the inner structure. (c) Image of the microcapsule surface showing the hexagonal alignment of pores. (d) Image of a split microcapsule shell showing the ordered pore structure inside.
the absence of Hg2+ the shell had a diffraction peak at 660 nm and showed a shiny red structural color, while the color and the diffraction peak gradually shifted to blue as the Hg2+ concentration increased (Figure 5a). In this process, the changes of both color and volume of the microcapsule shell were visually evident. Figure 5b shows the peak shift value of the PhC microcapsule shell as a function of Hg2+ concentration. The shift value increased increasing Hg2+ concentration and saturated at 10 µM Hg2+. In addition to the sensitivity, the detailed characteristics of the microcapsules (including selectivity, reusability and reproducibility) were also evaluated (Figures S3 and S4). These results indicated that the PhC microcapsules could self-sense a specific target without labeling the targets or using the labels. As with the label-free multiplex assay, three kinds of PhC microcapsules with core reflection peaks at 655 nm (M1), 542 nm(M2), and 497 nm(M3) that exhibited red, green, and blue colors were modified with three kinds of aptamers AHg, AAg, APb, respectively (as shown in Figure 6). These aptamermodified PhC microcapsules were then mixed and incubated in a multitarget solution of Hg2+ and Ag+. Because of the specific binding between the aptamers and their corresponding targets, we expected to observe the blue shift of the shell diffraction peak on the microcapsules only when their corresponding targets were present. Figure 6 shows the results of the multiplex analytes assay. The diffraction peak position of the shells showed significant blue shift only with the M1 and M2 microcapsules and no detectable peak shift was observed with the M3 microcapsules. These results were consistent with the content of the sample (only with Ag+ and Hg2+) to which the microcapsules were exposed. We next extensively tested the cross-reactivity to help ensure the specificity of each assay for its intended target. None of the microcapsules showed significant signal in response to nontarget ions (Table S2). One powerful feature of the PhC microcapsules for multiplex assays is that both the decoding and target detection are simple one-step measurements of the diffraction peak of the microcapsules, which simplifies the detection instruments and procedures.
Figure 4. (a-e) Reflection images of five kinds of PhC microcapsules prepared by silica nanoparticles with different sizes. The nanoparticle sizes were 290, 260, 240, 220, and 200 nm. (f) Reflection spectra of the five kinds of PhC microcapsules. All scale bars are 200 µm.
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Figure 5. (a) Reflection images (left) and optical response (right) of the PhC microcapsules incubated in different concentrations of target Hg2+. (b) Relationship between the reflection blue-shifts and the Hg2+ concentration.
In conclusion, we have developed a novel suspension array for multiplex label-free assays by using PhC microcapsules as encoding microcarriers. The PhC microcapsules had close-packed opal PhC cores and responsive inverse-opal PhC hydrogel shells. When used in multiplex targets analysis, the PhC cores of the microcapsules provided stable diffraction peaks for encoding, while the PhC hydrogel shells specifically recognized and reacted with the targets, causing the shells to shrink. This was detected as a corresponding blue shift in the
Bragg diffraction peak position of the PhC shells, which was used to estimate the amounts of the targets, quantitatively. The applications of the PhC microcapsules in aptamer-based ion detection demonstrate the flexibility and feasibility of the proposed method. In addition, the decoding and detection of reactions in our suspension array can be a one-step measurement of reflection which renders both the apparatus and the procedure simple. Therefore, this new type of suspension array is quite promising in overcoming many restrictions of current techniques. We anticipate that it will open new horizons in diverse areas such as forensic analysis, drug screening, and environmental monitoring.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This research was supported by the National Natural Science Foundation of China (Grants 21105011 and 50925309), the Natural Science Foundation of Jiangsu (Grant BK2012735), the Science and Technology Development Program of Suzhou (Grant SYN201307), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1222). Y.J.Z. thanks the Program for New Century Excellent Talents in University. Received: October 9, 2013 Revised: November 28, 2013 Published online:
Figure 6. (top) Scheme of multiplex analysis. The red, green, and blue microcapsules were immobilized with different aptamers. The optical signals only presented when the corresponding targets were present. (bottom) Bright-field microscopy images and reflection spectra of three kinds of microcapsules before (left) and after (right) multiplex assay. Before the assay, the microcapsules had different colors. They showed similar blue shell colors after the assay. The dashed lines and solid lines are the reflection spectra of the PhC microcapsules before and after exposure.
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[1] F. Deiss, C. N. LaFratta, M. Symer, T. M. Blicharz, N. Sojic, D. R. Walt, J. Am. Chem. Soc. 2009, 131, 6088. [2] C. T. Huang, W. W. Wong, L. H. Li, C. C. Chiang, B. D. Chen, S. Y. Li, J. Clin. Microbiol. 2008, 46, 1126. [3] D. Y. Wang, A. L. Rogach, F. Caruso, Nano Lett. 2002, 2, 857. [4] D. C. Pregibon, M. Toner, P. S. Doyle, Science 2007, 315, 1393. [5] B. He, S. J. Son, S. B. Lee, Anal. Chem. 2007, 79, 5257. [6] J. P. Nolan, L. A. Sklar, Trends Biotechnol. 2002, 20, 9.
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[7] R. Wilson, A. R. Cossins, D. G. Spiller, Angew. Chem. Int. Edit. 2006, 45, 6104. [8] X. A. Li, T. Q. Wang, J. H. Zhang, D. F. Zhu, X. Zhang, Y. Ning, H. Zhang, B. Yang, Acs Nano 2010, 4, 4350. [9] D. C. Appleyard, S. C. Chapin, R. L. Srinivas, P. S. Doyle, Nat. Protoc. 2011, 6, 1761. [10] Y. J. Zhao, H. C. Shum, H. S. Chen, L. L. A. Adams, Z. Z. Gu, D. A. Weitz, J. Am. Chem. Soc. 2011, 133, 8790. [11] B. J. Battersby, D. Bryant, W. Meutermans, D. Matthews, M. L. Smythe, M. Trau, J. Am. Chem. Soc. 2000, 122, 2138. [12] F. Cunin, T. A. Schmedake, J. R. Link, Y. Y. Li, J. Koh, S. N. Bhatia, M. J. Sailor, Nat. Mater. 2002, 1, 39. [13] Z. D. Lu, Y. D. Yin, Chem. Soc. Rev. 2012, 41, 6874. [14] Y. J. Zhao, Z. Y. Xie, H. C. Gu, C. Zhu, Z. Z. Gu, Chem. Soc. Rev. 2012, 41, 3297. [15] Y. J. Zhao, X. W. Zhao, Z. Z. Gu, Adv. Funct. Mater. 2010, 20, 2970. [16] Y. J. Zhao, X. W. Zhao, C. Sun, J. Li, R. Zhu, Z. Z. Gu, Anal. Chem. 2008, 80, 1598. [17] Y. J. Zhao, X. W. Zhao, J. Hu, M. Xu, W. J. Zhao, L. G. Sun, C. Zhu, H. Xu, Z. Z. Gu, Adv. Mater. 2009, 21, 569. [18] Y. J. Zhao, X. W. Zhao, J. Hu, J. Li, W. Y. Xu, Z. Z. Gu, Angew. Chem. Int. Edit. 2009, 48, 7350. [19] Y. J. Zhao, X. W. Zhao, B. C. Tang, W. Y. Xu, J. Li, L. Hu, Z. Z. Gu, Adv. Funct. Mater. 2010, 20, 976.
J. P. Ge, Y. D. Yin, Angew. Chem. Int. Edit. 2011, 50, 1492. H. Zhang, Y. Liu, D. Yao, B. Yang, Chem. Soc. Rev. 2012, 41, 6066. Z. W. Mao, H. L. Xu, D. Y. Wang, Adv.Funct. Mater. 2010, 20, 1053. O. D. Velev, S. Gupta, Adv. Mater. 2009, 21, 1897. K. D. Hermanson, S. O. Lumsdon, J. P. Williams, E. W. Kaler, O. D. Velev, Science 2001, 294, 1082. J. F. Galisteo-Lopez, M. Ibisate, R. Sapienza, L. S. Froufe-Perez, A. Blanco, C. Lopez, Adv. Mater. 2011, 23, 30. S. H. Kim, S. Y. Lee, S. M. Yang, G. R. Yi, NPG Asia Mater. 2011, 3, 25. J. X. Wang, Y. Z. Zhang, S. T. Wang, Y. L. Song, L. Jiang, Acc. Chem. Res. 2011, 44, 405. F. Gallego-Gomez, A. Blanco, C. Lopez, Adv. Mater. 2012, 24, 6204. B. F. Ye, F. Rong, H. C. Gu, Z. Y. Xie, Y. Cheng, Y. J. Zhao, Z. Z. Gu, Chem. Commun. 2013, 49, 5331. B. F. Ye, Y. J. Zhao, Y. Cheng, T. T. Li, Z. Y. Xie, X. W. Zhao, Z. Z. Gu, Nanoscale 2012, 4, 5998. H. L. Li, J. X. Wang, L. M. Yang, Y. L. Song, Adv. Funct. Mater. 2008, 18, 3258. S. Y. Lee, S. Yang, Angew. Chem. Int. Edit. 2013, 52, 8160. W. H. Tan, M. J. Donovan, J. H. Jiang, Chem. Rev. 2013, 113, 2842. X. H. Fang, W. H. Tan, Acc. Chem. Res. 2010, 43, 48. Z. Zhu, C. C. Wu, H. P. Liu, Y. Zou, X. L. Zhang, H. Z. Kang, C. J. Yang, W. H. Tan, Angew. Chem. Int. Edit. 2010, 49, 1052.
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Photonic Crystal Microcapsules for Label-free Multiplex Detection Baofen Ye, Haibo Ding, Yao Cheng, Hongcheng Gu, Yuanjin Zhao,* Zhuoying Xie,* and Zhongze Gu* In recent years, there has been immense interest in multiplex and high-throughput assays, which are very important for diagnosis, gene expression, drug screening, and so on.[1,2] One successful biotechnology developed in this field is suspension arrays, which use self-encoded microcarriers as elements for multiplexing.[3–5] Compared with conventional planar microarrays, suspension array-based assays show greater flexibility in the preparation of new assays, fast reaction because of radial diffusion, good reproducibility and high sensitivity.[6] Many encoding microcarriers have been proposed for suspension arrays, including segmented nanorods,[7,8] photopatterning particles,[9] semiconductor quantum dots (QDs),[10] fluorescent particles[11] and photonic crystal (PhC) particles.[12,13] In particular, PhC particles hold immense promise as the microcarriers of suspension arrays because of their excellent optical properties. These include minimal spectral width, remarkable stability and freedom from fluorescent background.[14,15] A key technique for using suspension arrays, including the photonic crystal particle arrays, is target analysis, which is generally realized by detecting fluorescence or absorbance labels attached to the target molecules.[16] As most labeled technologies involve complex labeling and/or require specific apparatus,[17–19] it is desirable to develop a simple, label-free suspension assay. Responsive photonic crystals, which can offer synchronous optical signals along with a change in the periodic lattice spacing or the refractive index contrast, are a good solution to this challenge.[20–26] They have been used successfully as self-reporting sensors to measure various physicochemical changes.[27–32] Thus, it is conceivable that the combination of responsive PhCs with encoding microcarriers, especially PhC particles, would form an unprecedented self-reporting suspension array technique. In this research, we present the desired suspension array with novel PhC microcapsules as its encoding microcarriers. The PhC capsule microcarriers have close-packed Dr. B. F. Ye, Dr. H. B. Ding, Dr. Y. Cheng, Dr. H. C. Gu, Prof. Y. J. Zhao, Prof. Z. Y. Xie, Prof. Z. Z. Gu State Key Laboratory of Bioelectronics Southeast University Nanjing 210096, China E-mail: [email protected]; [email protected]; [email protected] Dr. B. F. Ye Department of Analytical Chemistry China Pharmaceutical University Nanjing 210009, China Prof. Y. J. Zhao, Prof. Z. Y. Xie, Prof. Z. Z. Gu Laboratory of Environment and Biosafety Research Institute of Southeast University in Suzhou Suzhou 215123, China
DOI: 10.1002/adma.201305035
opal PhC cores as their encoding units and responsive inverseopal PhC hydrogel shells as their sensing units. When used in multiplex targets analyses, the PhC cores of the capsule microcarriers could offer stable diffraction peaks for encoding the cross-linked probes in the capsule shells and distinguishing the targets that correspond to the probes, while the PhC hydrogel shells could specifically recognize and react with the targets, causing the shells to shrink. This could be detected as a corresponding blue shift in the Bragg diffraction peak position of the PhC shell, which can be used for quantitatively estimating the amount of the target (as indicated in Figure 1). This feature of the PhC microcapsules makes them ideal encoding microcarriers of suspension arrays for label-free multiplex detection. The PhC microcapsules were fabricated by the template replication of silica colloidal crystal beads (SCCBs), as indicated in Figure 2a. SCCB templates with high monodispersity and brilliant structural colors were prepared by self-assembling of silica nanoparticles in microfluidic droplets, as shown in Figure 2b. The structural colors of the SCCBs originate from the ordered arrangement of the spherical silica nanoparticles composing them. These packed spherical nanoparticles form connected nanopores throughout the SCCBs. The pre-gel solution entered these nanopores and filled all the void spaces between the silica nanoparticles of the SCCBs by capillary force. The mixture was then exposed to UV light to polymerize the pre-gel solution around and between the SCCBs. Then the polymerized hydrogel was mechanically disrupted whilst immersed in a buffer solution, and the hybrid SCCBs at the split could fall off from the hydrogel body. Removal of the silica nanoparticles of the hybrid SCCBs yielded inverse opaline PhC hydrogel beads (Figure 2c). Because of the different relative refractive indexes of the SCCBs and the PhC hydrogel beads, the reflection peak of the hydrogel beads shifted towards blue from 660 nm to 620 nm (Figure 2e and 2f). The change of the reflection peak positions before and after etching the silica nanoparticles implied that we could obtain PhC beads with two reflection peaks if the selected etching was carried out. We found that a novel PhC microcapsule was formed when the etching conditions were controlled. The different relative refractive indexes of the shell and core created two reflection peaks in the PhC microcapsule (Figure 2d and 2g). We found that the relative intensity of the reflection peaks of the shell and the core was influenced by the thickness of the PhC microcapsule shells (Figure S1b). Thin shell were difficult to detect, while thicker shells prevented recording of the reflection peak of the PhC microcapsule core. The shell thickness could be controlled by the etching time; the relationship is shown in Figure S1a. Therefore, an optimized etching time of about 5 min was used to fabricate the microcapsules for the following experiments. Under this condition, microcapsules
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from the SCCB templates should have a similar highly ordered inverse opal structure. Figures 3c and 3d show the SEM images of the microcapsules. It can be observed that the PhC microcapsules have porous surfaces. As expected, the pores form a mainly hexagonal symmetry (Figure 3c). The pores were also interconnected and extended inside the inverse-opaline hydrogel shell (Figure 3d), which offered easier accessibility for the targets to diffuse into the cross-linked probes. Characteristic reflection peaks are a remarkable property of the PhC microcapsules. Under normal incidence, the peak positions λ of the microcapsules can be estimated by Bragg's equation:
λ = 1.633 dnaverage
(1)
where d is the center-to-center distance between two neighboring nanopores or silica nanoparticles, and naverage is the average refractive index of the system. Therefore, by changing the diameters of the silica nanoparticles and their derived pores, a series of PhC microcapsules that show the same size (Figure S2) with different diffraction-peak positions and colors could be obtained (Figure 4). In these microcapsules, the reflection peaks of the photonic cores originated from the close-packed silica nanoparticles, and thus they were very stable against the environmental variation and could be employed for encoding,
Figure 1. Schematic illustration of the PhC microcapsules for label-free multiplex detection.
with shells of uniform thickness and distinguishable core and shell reflections could be achieved. The microstructures of the PhC microcapsules were characterized by a scanning electron microscope (SEM), as shown in Figure 3. It can be observed that the nanoparticles on the surface of the SCCB templates formed a hexagonal alignment (Figure 3a), and this structure extended to the inside of the beads (Figure 3b). Thus, the PhC microcapsules replicated
Figure 2. (a) Schematic illustration of the fabrication of the microcapsules. (b-g) The reflection image (top) and the reflection spectra (bottom) of the SCCBs (b, e), PhC hydrogel beads (c, f) and PhC microcapsules (d, g). Scale bars are 200 µm.
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while the reflection peaks from the inverse-opaline hydrogel shells of the PhC microcapsules were flexible and changeable when the hydrogel was integrated with stimulus-responsive elements. This shell feature gave the PhC microcapsules the function of self-sensing physicochemical stimuli around them. To demonstrate the self-sensing capability of the PhC microcapsules, an aptamer was selected as the target recognition unit to construct the stimulus-responsive hydrogel.[33,34] The specific binding interaction between the aptamer and its target changes the aptamer’s conformation and so triggers the shrinkage of the hydrogel.[35] Here, an Hg2+-responsive aptamer cross-linked hydrogel was employed as the scaffold polymer to construct the microcapsules. Hence, the reflection peak of the PhC microcapsule shell shifted towards blue when the microcapsules were in an Hg2+ solution. The quantitative behavior of the microcapsules was assessed using different concentrations of Hg2+. In
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Figure 3. (a) SEM image of the template SCCB surface showing the hexagonal alignment of nanoparticles. (b) Image of a split bead showing the inner structure. (c) Image of the microcapsule surface showing the hexagonal alignment of pores. (d) Image of a split microcapsule shell showing the ordered pore structure inside.
the absence of Hg2+ the shell had a diffraction peak at 660 nm and showed a shiny red structural color, while the color and the diffraction peak gradually shifted to blue as the Hg2+ concentration increased (Figure 5a). In this process, the changes of both color and volume of the microcapsule shell were visually evident. Figure 5b shows the peak shift value of the PhC microcapsule shell as a function of Hg2+ concentration. The shift value increased increasing Hg2+ concentration and saturated at 10 µM Hg2+. In addition to the sensitivity, the detailed characteristics of the microcapsules (including selectivity, reusability and reproducibility) were also evaluated (Figures S3 and S4). These results indicated that the PhC microcapsules could self-sense a specific target without labeling the targets or using the labels. As with the label-free multiplex assay, three kinds of PhC microcapsules with core reflection peaks at 655 nm (M1), 542 nm(M2), and 497 nm(M3) that exhibited red, green, and blue colors were modified with three kinds of aptamers AHg, AAg, APb, respectively (as shown in Figure 6). These aptamermodified PhC microcapsules were then mixed and incubated in a multitarget solution of Hg2+ and Ag+. Because of the specific binding between the aptamers and their corresponding targets, we expected to observe the blue shift of the shell diffraction peak on the microcapsules only when their corresponding targets were present. Figure 6 shows the results of the multiplex analytes assay. The diffraction peak position of the shells showed significant blue shift only with the M1 and M2 microcapsules and no detectable peak shift was observed with the M3 microcapsules. These results were consistent with the content of the sample (only with Ag+ and Hg2+) to which the microcapsules were exposed. We next extensively tested the cross-reactivity to help ensure the specificity of each assay for its intended target. None of the microcapsules showed significant signal in response to nontarget ions (Table S2). One powerful feature of the PhC microcapsules for multiplex assays is that both the decoding and target detection are simple one-step measurements of the diffraction peak of the microcapsules, which simplifies the detection instruments and procedures.
Figure 4. (a-e) Reflection images of five kinds of PhC microcapsules prepared by silica nanoparticles with different sizes. The nanoparticle sizes were 290, 260, 240, 220, and 200 nm. (f) Reflection spectra of the five kinds of PhC microcapsules. All scale bars are 200 µm.
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Figure 5. (a) Reflection images (left) and optical response (right) of the PhC microcapsules incubated in different concentrations of target Hg2+. (b) Relationship between the reflection blue-shifts and the Hg2+ concentration.
In conclusion, we have developed a novel suspension array for multiplex label-free assays by using PhC microcapsules as encoding microcarriers. The PhC microcapsules had close-packed opal PhC cores and responsive inverse-opal PhC hydrogel shells. When used in multiplex targets analysis, the PhC cores of the microcapsules provided stable diffraction peaks for encoding, while the PhC hydrogel shells specifically recognized and reacted with the targets, causing the shells to shrink. This was detected as a corresponding blue shift in the
Bragg diffraction peak position of the PhC shells, which was used to estimate the amounts of the targets, quantitatively. The applications of the PhC microcapsules in aptamer-based ion detection demonstrate the flexibility and feasibility of the proposed method. In addition, the decoding and detection of reactions in our suspension array can be a one-step measurement of reflection which renders both the apparatus and the procedure simple. Therefore, this new type of suspension array is quite promising in overcoming many restrictions of current techniques. We anticipate that it will open new horizons in diverse areas such as forensic analysis, drug screening, and environmental monitoring.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This research was supported by the National Natural Science Foundation of China (Grants 21105011 and 50925309), the Natural Science Foundation of Jiangsu (Grant BK2012735), the Science and Technology Development Program of Suzhou (Grant SYN201307), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1222). Y.J.Z. thanks the Program for New Century Excellent Talents in University. Received: October 9, 2013 Revised: November 28, 2013 Published online:
Figure 6. (top) Scheme of multiplex analysis. The red, green, and blue microcapsules were immobilized with different aptamers. The optical signals only presented when the corresponding targets were present. (bottom) Bright-field microscopy images and reflection spectra of three kinds of microcapsules before (left) and after (right) multiplex assay. Before the assay, the microcapsules had different colors. They showed similar blue shell colors after the assay. The dashed lines and solid lines are the reflection spectra of the PhC microcapsules before and after exposure.
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