Article pubs.acs.org/Langmuir
Microfluidic Generation of Multicolor Quantum-Dot-Encoded CoreShell Microparticles with Precise Coding and Enhanced Stability Yang Chen,† Peng-Fei Dong,† Jian-Hong Xu,* and Guang-Sheng Luo The State Key Lab of Chemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China S Supporting Information *
ABSTRACT: A novel microfluidic approach is developed to prepare multicolor QDs-encoded core-shell microparticles with precise and various barcode and enhanced stability performance. With the protection of the hydrogel shell, the leakage of QDs is avoided and the fluorescent stability is enhanced greatly. By embedding different QDs into different cores, no interaction between different QDs existed and the fluorescence spectrum of each kind of QDs can be recorded, respectively. Compared with QDs mixtures in a single particle, it is unnecessary to separate the emissions of QDs in different colors, and deconvolution algorithms are not needed. Therefore, it still maintains precise coding even if QDs with approximate emission wavelengths are used.
microparticles with the help of in situ solidification.17−19 With microfluidic techniques, monodispersed microparticles are manufactured in a large number with low costs.20 In most of the microfluidic generations of QDs-encoded microparticles,2,6,9,21−23 different QDs were incorporated into a single particle directly, which resulted in Forster resonance energy transfer (FRET) of different QDs aggregated in the polymer matrix,21,22 and thus the fluorescence spectra of these QDdoped microparticles became different from those of the QDs mixtures before incorporation. In this study, we demonstrate a simple approach to prepare multicolor QD-encoded core-shell microparticles with precise coding and enhanced stability by using capillary microfluidics. These microparticles contain QDs-trapped polymer cores and hydrogel shells. With O/W/O double emulsions with multicores as templates, different QDs were dispersed in different inner droplets and then embedded in different polymer cores of microparticles after UV polymerization, respectively. As a result, no interactions between different QDs existed, and the fluorescent coding of microparticles remained precise. Furthermore, with the protection of the hydrogel shells, the leakage of QDs was prevented, and the stability of the fluorescent microparticles was improved significantly. These features make these QD-encoded microparticles more applicable in biomedical applications.
1. INTRODUCTION High-throughput multiplexed detection, which has been widely used in many biomedical applications such as drug discovery, clinical diagnostic, genetic analysis, and so on1,2 necessitates effective encoding schemes for molecular identification.2−4 Among those schemes, optical barcoding technology based on fluorescence-encoded microparticles, which can be rapidly processed using conventional flow cytometry,3,5−8 has become an emerging platform. Compared with traditional fluorescent dyes, semiconductor quantum dots (QDs) are ideal fluorophores because of their excellent optical properties for encoding, including size-tunable fluorescence, minimal emission width, broad excitation range, and remarkable photostability against bleaching.9−11 Furthermore, by embedding QDs with different sizes at precisely controlled concentrations, microparticles with large number of unique spectral barcodes can be produced, which can be used to identify the recognition molecules conjugated on their surfaces.4 However, the application of coded microparticles requires properties of monodispersity, stability, and recognizable and diversified coding. QDs can be incorporated into particles by swelling method to generate precise barcodes.4 Also, the QDs can be applied by layer-by-layer assembly approach.12,13 In this method, the interaction different QDs are avoided by separating QDs in different layer. However, it suffers from leakage of QDs. To improve QDs microparticles stability, many efforts have been done, including polymerizable QDs encapsulation,14 growing metal nanoshells on the surface of QD barcodes,15 and self-healing encapsulation strategy.16 Owing to the ability to precisely control the structures of the emulsion droplets and easily incorporate functional additions, microfluidics have emerged as a promising and versatile technique for generating monodispersed functional polymer © 2014 American Chemical Society
2. EXPERIMENTAL SECTION 2.1. Materials. Ethoxylated trimethylolpropane triacrylate (ETPTA) solutions consist of QDs and are injected as inner phase fluid. The middle phase fluid is a poly(ethylene glycol) diacrylate Received: January 7, 2014 Published: June 23, 2014 8538
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(PEG-DA) solution with surfactants and photoinitiator. The outer phase fluid is made up of hexadecane and ABIL EM90 surfactant. Core-shell CdSe/ZnS QDs were purchased from Wuhan Jiayuan Quantum Dots (China). They were coated with a mixture of HDA and TOPO and dispersed in n-hexane. O/W/O Double-Emulsion Droplets. The outer phase was hexadecane containing 2.5% w/w ABIL EM90. The middle phase was an aqueous solution of 15% w/w PEG-DA, 0.25% w/w SDS, 0.25% w/w Pluronic F108, 1% w/w MAA, and 1% w/w photoinitiator HMPF. The inner phase was ETPTA with 0.25% v/v photoinitiator HMPF. QDs were CdSe/ZnS nanocrystal with emission wavelength of 558 and 607 nm. Multicore Emulsion Droplets. Inner phase was ETPTA containing 0.25% v/v HMPF. The middle phase contained 15% w/w PEG-DA, 0.25% w/w SDS, 0.25% w/w Pluronic F108, 6% w/w Trion X-100, 1% w/w MAA, and 1% w/w HMPF. The outer phase was liquid paraffin. 2.2. Microfluidic Devices. For generating single-core double emulsions, the round glass capillary tubes with outer and inner diameters of 1.0 mm and 700 μm, respectively, were tapered to the desired orifice of 35 and 150 μm for the inner phases and for collecting the final emulsions, respectively, using a capillary puller (Sutter Instrument, P-97). The two capillaries were assembled into a square tube with inner diameter 1.0 mm. The square capillary was coated with a hydrophobic reagent (hexamethyldisilazane, Sinoreagent). For generating multicore double emulsions, syringe needles (outer diameter 0.2 mm and inner diameter 0.06 mm) were used for inner phases and assembled into a capillary with orifice of 420 μm for middle phase. The collecting tube was a capillary with inner diameter 700 μm. 2.3. Preparation of Monodispersed Fluorescent Microparticles. The capillary microfluidic device used to generate double emulsion is assembled by two coaxial tapered cylindrical capillaries and a square capillary, as shown in Figure 1a. Ethoxylated trimethylolpropane triacrylate (ETPTA) solutions consist of QDs are injected as inner phase fluid. The middle phase fluid is a poly(ethylene glycol) diacrylate (PEG-DA) solution with surfactants and photoinitiator. The outer phase fluid is made up of hexadecane and ABIL EM90 surfactant. The inner droplets containing QDs are generated in coflow dripping regime from the tip of the inner capillary, while the middle phase containing inner droplets is flow-focused by outer phase flowing in the opposite direction and breaks up into monodisperse O/W/O doubleemulsion droplets at the orifice of the collection capillary. (The typical formation process of double emulsions can be seen in Movie S1 in the Supporting Information.) By adjusting the flow rate, double-emulsion diameter and shell thickness can be adjusted from 100 to 200 μm and 20 to 80 μm, respectively (Figure S1 in the Supporting Information). By photopolymerizing these double-emulsion droplets in situ with UV light, monodispersed fluorescent microparticles with QD-trapped ETPTA cores and PEG hydrogel shells are produced controllably, as shown in Figure 1b.
3. RESULTS AND DISCUSSION 3.1. Fluorescent Stability. Among the requirements for biomedical applications, the fluorescent stability of the particles is a major concern. QDs would leak out of the particles to the solvents as a result of solubility,24 which will reduce the brightness of the particles and affect the sensitivity of analytic detection. The hydrogel shells of the microparticles, which are quite stable in aqueous solution, would provide firm protection for the fluorescent microparticles. To compare the fluorescence stability between the core-shell microparticles and those without shells, microparticles were produced in a same device by suspending the middle-phase flow rate and then washed and suspended in PBS buffer. As we can see in Figure 1c−h, the QDs were uniformly dispersed in the polymer microparticles, and no QDs were leaked out into the hydrogel shells, as confirmed by the fluorescence intensity over the microparticles. Fluorescence intensity was measured by laser scanning confocal microscope to test the stability of microparticles quantitatively.
Figure 1. (a) Formation of O/W/O double emulsions in a capillary microfluidic device. (b) Optical micrograph of the core-shell microparticles with QD-tagged polymer cores and PEG hydrogel shells. The scale bar is 200 μm. (c−e) Laser scanning confocal microscope (LSCM) image, bright-field microscope image, and the combination of above images of the QD-tagged microparticle. (f−h) (LSCM) image, bright-field microscope image, and the combined image of QD-tagged core-shell microparticle. (i) Stability of QDtagged microparticles in PBS buffer.
As shown in Figure 1i, the fluorescence intensity of the microparticles without shells decreased ∼60% after 35 days, which meant nearly 60% of QDs leaked out of the 8539
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Figure 2. (a−e) Representative spectra of single-core QDs-encoded microparticles containing two types of QDs mixed in ratios of 1:1, 2:1, 3:1, 4:1, and 5:1, in which QDs-565 were used by concentration 0.8, 1.6, 2.4, 3.2, and 4.0 μmol/L and QDs-605 were used by concentration 0.8 μmol/L. (f) Fluorescence intensities of single-core QDs-encoded microparticles containing QDs-565 by concentration 0.8, 1.6, 2.4, 3.2, and 4.0 μmol/L (from numbers 1 to 5) and QDs-605 by concentration 0.8 μmol/L, respectively. (g) Fluorescence intensities of single-core QDs-encoded microparticles containing two types of QDs mixed in ratios of 1:1, 2:1, 3:1, 4:1, and 5:1.
microparticles. In contrast, it presented a nearly constant fluorescence intensity for the core-shell microparticles, showing excellent fluorescent stability. The enhanced stability can be attributed to the hydrogel shells of the microparticles. Because QDs as a kind of nanoparticles tended to concentrate near the surface25 (Figure S2 in the Supporting Information), the shells acted as an effective barrier between the QDs and the solvent, avoiding QDs near the surface leaking out. 3.2. Influence of FRET. The interaction between different QDs would affect the fluorescent spectra of the QDs encapsulated, which is a critical problem in the generation of multicolor QD-encoded microparticles. Furthermore, even though a large number of unique codes can be achieved by embedding different QDs at different concentrations theoretically, the achievable codes in reality are limited because of spectral overlapping problem.3,4 We added QDs emitting at 565 nm (QDs-565) by concentration 0.8, 1.6, 2.4, 3.2, and 4.0 μmol/L and QDs-605 by concentration 0.8 μmol/L into inner phase fluids, respectively. With UV polymerization of the emulsions in situ, single-color QDs-encoded microparticles were fabricated. The relationship between photoluminescence intensity and concentration is linear (QDs-565), as shown in Figure 2f. Then, we mixed QDs-565 and QDs-605 into inner phase fluids at precisely controlled ratios of 1:1, 2:1, 3:1, 4:1, and 5:1, in which QDs-565 were used, respectively, by concentration 0.8, 1.6, 2.4, 3.2, and 4.0 μmol/L and QDs-605 were used by concentration 0.8 μmol/L. With UV polymerization of the emulations in situ, bicolor QDs-encoded microparticles were
fabricated. Through the professional peak-fit software (XPSpeak4.1), the decoded peaks of the original encoded fluorescence spectrum were analyzed (Figure 2a−e). However, the peak intensities of the encoded microparticles were not in accordance with the original encoding ratios. The relation between photoluminescence intensity and concentration is nonlinear (QDs-565), as shown in Figure 2g. Otherwise, with concentration of QDs-565 increasing, the fluorescence intensity of QDs-605 in bicolor microparticles increased appreciably, which meant FRET occurred and significantly altered the fluorescence spectrum. 3.3. Precise Coding. To solve the above problems, we generated microparticles with multicores by using a capillary device with several inner phase injection capillaries (as shown in Figure 3a,b), in which different QDs were encapsulated in different cores and could be detected separately. For example, microparticles with two kinds of QDs in two cores at various ratios, respectively, were generated by mixing QDs with emission wavelength of 558 and 667 nm into two inner phase fluids at precisely controlled ratios of 1.75:1, 1.17:1, and 0.58:1. By adding appropriate surfactants (SDS, Pluronic F108, and Triton X-100 in this case) in the middle phase, the inner phases could flow separately, and the droplets generated can keep quite stable without coalescence with each other. Double emulsions with different sizes and structures could be generated by changing the flow rates (as shown in Figure S3 in the Supporting Information, and the typical formation process of double emulsions can be seen in Movie S2 in the Supporting Information), and the emulsions remain stable for a long time 8540
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method of embedding different QDs into different cores, the fluorescence spectrum of the whole microparticles fitted well with those of the cores, which meant that no interaction existed between the two kinds of QDs. In particular, the peak intensities of the encoded microparticles were in good accordance with the original encoding ratios. The fluorescence intensities of each core were recorded, showing a linear relationship between the fluorescence intensity and the concentration of embedded QDs, as shown in Figure 3i. These results indicated that QDs dispersed in the inner phases were embedded into the microparticle cores completely, and the embedded QDs had almost the same optical properties as free QDs. Furthermore, the fluorescence spectrum of each kind of QDs can be recorded, respectively. Compared with QD mixtures, it is unnecessary to separate the emissions of QDs in different colors, and deconvolution algorithms are not needed. Therefore, it still maintains precise coding even if QDs with approximate emission wavelengths are used. 3.4. Tricore Microparticles. Furthermore, tricolor QDsencoded microparticles with three different cores were generated, and the fluorescence spectrum of microparticles was also in good accordance with that of each core, as shown in Figure 4 (The typical formation process of double emulsions
Figure 3. (a) Scheme of the microfluidic device for bicore double emulsions preparation. (b) High-speed micrograph of the formation of the emulsions. (c) Optical micrograph of the core-shell microparticles with bicores. (d−g) Laser scanning confocal microscope (LSCM) images in 558 and 607 nm; bright-field microscope image; combination of the above images of the core-shell microparticles with bicores. (h) Representative spectra of multicolor QDs-encoded microparticles containing two types of QDs mixed in ratios of 1.75:1, 1.17:1, and 0.58:1. (i) Fluorescence intensities as a function of the concentration of QDs in the QD-encoded microparticles.
Figure 4. (a) Optical micrograph of the tricolor core-shell microparticles. (b) Laser scanning confocal microscope (LSCM) image of 516 nm. (c) (LSCM) image of 558 nm. (d) (LSCM) image of 607 nm. (e) Combination of the above images. (f) Representative spectra of tricolor QD-encoded core-shell microparticles.
by adjusting the interface tensions.26 Then, with UV polymerization of the emulations in situ, bicolor QDs-encoded microparticles with two different cores were fabricated, as shown in Figure 3c−g. The prepared QD-encoded microparticles have outstanding optical properties that make them an excellent choice as encoded microparticles for biomedical applications. As we can see in Figure 3d−g, QDs were uniformly dispersed in the cores of the microparticles, and the strongest fluorescence emission intensity of the two cores under the excitation of 405 nm light appeared at 558 and 607 nm, respectively, which can be confirmed by the fluorescence spectrum of the microparticles, as shown in Figure 3h. With the
can be seen in Movie S3 in the Supporting Information). Thus, by changing the core number and the QDs concentration in each core, large number of precise codes could be generated. Theoretically, with N intensity levels with M colors, we could get [(M3 ) × N3 − 1] types of unique codes. For instance, 31 999 recognizable codes can be generated by using 4 types of QDs with 20 intensities for each type of QDs (that is, N = 20 and M = 4). Significantly, by detecting each core in which only one kind of QDs dispersed, respectively, the fluorescence spectrum 8541
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with distinct quantum dot barcodes. J. Mater. Chem. 2011, 21 (35), 13380−13387. (7) Wang, G.; Leng, Y.; Dou, H.; Wang, L.; Li, W.; Wang, X.; Sun, K.; Shen, L.; Yuan, X.; Li, J.; Sun, K.; Han, J.; Xiao, H.; Li, Y. Highly Efficient Preparation of Multiscaled Quantum Dot Barcodes for Multiplexed Hepatitis B Detection. ACS Nano 2013, 7 (1), 471−481. (8) Giri, S.; Sykes, E. A.; Jennings, T. L.; Chan, W. C. W. Rapid Screening of Genetic Biomarkers of Infectious Agents Using Quantum Dot Barcodes. ACS Nano 2011, 5 (3), 1580−1587. (9) Ji, X. H.; Cheng, W.; Guo, F.; Liu, W.; Guo, S. S.; He, Z. K.; Zhao, X. Z. On-demand preparation of quantum dot-encoded microparticles using a droplet microfluidic system. Lab Chip 2011, 11 (15), 2561−2568. (10) Kuang, H.; Zhao, Y.; Ma, W.; Xu, L. G.; Wang, L. B.; Xu, C. L. Recent developments in analytical applications of quantum dots. TrAC, Trends Anal. Chem.Chemistry 2011, 30 (10), 1620−1636. (11) Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Quantum dots versus organic dyes as fluorescent labels. Nat. Methods 2008, 5 (9), 763−775. (12) Li, J.; Zhao, X. W.; Zhao, Y. J.; Gu, Z. Z. Quantum-dot-coated encoded silica colloidal crystals beads for multiplex coding. Chem. Commun. 2009, No. 17, 2329−2331. (13) Wang, D. Y.; Rogach, A. L.; Caruso, F. Semiconductor quantum dot-labeled microsphere bioconjugates prepared by stepwise selfassembly. Nano Lett. 2002, 2 (8), 857−861. (14) Yang, Y. H.; Wen, Z. K.; Dong, Y. P.; Gao, M. Y. Incorporating CdTe nanocrystals into polystyrene microspheres: Towards robust fluorescent beads. Small 2006, 2 (7), 898−901. (15) Chen, K.; Chou, L. Y. T.; Song, F.; Chan, W. C. W. Fabrication of metal nanoshell quantum-dot barcodes for biomolecular detection. Nano Today 2013, 8 (3), 228−234. (16) Song, T.; Liu, J.; Li, W.; Li, Y.; Yang, Q.; Gong, X.; Xuan, L.; Chang, J. Self-healing Encapsulation Strategy for Preparing Highly Stable, Functionalized Quantum-Dot Barcodes. ACS Appl. Mater. Interfaces 2014, 6 (4), 2744−2751. (17) Serra, C. A.; Chang, Z. Q. Microfluidic-assisted synthesis of polymer particles. Chem. Eng. Technol. 2008, 31 (8), 1099−1115. (18) Marre, S.; Jensen, K. F. Synthesis of micro and nanostructures in microfluidic systems. Chem. Soc. Rev. 2010, 39 (3), 1183−1202. (19) Shah, R. K.; Shum, H. C.; Rowat, A. C.; Lee, D.; Agresti, J. J.; Utada, A. S.; Chu, L. Y.; Kim, J. W.; Fernandez-Nieves, A.; Martinez, C. J.; Weitz, D. A. Designer emulsions using microfluidics. Mater. Today 2008, 11 (4), 18−27. (20) Fournier-Bidoz, S.; Jennings, T. L.; Klostranec, J. M.; Fung, W.; Rhee, A.; Li, D.; Chan, W. C. W. Facile and rapid one-step mass preparation of quantum-dot barcodes. Angew. Chem., Int. Ed. 2008, 47 (30), 5577−5581. (21) Shojaei-Zadeh, S.; Morris, J. F.; Couzis, A.; Maldarelli, C. Highly crosslinked poly(dimethylsiloxane) microbeads with uniformly dispersed quantum dot nanocrystals. J. Colloid Interface Sci. 2011, 363 (1), 25−33. (22) Zhao, Y.; Chen, W.; Peng, C. F.; Liu, L. Q.; Xue, F.; Zhu, S. F.; Kuang, H.; Xu, C. L. Facile preparation of fluorescence-encoded microspheres based on microfluidic system. J. Colloid Interface Sci. 2010, 352 (2), 337−342. (23) Bo, W.; Hai-Qing, G. Fluorescence-profile pre-definable quantum-dot barcodes in liquid-core microcapsules. Microfluid. Nanofluid. 2012, 13 (6), 909−917. (24) Yang, Q. H.; Li, Y. H.; Song, T.; Chang, J. Facile single step preparation of high-performance quantum dot barcodes. J. Mater. Chem. 2012, 22 (14), 7043−7049. (25) Balazs, A. C.; Emrick, T.; Russell, T. P. Nanoparticle polymer composites: Where two small worlds meet. Science 2006, 314 (5802), 1107−1110. (26) Torza, S.; Mason, S. G. Coalescence of 2 Immiscible Liquid Drops. Science 1969, 163 (3869), 813−814.
of each kind of QDs could be recorded easily and precisely, without the help of spectral deconvolution or signal processing methods. As a result, the problem of spectral overlapping could be avoided and the kind of embedded QDs could be increased. Therefore, the QD-encoded core-shell microparticles with multi cores generated by this method are more applicable for high-throughput multiplexed detection.
4. CONCLUSIONS In summary, we have demonstrated an effective and novel microfluidic approach to generate multicolor QD-encoded core-shell microparticles with precise coding and enhanced stability by using double emulsions with multi cores as template. With the protection of the hydrogel shells, the leakage of QDs was avoided and the fluorescence stability of the particles was enhanced greatly. Significantly, by embedding different QDs into different cores, no interaction between different QDs such as resonance energy transfer existed, and the fluorescence spectrum of each kind of QDs could be recorded, respectively. This article provides a novel method to overcome the difficulties in the synthesis of multicolor QDencoded microparticles.
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ASSOCIATED CONTENT
S Supporting Information *
Details of experiments, movies, and the size and structure control of double emulsions. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Author Contributions †
Y.C. and P.-F.D contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the support of the National Natural Science Foundation of China (21322604, 21136006), National Basic Research Program of China (2012CBA01203), and A Foundation for the Author of National Excellent Doctoral Dissertation of PR China (FANEDD 201053).
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REFERENCES
(1) Wilson, R.; Cossins, A. R.; Spiller, D. G. Encoded microcarriers for high-throughput multiplexed detection. Angew. Chem., Int. Ed. 2006, 45 (37), 6104−6117. (2) Zhao, Y. J.; Shum, H. C.; Chen, H. S.; Adams, L. L. A.; Gu, Z. Z.; Weitz, D. A. Microfluidic Generation of Multifunctional Quantum Dot Barcode Particles. J. Am. Chem. Soc. 2011, 133 (23), 8790−8793. (3) Pregibon, D. C.; Toner, M.; Doyle, P. S. Multifunctional encoded particles for high-throughput biomolecule analysis. Science 2007, 315 (5817), 1393−1396. (4) Han, M. Y.; Gao, X. H.; Su, J. Z.; Nie, S. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat. Biotechnol. 2001, 19 (7), 631−635. (5) Battersby, B. J.; Bryant, D.; Meutermans, W.; Matthews, D.; Smythe, M. L.; Trau, M. Toward larger chemical libraries: Encoding with fluorescent colloids in combinatorial chemistry. J. Am. Chem. Soc. 2000, 122 (9), 2138−2139. (6) Ji, X. H.; Zhang, N. G.; Cheng, W.; Guo, F.; Liu, W.; Guo, S. S.; He, Z. K.; Zhao, X. Z. Integrated parallel microfluidic device for simultaneous preparation of multiplex optical-encoded microbeads 8542
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Microfluidic Generation of Multicolor Quantum-Dot-Encoded CoreShell Microparticles with Precise Coding and Enhanced Stability Yang Chen,† Peng-Fei Dong,† Jian-Hong Xu,* and Guang-Sheng Luo The State Key Lab of Chemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China S Supporting Information *
ABSTRACT: A novel microfluidic approach is developed to prepare multicolor QDs-encoded core-shell microparticles with precise and various barcode and enhanced stability performance. With the protection of the hydrogel shell, the leakage of QDs is avoided and the fluorescent stability is enhanced greatly. By embedding different QDs into different cores, no interaction between different QDs existed and the fluorescence spectrum of each kind of QDs can be recorded, respectively. Compared with QDs mixtures in a single particle, it is unnecessary to separate the emissions of QDs in different colors, and deconvolution algorithms are not needed. Therefore, it still maintains precise coding even if QDs with approximate emission wavelengths are used.
microparticles with the help of in situ solidification.17−19 With microfluidic techniques, monodispersed microparticles are manufactured in a large number with low costs.20 In most of the microfluidic generations of QDs-encoded microparticles,2,6,9,21−23 different QDs were incorporated into a single particle directly, which resulted in Forster resonance energy transfer (FRET) of different QDs aggregated in the polymer matrix,21,22 and thus the fluorescence spectra of these QDdoped microparticles became different from those of the QDs mixtures before incorporation. In this study, we demonstrate a simple approach to prepare multicolor QD-encoded core-shell microparticles with precise coding and enhanced stability by using capillary microfluidics. These microparticles contain QDs-trapped polymer cores and hydrogel shells. With O/W/O double emulsions with multicores as templates, different QDs were dispersed in different inner droplets and then embedded in different polymer cores of microparticles after UV polymerization, respectively. As a result, no interactions between different QDs existed, and the fluorescent coding of microparticles remained precise. Furthermore, with the protection of the hydrogel shells, the leakage of QDs was prevented, and the stability of the fluorescent microparticles was improved significantly. These features make these QD-encoded microparticles more applicable in biomedical applications.
1. INTRODUCTION High-throughput multiplexed detection, which has been widely used in many biomedical applications such as drug discovery, clinical diagnostic, genetic analysis, and so on1,2 necessitates effective encoding schemes for molecular identification.2−4 Among those schemes, optical barcoding technology based on fluorescence-encoded microparticles, which can be rapidly processed using conventional flow cytometry,3,5−8 has become an emerging platform. Compared with traditional fluorescent dyes, semiconductor quantum dots (QDs) are ideal fluorophores because of their excellent optical properties for encoding, including size-tunable fluorescence, minimal emission width, broad excitation range, and remarkable photostability against bleaching.9−11 Furthermore, by embedding QDs with different sizes at precisely controlled concentrations, microparticles with large number of unique spectral barcodes can be produced, which can be used to identify the recognition molecules conjugated on their surfaces.4 However, the application of coded microparticles requires properties of monodispersity, stability, and recognizable and diversified coding. QDs can be incorporated into particles by swelling method to generate precise barcodes.4 Also, the QDs can be applied by layer-by-layer assembly approach.12,13 In this method, the interaction different QDs are avoided by separating QDs in different layer. However, it suffers from leakage of QDs. To improve QDs microparticles stability, many efforts have been done, including polymerizable QDs encapsulation,14 growing metal nanoshells on the surface of QD barcodes,15 and self-healing encapsulation strategy.16 Owing to the ability to precisely control the structures of the emulsion droplets and easily incorporate functional additions, microfluidics have emerged as a promising and versatile technique for generating monodispersed functional polymer © 2014 American Chemical Society
2. EXPERIMENTAL SECTION 2.1. Materials. Ethoxylated trimethylolpropane triacrylate (ETPTA) solutions consist of QDs and are injected as inner phase fluid. The middle phase fluid is a poly(ethylene glycol) diacrylate Received: January 7, 2014 Published: June 23, 2014 8538
dx.doi.org/10.1021/la501692h | Langmuir 2014, 30, 8538−8542
Langmuir
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(PEG-DA) solution with surfactants and photoinitiator. The outer phase fluid is made up of hexadecane and ABIL EM90 surfactant. Core-shell CdSe/ZnS QDs were purchased from Wuhan Jiayuan Quantum Dots (China). They were coated with a mixture of HDA and TOPO and dispersed in n-hexane. O/W/O Double-Emulsion Droplets. The outer phase was hexadecane containing 2.5% w/w ABIL EM90. The middle phase was an aqueous solution of 15% w/w PEG-DA, 0.25% w/w SDS, 0.25% w/w Pluronic F108, 1% w/w MAA, and 1% w/w photoinitiator HMPF. The inner phase was ETPTA with 0.25% v/v photoinitiator HMPF. QDs were CdSe/ZnS nanocrystal with emission wavelength of 558 and 607 nm. Multicore Emulsion Droplets. Inner phase was ETPTA containing 0.25% v/v HMPF. The middle phase contained 15% w/w PEG-DA, 0.25% w/w SDS, 0.25% w/w Pluronic F108, 6% w/w Trion X-100, 1% w/w MAA, and 1% w/w HMPF. The outer phase was liquid paraffin. 2.2. Microfluidic Devices. For generating single-core double emulsions, the round glass capillary tubes with outer and inner diameters of 1.0 mm and 700 μm, respectively, were tapered to the desired orifice of 35 and 150 μm for the inner phases and for collecting the final emulsions, respectively, using a capillary puller (Sutter Instrument, P-97). The two capillaries were assembled into a square tube with inner diameter 1.0 mm. The square capillary was coated with a hydrophobic reagent (hexamethyldisilazane, Sinoreagent). For generating multicore double emulsions, syringe needles (outer diameter 0.2 mm and inner diameter 0.06 mm) were used for inner phases and assembled into a capillary with orifice of 420 μm for middle phase. The collecting tube was a capillary with inner diameter 700 μm. 2.3. Preparation of Monodispersed Fluorescent Microparticles. The capillary microfluidic device used to generate double emulsion is assembled by two coaxial tapered cylindrical capillaries and a square capillary, as shown in Figure 1a. Ethoxylated trimethylolpropane triacrylate (ETPTA) solutions consist of QDs are injected as inner phase fluid. The middle phase fluid is a poly(ethylene glycol) diacrylate (PEG-DA) solution with surfactants and photoinitiator. The outer phase fluid is made up of hexadecane and ABIL EM90 surfactant. The inner droplets containing QDs are generated in coflow dripping regime from the tip of the inner capillary, while the middle phase containing inner droplets is flow-focused by outer phase flowing in the opposite direction and breaks up into monodisperse O/W/O doubleemulsion droplets at the orifice of the collection capillary. (The typical formation process of double emulsions can be seen in Movie S1 in the Supporting Information.) By adjusting the flow rate, double-emulsion diameter and shell thickness can be adjusted from 100 to 200 μm and 20 to 80 μm, respectively (Figure S1 in the Supporting Information). By photopolymerizing these double-emulsion droplets in situ with UV light, monodispersed fluorescent microparticles with QD-trapped ETPTA cores and PEG hydrogel shells are produced controllably, as shown in Figure 1b.
3. RESULTS AND DISCUSSION 3.1. Fluorescent Stability. Among the requirements for biomedical applications, the fluorescent stability of the particles is a major concern. QDs would leak out of the particles to the solvents as a result of solubility,24 which will reduce the brightness of the particles and affect the sensitivity of analytic detection. The hydrogel shells of the microparticles, which are quite stable in aqueous solution, would provide firm protection for the fluorescent microparticles. To compare the fluorescence stability between the core-shell microparticles and those without shells, microparticles were produced in a same device by suspending the middle-phase flow rate and then washed and suspended in PBS buffer. As we can see in Figure 1c−h, the QDs were uniformly dispersed in the polymer microparticles, and no QDs were leaked out into the hydrogel shells, as confirmed by the fluorescence intensity over the microparticles. Fluorescence intensity was measured by laser scanning confocal microscope to test the stability of microparticles quantitatively.
Figure 1. (a) Formation of O/W/O double emulsions in a capillary microfluidic device. (b) Optical micrograph of the core-shell microparticles with QD-tagged polymer cores and PEG hydrogel shells. The scale bar is 200 μm. (c−e) Laser scanning confocal microscope (LSCM) image, bright-field microscope image, and the combination of above images of the QD-tagged microparticle. (f−h) (LSCM) image, bright-field microscope image, and the combined image of QD-tagged core-shell microparticle. (i) Stability of QDtagged microparticles in PBS buffer.
As shown in Figure 1i, the fluorescence intensity of the microparticles without shells decreased ∼60% after 35 days, which meant nearly 60% of QDs leaked out of the 8539
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Figure 2. (a−e) Representative spectra of single-core QDs-encoded microparticles containing two types of QDs mixed in ratios of 1:1, 2:1, 3:1, 4:1, and 5:1, in which QDs-565 were used by concentration 0.8, 1.6, 2.4, 3.2, and 4.0 μmol/L and QDs-605 were used by concentration 0.8 μmol/L. (f) Fluorescence intensities of single-core QDs-encoded microparticles containing QDs-565 by concentration 0.8, 1.6, 2.4, 3.2, and 4.0 μmol/L (from numbers 1 to 5) and QDs-605 by concentration 0.8 μmol/L, respectively. (g) Fluorescence intensities of single-core QDs-encoded microparticles containing two types of QDs mixed in ratios of 1:1, 2:1, 3:1, 4:1, and 5:1.
microparticles. In contrast, it presented a nearly constant fluorescence intensity for the core-shell microparticles, showing excellent fluorescent stability. The enhanced stability can be attributed to the hydrogel shells of the microparticles. Because QDs as a kind of nanoparticles tended to concentrate near the surface25 (Figure S2 in the Supporting Information), the shells acted as an effective barrier between the QDs and the solvent, avoiding QDs near the surface leaking out. 3.2. Influence of FRET. The interaction between different QDs would affect the fluorescent spectra of the QDs encapsulated, which is a critical problem in the generation of multicolor QD-encoded microparticles. Furthermore, even though a large number of unique codes can be achieved by embedding different QDs at different concentrations theoretically, the achievable codes in reality are limited because of spectral overlapping problem.3,4 We added QDs emitting at 565 nm (QDs-565) by concentration 0.8, 1.6, 2.4, 3.2, and 4.0 μmol/L and QDs-605 by concentration 0.8 μmol/L into inner phase fluids, respectively. With UV polymerization of the emulsions in situ, single-color QDs-encoded microparticles were fabricated. The relationship between photoluminescence intensity and concentration is linear (QDs-565), as shown in Figure 2f. Then, we mixed QDs-565 and QDs-605 into inner phase fluids at precisely controlled ratios of 1:1, 2:1, 3:1, 4:1, and 5:1, in which QDs-565 were used, respectively, by concentration 0.8, 1.6, 2.4, 3.2, and 4.0 μmol/L and QDs-605 were used by concentration 0.8 μmol/L. With UV polymerization of the emulations in situ, bicolor QDs-encoded microparticles were
fabricated. Through the professional peak-fit software (XPSpeak4.1), the decoded peaks of the original encoded fluorescence spectrum were analyzed (Figure 2a−e). However, the peak intensities of the encoded microparticles were not in accordance with the original encoding ratios. The relation between photoluminescence intensity and concentration is nonlinear (QDs-565), as shown in Figure 2g. Otherwise, with concentration of QDs-565 increasing, the fluorescence intensity of QDs-605 in bicolor microparticles increased appreciably, which meant FRET occurred and significantly altered the fluorescence spectrum. 3.3. Precise Coding. To solve the above problems, we generated microparticles with multicores by using a capillary device with several inner phase injection capillaries (as shown in Figure 3a,b), in which different QDs were encapsulated in different cores and could be detected separately. For example, microparticles with two kinds of QDs in two cores at various ratios, respectively, were generated by mixing QDs with emission wavelength of 558 and 667 nm into two inner phase fluids at precisely controlled ratios of 1.75:1, 1.17:1, and 0.58:1. By adding appropriate surfactants (SDS, Pluronic F108, and Triton X-100 in this case) in the middle phase, the inner phases could flow separately, and the droplets generated can keep quite stable without coalescence with each other. Double emulsions with different sizes and structures could be generated by changing the flow rates (as shown in Figure S3 in the Supporting Information, and the typical formation process of double emulsions can be seen in Movie S2 in the Supporting Information), and the emulsions remain stable for a long time 8540
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method of embedding different QDs into different cores, the fluorescence spectrum of the whole microparticles fitted well with those of the cores, which meant that no interaction existed between the two kinds of QDs. In particular, the peak intensities of the encoded microparticles were in good accordance with the original encoding ratios. The fluorescence intensities of each core were recorded, showing a linear relationship between the fluorescence intensity and the concentration of embedded QDs, as shown in Figure 3i. These results indicated that QDs dispersed in the inner phases were embedded into the microparticle cores completely, and the embedded QDs had almost the same optical properties as free QDs. Furthermore, the fluorescence spectrum of each kind of QDs can be recorded, respectively. Compared with QD mixtures, it is unnecessary to separate the emissions of QDs in different colors, and deconvolution algorithms are not needed. Therefore, it still maintains precise coding even if QDs with approximate emission wavelengths are used. 3.4. Tricore Microparticles. Furthermore, tricolor QDsencoded microparticles with three different cores were generated, and the fluorescence spectrum of microparticles was also in good accordance with that of each core, as shown in Figure 4 (The typical formation process of double emulsions
Figure 3. (a) Scheme of the microfluidic device for bicore double emulsions preparation. (b) High-speed micrograph of the formation of the emulsions. (c) Optical micrograph of the core-shell microparticles with bicores. (d−g) Laser scanning confocal microscope (LSCM) images in 558 and 607 nm; bright-field microscope image; combination of the above images of the core-shell microparticles with bicores. (h) Representative spectra of multicolor QDs-encoded microparticles containing two types of QDs mixed in ratios of 1.75:1, 1.17:1, and 0.58:1. (i) Fluorescence intensities as a function of the concentration of QDs in the QD-encoded microparticles.
Figure 4. (a) Optical micrograph of the tricolor core-shell microparticles. (b) Laser scanning confocal microscope (LSCM) image of 516 nm. (c) (LSCM) image of 558 nm. (d) (LSCM) image of 607 nm. (e) Combination of the above images. (f) Representative spectra of tricolor QD-encoded core-shell microparticles.
by adjusting the interface tensions.26 Then, with UV polymerization of the emulations in situ, bicolor QDs-encoded microparticles with two different cores were fabricated, as shown in Figure 3c−g. The prepared QD-encoded microparticles have outstanding optical properties that make them an excellent choice as encoded microparticles for biomedical applications. As we can see in Figure 3d−g, QDs were uniformly dispersed in the cores of the microparticles, and the strongest fluorescence emission intensity of the two cores under the excitation of 405 nm light appeared at 558 and 607 nm, respectively, which can be confirmed by the fluorescence spectrum of the microparticles, as shown in Figure 3h. With the
can be seen in Movie S3 in the Supporting Information). Thus, by changing the core number and the QDs concentration in each core, large number of precise codes could be generated. Theoretically, with N intensity levels with M colors, we could get [(M3 ) × N3 − 1] types of unique codes. For instance, 31 999 recognizable codes can be generated by using 4 types of QDs with 20 intensities for each type of QDs (that is, N = 20 and M = 4). Significantly, by detecting each core in which only one kind of QDs dispersed, respectively, the fluorescence spectrum 8541
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of each kind of QDs could be recorded easily and precisely, without the help of spectral deconvolution or signal processing methods. As a result, the problem of spectral overlapping could be avoided and the kind of embedded QDs could be increased. Therefore, the QD-encoded core-shell microparticles with multi cores generated by this method are more applicable for high-throughput multiplexed detection.
4. CONCLUSIONS In summary, we have demonstrated an effective and novel microfluidic approach to generate multicolor QD-encoded core-shell microparticles with precise coding and enhanced stability by using double emulsions with multi cores as template. With the protection of the hydrogel shells, the leakage of QDs was avoided and the fluorescence stability of the particles was enhanced greatly. Significantly, by embedding different QDs into different cores, no interaction between different QDs such as resonance energy transfer existed, and the fluorescence spectrum of each kind of QDs could be recorded, respectively. This article provides a novel method to overcome the difficulties in the synthesis of multicolor QDencoded microparticles.
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ASSOCIATED CONTENT
S Supporting Information *
Details of experiments, movies, and the size and structure control of double emulsions. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Author Contributions †
Y.C. and P.-F.D contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the support of the National Natural Science Foundation of China (21322604, 21136006), National Basic Research Program of China (2012CBA01203), and A Foundation for the Author of National Excellent Doctoral Dissertation of PR China (FANEDD 201053).
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REFERENCES
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