Multiphasic Sensory Alginate Particle Having Polydiacetylene

Do Hyun Kang , Ho-Sup Jung , Namyoung Ahn , Su Min Yang , Sungbaek Seo , Kahp-Yang Suh , Pahn-Shick Chang , Noo Li Jeon , Jinsang Kim , and Keesung ...
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Multiphasic Sensory Alginate Particle Having Polydiacetylene Liposome for Selective and More Sensitive Multitargeting Detection Jiseok Lee† and Jinsang Kim*,†,‡,§,∥ †

Macromolecular Science and Engineering, ‡Departments of Materials Science and Engineering, §Chemical Engineering, ∥Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States ABSTRACT: A novel fabrication method of monophasic, biphasic, and triphasic alginate microparticles having sensory polydiacetylene (PDA) liposomes has been developed to achieve selective and more sensitive multitargeting detection in solution. In this alginate microparticle based detection system, the sensory PDA liposomes are concentrated in the particles rather than being diluted in a solution, which is the case of a conventional solution based detection system, providing superior sensitivity and stability. The biphasic nature of the alginate microparticles was realized by coinjecting two different PDA liposome/alginate mixture solutions into a CaCl2 solution using a simple combined needle injection system. The size and the constituent of the Janus particles and the extended triphasic particles could be independently manipulated by controlling a centrifugal force and formulating the composition of the PDA liposome solutions, respectively. The multitargeting capability of such mutiphasic alginate particles was demonstrated by fluorescence microscopy. The presented particle-based detection system has a great potential to be combined with a microfluidic device for the development of advanced biosensors having a high throughput screening capability. KEYWORDS: polydiacetylene liposomes, sensor, Janus particle

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INTRODUCTION Conjugated polydiacetylenes (PDA) have attracted great attention in the sensor application due to their unique colorimetric/fluorescence dual detection capability as well as the convenient molecular self-assembly based fabrication method.1−6 In general, diacetylene (DA) monomers having a rationally designed probe molecule are dispersed by sonication in an aqueous solution to form self-assembled PDA liposomes. Alternatively, DA monomers can also be transferred onto a solid substrate via the Langmuir−Blodgett (LB) or Langmuir− Schaefer (LS) film transfer method to build well-defined selfassembled monolayer7 or multilayers.8 These well-ordered DA monomers are then subsequently photopolymerized by UV irradiation. PDAs undergo dramatic blue to red chromatic transitions and also develop red fluorescence emissions upon being exposed to various external stimuli such as heat,9−11 pH,12 organic solvent,13−15 and mechanical stress.16,17 It is wellknown that such external stimuli exert stress on the ene-yne conjugated backbone of PDA, which widens the band gap of PDA via backbone twisting.18 PDAs have also been widely investigated for bio- and chemosensor applications due to their self-signaling capability.19−21 Many successful PDA biosensor systems have been developed in the form of aqueous suspensions, thin films on a solid substrate, and microarrays.22,23,8,24 PDA sensor systems in the form of aqueous suspension are practically very useful for the detection of biological molecules in an aqueous phase but have some undesirable limitations. First, PDA liposomes tend to aggregate and/or polymerize in an ambient condition for a long period of time. In addition, most PDA aqueous sensory systems can recognize only one © 2012 American Chemical Society

type of target, therefore lacking a multitargeting capability. Moreover, in this homogeneous solution detection scheme, sensory PDA liposomes are dispersed in a solution to which analytes are introduced, resulting in rather limited sensitivity due to the homogeneous dilution. In order to overcome these limitations, several new approaches have been developed, such as the encapsulation of the probe PDA liposomes into a host matrix, such as poly(vinyl alcohol) (PVA),25,26 agar scaffold,27 hybrid sol−gel matrix,28,29 silica nanocomposite,30 microbubbling,31 and electrospining32 besides the solid state PDA liposome microarrays.33,34 Indeed those new approaches enhance the long-term stability of PDA liposomes, and the microarray format provides a possible multitargeting capability. However, a simultaneous detection of multiple targets in a homogeneous solution has not been realized even though such a system has a great potential to be combined with a microfluidic device for various biosensor applications. The above-mentioned dilution effect should also be systematically investigated by comparing the conventional solution-based detection scheme with a particle-based novel solution detection scheme where sensory molecules are concentrated in the particle rather than being homogeneously diluted in the solution. In this contribution, we present a new concept and fabrication principle of biphasic and triphasic Janus particles loaded with PDA liposomes. We systematically investigated the sensitivity, potential multitargeting capability, and long-term Received: May 16, 2012 Revised: June 21, 2012 Published: July 9, 2012 2817

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stability of the new multiphasic particle-based solution detection scheme. The biphasic feature was achieved by simple centrifugation of two different PDA liposome/alginate mixture solutions into a CaCl2 solution as schematically illustrated in Scheme 1. The size of the particle and the fraction and

Article

EXPERIMENTAL SECTION

Materials and Method. All solvents were purchased from SigmaAldrich Chemicals. 10,12-Pentacosadiynoic acid (PCDA) was purchased from GFS Chemicals. Dicyclohexylcarbodiimide, 4dimethylaminopyridine, cyanuric acid (CA), 1,8-diazabicycloundec-7ene, carbon tetrabromide, triphenylphosphine, 6-bromohexan-1-ol, and tri(ethylene glycol) were purchased from Sigma-Aldrich Chemical Co. UV/vis absorption spectra were taken on a Varian Cary50 UV/vis spectrophotometer. Fluorescence spectra were obtained using PTI QuantaMasterTM spectrofluorometers equipped with an integrating sphere. Fluorescence images were taken by Olympus BX51 W/DP71 fluorescent microscope. Synthesis of PCDA-EG-CA. 2-(2-(2-Bromoethoxy)ethoxy)ethanol. To a solution of tri(ethylene glycol) (4.08 g, 27.14 mmol) in dichloromethane (50 mL) at 0 °C was added carbon tetrabromide (3.00 g, 9.05 mmol) and triphenylphosphine (2.61 g, 9.95 mmol). The reaction mixture was stirred at room temperature for 2 h, and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1/2) to give 1.61 g (83%) of desired monomer 2-(2-(2-bromoethoxy)ethoxy)ethanol as a yellowish oil (1.61 g). 1H NMR (400 MHz, CDCl3): δ 2.18 (brs, 1H), 3.44 (t, J = 6.4 Hz, 2H), 3.58 (m, 2H), 3.64 (s, 4H), 3.70 (m, 2H), 3.78 (t, J = 6.2 Hz, 2H). 2-(2-(2-Bromoethoxy)ethoxy)ethyl pentacosa-10,12-diynoate. To a solution of 10,12-pentacosadiynoic acid (2 g, 5.34 mmol)) in dichloromethane (30 mL) at 0 °C was added 2-(2-(2-bromoethoxy)ethoxy)ethanol (1.32 g, 5.34 mmol), dicyclohexylcarbodiimide (1.32 g, 6.41 mmol), and 4-dimethylaminopyridine (0.012 g, 0.10 mmol). After the reaction mixture was vigorously stirred for 2 h, 20 mL of hexane was poured into the mixture, and the white urea solid was filtered off. The solvent was removed in vacuo. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 10/1) to give 2.7 g (89%) of desired diacetylene monomer 2-(2-(2bromoethoxy)ethoxy)ethyl pentacosa-10,12-diynoate as a colorless oil. 1H NMR (400 MHz, CDCl3): δ0.84 (t, J = 6.8 Hz, 3H), 1.21−1.32 (m, 26H), 1.43−1.59 (m, 6H), 2.20 (t, J = 6.8 Hz, 4H), 2.29 (t, J = 7.6 Hz, 2H), 3.43 (t, J = 6.2 Hz, 2H), 3.62−3.68 (m, 6H), 3.77 (t, J = 6.2 Hz, 2H), 4.19 (t, J = 4.8 Hz, 2H). PCDA-EG-CA. To a solution of 2-(2-(2-bromoethoxy)ethoxy)ethyl pentacosa-10,12-diynoate (1.9 g, 3.33 mmol) in dimethylformamide (50 mL) was added cyanuric acid (4.30 g, 33.33 mmol) and 1,8-diazabicycloundec-7-ene (0.50 mL, 3.33 mmol). The reaction mixture was heated under 60 °C for 8 h, poured into the water, and extracted with ethyl acetate. The organic layer was washed 3 times with water to eliminate the excess cyanuric acid, dried with MgSO4, and filtered. Most of the solvent was removed under vacuo. The resulting solution was recrystallized from methyl alcohol and ethyl acetate to give 1.56 g (76%) of desired diacetylene monomer PCDAEG-CA as a white solid. 1H NMR (400 MHz, CDCl3): δ 0.875 (t, J = 7.0 Hz, 3H), 1.25−1.47 (m, 26H), 1.49−1.57 (m, 4H), 1.60 (t, J = 7.0 Hz, 2H), 2.22−2.25 (m, 4H), 2.32 (t, J = 7.6 Hz, 2H), 3.65−3.69 (m, 6H), 3.76 (t, J = 5.2 Hz, 2H), 4.07 (t, J = 5.2 Hz, 2H), 4.22 (t, J = 4.8 Hz, 2H), 9.49 (brs, 2H) . 13C NMR (100 MHz, CDCl3): 14.07, 19.15, 22.63, 24.78, 28.27, 28.30, 28.73, 28.81, 28.86, 29.05, 29.29, 29.42, 29.59, 31.86, 34.12, 40.46, 63.27, 65.17, 65.24, 67.32, 69.00, 69.80, 70.42, 77.38, 77.55, 148.24, 149.22, 173.97. MS (ES): Calcd, 617.4; found (M+Na)+, 640.4. PCDA-NHS. To a solution containing 1.00 g (2.67 mmol) of 10,12pentacosadiynoic acid in 10 mL of methylene chloride was added 0.38 g (3.47 mmol) of N-hydroxysuccinimide and 0.38 g (4.01 mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride at room temperature. The resulting solution was stirred at room temperature for 2 h. The solvent was removed in vacuo, and the reside was purified by extraction with ethyl acetate to give 1.08 g (86.2%) of the desired diacetylene monomer PCDA-NHS as a white solid. 1H NMR (300 MHz, CDCl3): δ 0.85 (t, 3H), 1.20−1.62 (m, 36H), 2.21 (t, 4H), 2.60 (t, 2H), 2.85 (s, 4H), 7.18 (brs, 1H). PCDA-EDEA. To a solution containing 1.055 mL (7.21 mmol) of 2,2′-(ethylenedioxy)bis(ethylamine) in 60 mL of methylene chloride

Scheme 1. (A) Chemical Structures of the Investigated PDA Molecules and (B) the Schematic Illustration of the Fabrication of Biphasic Alginate Particle Having Embedded PDA Liposomes: (a) Centrifugation of PDA/Alginate Mixture Solution into a CaCl2 Solution, (b) Photopolymerization of the Biphasic Alginate Particle Loaded with PDA Liposomes, (c) Melamine Detection Manifested by Hemispherical Color Transition and Fluorescence Emission, and (d) Additional Hemispherical Green Fluorescence Emission upon the Addition of AvidinFITC, Demonstrating a Possible Multitargeting Capability

composition of each component can be independently manipulated by controlling the centrifugal force and the type of PDA liposomes, which was confirmed by fluorescence microscopy and optical microscopy. Efficient fabrication of triphasic microparticles was also demonstrated toward simultaneous screening of multiple targets in a homogeneous aqueous environment. Alginate, also called alginic acid, is an anionic polysaccharide constituent of the algae cell membrane. Because of its biocompatibility, porosity, and gelation property with multivalent cations, such as Ca2+ ions, it has been widely used for the encapsulation of various chemicals and drugs.35 We applied the gelation and porous nature of a cross-linked alginate microparticle to the encapsulation of our sensory PDA liposomes. The porous nature of the encapsulating host material, alginate, is very important to maintain the sensory property of the embedded PDA liposomes because in order to produce a sensory signal, analytes should be able to freely access the embedded PDA. Rapid and efficient cross-linking in a mild condition is also important to successfully create a biphasic or multiphasic structure without mixing component PDA liposomes and without disrupting the self-assembled ordered structure of the embedded PDA liposomes. Much effort has been devoted to develop biphasic polymeric structures including the microcontact printing,36 electrified jetting,37 and microfluidic systems.38−42 However, it is difficult to use these techniques in a practical way, as they require serious investigation and experienced skills for the device fabrication in order to control the size, homogeneity, and the biphasic property of the particles. 2818

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was added dropwise 1.00 g (2.12 mmol) of PCDA-NHS for 3 h at room temperature. The resulting mixture was allowed to stir for 2 h at room temperature. The resulting mixture was concentrated in vacuo, and the residue was purified by column chromatography (9:1 chloroform:methanol) to give 0.51 g (46.7%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 0.89 (t, 3H), 1.27−1.76 (m, 35H), 2.23 (m, 6H), 2.91 (t, 2H), 3.42−3.63 (m, 12H), 6.21 (t, 1H), 7.27 (brs, 1H). PCDA-Biotin. To a solution containing 0.23 g (0.94 mmol) of biotin in 5 mL of DMF was added dropwise 0.40 g (0.79 mmol) of PCDAEDEA in 5 mL of methylene chloride. The mixture solution was allowed to stir for overnight at room temperature. The solvent was removed in vacuo, and the residue was purified by column chromatography (9:1 chloroform:methanol) to give 0.18 g (33.9%) of the desired diacetylene monomer PCDA-EDEA-biotin as a white solid. 1H NMR (300 MHz, CDCl3): δ 0.89 (t, 3H), 1.27−1.76 (m, 41H), 2.23 (m, 8H), 2.74 (d, 1H), 2.94 (m, 1H), 3.17 (m, 1H), 3.42− 3.65 (m, 13H), 4.32 (q, 1H), 4.54 (q, 1H), 4.92 (s, 1H), 5.87 (s, 1H), 6.27 (t, 1H), 6.31(t, 1H), 7.27 (brs, 1H). PDA Liposomes Formation. A mixture of the PCDA-EG-CA and PCDA (9:1 mol ratio) monomer mixture was dissolved in 100 μL of tetrahydrofuran. The PDA mixture solution was injected rapidly into 20 mL of 50 mM HEPES buffer pH 7.0 and was sonicated for 20 min. The suspension was filtered with 0.8 μm syringe filter and stored for 4 h at 5 °C (final concentration of liposomes: 1 mM). Biotin functionalized PDA liposomes were generated in the diwater using PCDA-biotin, PCDA-EDEA, and PCDA (3:1:6 mol ratio) monomer with the same protocol. Monophase PDA Liposomes Loaded Alginate Microparticles. One mM of PCDA-EG-CA/PCDA (9/1) liposomes solution in 50 mM of HEPES buffer pH 7.0 was added to the 4 wt % of alginic acid solution by 2:1 ratio. The PDA liposomes/alginate mixture solution was injected into the 2.5 wt % CaCl2 solution by centrifugation (needle size: 25G, centrifuge force: 21G). The PDA liposomes loaded alginate microparticles were stayed in the CaCl2 solution for an additional 30 min. Biotin functionalized PDA liposomes loaded alginate particles were generated with the same protocol. The red fluorescence image was taken using a fluorescence microscope (Olympus BX51) after 2 h incubation in the desired concentration of melamine or avidin-FITC solution at room temperature. Biphasic and Triphasic Phase PDA Liposomes Loaded Alginate Microparticles. The experimental setup for Janus particle was similar to that of a monophase particle. Briefly, PCDA-EG-CA/PCDA (9/1) liposomes and PCDA-biotin/PCAD-EDEA/PCDA (3/1/6) liposomes solution were separately added to the 4 wt % alginic acid solution by a 2:1 ratio, and then each mixture solution was injected together into the 2.5 wt % CaCl2 solution through the combined needle by the centrifugation for 1 h. The PDA liposomes loaded alginate microparticles were stayed in the CaCl2 solution for additional 30 min. The red/green biphasic fluorescence image was taken after 2 h incubation in the melamine (50 ppm)/avidin-FITC (1 mg/1 mL) mixture solution using a fluorescence microscope (Olympus BX51). Triphasic phase PDA liposomes loaded particles were generated with the same protocol by attaching additional syringe containing the PCDA liposomes/alginate mixture solution.

phases can be readily achieved by using differently formulated PDA liposomes for each phase, which can be extended to multiphasic microparticles for many promising potential applications. Melamine Detection Using PDA Liposomes Embedded Alginate Microparticles. We applied the single-phase alginate particle system to our recent development of a melamine detection sensor to investigate its applicability as a sensitive sensory platform.22 It is well-known that melamine can cause kidney damage and therefore the melamine content in food products has been strictly regulated by the international health organizations. We recently invented a solution based colorimetric sensory system for selective and sensitive melamine detection by adapting the strong multiple hydrogen bondings between melamine and cyanuric acid (CA) to our sensor design.22 We prepared the melamine detecting 1 mM PCDA-EG-CA/PCDA (9/1 mol %) liposome solution and mixed the liposome solution with a 4 wt % alginate solution by a 2/1(v/v) ratio and injected the mixture solution dropwise to a CaCl2 solution (2.5 wt %) by means of the centrifugation (Scheme 1). The alginate droplets having PDA liposomes were physically cross-linked by Ca2+ ions in the CaCl2 solution. As demonstrated in Figure 1, the size of the produced micro-

Figure 1. (A) Optical microscope images of alginate microparticles having embedded PCDA-EG-CA/PCDA (9/1) liposomes (scale bar: 500 μm). (B) Fluorescence microscope images of the same alginate microparticles after heating (Excitation at 550 nm and a long-pass emission filter with 600 nm cutoff were used.). (C) A correlation curve between the diameter of the alginate microparticles and the G-force (A 1.3 wt % alginate solution and a 2.5 wt % CaCl2 solution were used.).

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RESULTS AND DISCUSSION Our designed system provides a convenient fabrication protocol for assembling biphasic particles. First, by using single-phase alginate particles having single component PDA liposomes, we investigated the optimum conditions, such as concentration of the PDA liposomes/alginate mixture solution and the G-force in order to achieve homogeneous particle formation and control the particle size. Biphasic alginate particles loaded with PDA liposomes were then produced by the simultaneous parallel injection of two alginate/PDA liposome mixture solutions into a CaCl2 solution as schematically illustrated in Scheme 1. Variety combinations of the two

particles decreased approximately from 750 to 250 μm as the centrifugal force increased. The alginate microparticles having the PCDA-EG-CA/PCDA (9/1) liposomes were subsequently photopolymerized. Strong blue color from the alginate particles confirms that the PDA liposomes were entrapped uniformly in the alginate matrices and effectively photopolymerized. In addition, the blue intensity of the single-phase particles did not change over 30 days of storage, demonstrating the superior 2819

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that a quantitative analysis of an unknown melamine concentration is also achievable by simply counting the number of the red particles. Moreover, the results show that the particle-based sensory platform provides a much better sensitivity by detecting a much lower melamine concentration (0.05 ppm) compared to the solution (1 ppm) and the microarray based detection system (0.5 ppm).11 As we briefly discussed the dilution effect in the Introduction, we believe that the improved sensitivity is due to the highly localized PDA liposomes in the confined alginate particle interior. In the case of our alginate particle-based detection scheme, the total number of receptor molecules (cyanuric acid) for the recognition of melamine is smaller and located nearby each other in a more concentrated fashion. Therefore, the chance for adjacent receptors to be occupied by melamine is higher because the smaller number of receptors in a confined volume will more effectively sweep the analyte solution for melamine than homogeneously dispersed receptors in the conventional solution-based detection scheme. Synthesis of the Biphasic Alginate Microparticles. After demonstrating the more sensitive detection capability of the particle-based detection system than a conventional solution scheme, we directed our development toward a multitargeting capability. We designed Janus alginate PDA particles having a melamine sensor phase and a negative control phase. For that, we prepared nonfunctionalized PDA liposomes/alginate mixture solution and the PCDA-EG-CA/ PCDA (9/1) liposomes/alginate mixture solution having the same viscosity. The two syringe needles were combined together in such a way that a droplet of each PDA liposome/ alginate mixture solution met together at the tip of the two needles to form the biphasic particles (Scheme 1B). Microparticles having 300 μm diameter were obtained after 15 min centrifugation at a G-force of 250G, and the imbedded liposomes were subsequently polymerized under 254 nm UV light. As shown in Figure 4A, only the half sphere changed its

stability of the PDA liposomes in the alginate particles to that in the conventional solution phase. We expound on the sensitivity of the alginate particles having the PCDA-EG-CA/PCDA (9/1) liposomes toward melamine. The cross-linked alginate microparticles were very stable and could be manipulated one by one with a micropipet or a tweezer. We counted approximately 100 particles using a micropipet and incubated the particles in a 50 ppm melamine solution. As can be seen in Figure 2, approximately 70% of the

Figure 2. (A) Optical microscope image showing colorimetric transition and (B) red fluorescent emission image of alginate microparticles having PCDA-EG-CA/PCDA (9/1) liposomes after incubation in a 50 ppm melamine solution.

particles showed the blue to red color transition and red fluorescence emission after 2 h of incubation in the melamine solution, demonstrating that the sensory property of the PDA was maintained in the alginate particles. We further conducted a detection limit study with 30 particles in various concentrations of melamine. All of the particles changed their color from blue to red in the 50 ppm and 25 ppm of melamine solutions. The number of the color changed red particles decreased from 30 to 4 as the concentration of melamine decreased to 0.05 ppm. The correlation curve between the number of red particles and the amount of melamine is shown in Figure 3A, demonstrating

Figure 4. A) Optical microscope image showing hemispherical colorimetric transition and (B) red fluorescence emission image of the Janus alginate particles of PCDA-EG-CA/PCDA (9/1) upon exposure to melamine.

color to red and produced red fluorescence emission after being incubated in a 50 ppm melamine, while the other half side remained in blue as a negative control, which clearly demonstrates that the biphasic alginate particles having a sensory unit and a negative control could be easily fabricated to improve the fidelity of the detection as well as sensitivity. We further conducted a series of experiments to devise biphasic alginate microparticles having a multitargeting capability. First, we synthesized the biotin-functionalized PDA monomer shown in Scheme 1. It is well-known that biotin has a strong affinity (Kd of ∼10−14 mol/L) to avidin.43 We prepared single-phase alginate particles containing the biotin-PCDA

Figure 3. (A) Colorimetric transition of alginate microparticles having PCDA-EG-CA/PCDA (9/1) liposomes after incubation in various concentration of melamine solution. (B) Correlation curve between the number of red colored particles and the concentration of melamine. 2820

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liposomes. We incubated the alginate particles in a FITClabeled avidin solution (1 mg/mL) and washed them with diwater. Although the biotin-PCDA liposomes in the alginate particles successfully interacted with the avidin-FITC and produced green fluorescence due to FITC, neither blue to red color change nor red fluorescence emission developed from the avidin/biotin interaction (Figure 5). We postulate that the

Figure 6. (A) Fluorescence microscope image of the biphasic Janus alginate microparticles having two different PDA liposomes (PCDAEG-CA/PCDA (9/1) and PCDA/PCDA-EDEA/PCDA-biotin (6/1/ 3)) after the incubation in a 50 ppm melamine solution. (B) Green fluorescence emission after the incubation in an avidin-FITC solution (1 mg/mL in 1xPBS buffer). (C) The overlay of the two fluorescence images representing the biphasic property of the Janus alginate particles (scale bar: 500 mm). (D) Triphasic image of PDA liposomes loaded with alginate particles after incubation with avidin-FITC. (E) Triphasic image of PDA liposomes loaded with alginate particles after incubation with avidin-FITC and subsequently with melamine solution.

Figure 5. Optical microscope images showing colorimetric transition of alginate particles having the biotin-PCDA liposomes (PCDA/ PCDA-EDEA/PCDA-biotin (6/1/3)) (top row) and PCDA-EG-CA/ PCDA (9/1) (bottom row) before (a) and after addition of avidinFITC (b) and melamine (c). (d) Fluorescence microscope image of the alginate particles having the biotin-PCDA liposomes after addition of avidin-FITC (top) and that of the alginate particles having the PCDA-EG-CA/PCDA (9/1) after addition of melamine (bottom).

development. The high throughput screening capability is very important in that one can selectively and efficiently identify multiple targets at the same time with a good cross selectivity. The three syringe needles were combined together in the same manner with the biphasic Janus particle generation protocol. Three different PDA liposome/alginate mixture solutions (PCDA, biotin-PCDA, PCDA-EG-CA/PCDA (9/1) liposomes) were coinjected to the CaCl2 solution by means of the combined needle injection system. The viscosity of each PDA liposomes/alginate mixture solution was precisely matched to achieve the desired accurate triphasic structure. As shown in Figure 6D, we observed that 1/3 of alginate particles represented green fluorescence after the incubation of the resulting alginate PDA particles in an avidin-FITC solution (1 mg/mL). After subsequent incubation in a 50 ppm melamine solution, additional red fluorescence was developed in the adjacent 1/3 fraction of the alginate particles by the melamine detection, but the last 1/3 fraction of the particles having PCDA liposomes remained in the dark as a negative control (Figure 6E). We successfully demonstrated triphasic alginate particles having a sensor, an indicator, and a negative control unit. Therefore, it is possible to further expand the multitargeting capability by loading various fluorescence dye or colorimetric pigments as an indicator and also imbedding multiple types of probe PDA liposomes as a sensor unit.

cross-linked structure of alginate particles may decrease the mobility of the bound avidin-FITC, resulting in a less effective conformational change of the biotin-PCDA liposomes. We subsequently incubated the alginate particles in a melamine solution (50 ppm) and did not observe any change, confirming a good selectivity. We also made single-phase alginate particles containing the PCDA-EG-CA/PCDA (9/1) liposomes. The alginate particles changed color and developed red fluorescence only after being incubated in a melamine solution (50 ppm). These experiments demonstrated cross-selectivity and imply a possible multidetection capability. We built biphasic Janus alginate particles using the biotinPCDA liposomes and the PCDA-EG-CA/PCDA (9/1) liposomes. The biotin-PCDA liposomes/alginate mixture solution and the PCDA-EG-CA/PCDA (9/1) liposomes/alginate mixture solution having the same viscosity were injected together into a CaCl2 solution using the combined needle injection system. To confirm the biphasic property of the resulting alginate particles, we incubated the obtained microparticles in a melamine/avidin-FITC mixture solution for 2 h and rinsed the particles with diwater. We observed the hemispherical red/green fluorescence originated from the melamine and avidin-FITC interaction, respectively (Figure 6A-C), which confirmed that each constituent liposome of the biphasic alginate particles selectively interacted with its desired target without interference from the other. Alginate Janus particles having exact half−half hemispherical red/green fluorescent were reproduced by closely matching the viscosity of each PDA/alginate mixture solution to prevent convective mixing of the two solutions. This result is very attractive in that the process is very simple and convenient, requiring no complicated device but just a centrifuge and the simple combined needle injection system. Synthesis of the Multiphasic Alginate Microparticles. We finally extended our study to multiphasic alginate particle

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CONCLUSIONS We have successfully demonstrated a convenient and efficient fabrication method of biphasic and multiphasic alginate microparticles having sensory PDA liposomes. The developed microparticles based detection system showed selective and sensitive melamine detection down to the 50 ppb level in a homogeneous aqueous solution, which is a 20-times better detection limit compared to the 1 ppm level of the conventional solution phase detection system. We also demonstrated a promising multitargeting capability by developing the multiphasic alginate/PDA microparticles. The 2821

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Chemistry of Materials

Article

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presented particle-based detection system has a great potential to be combined with a microfluidic device for the development of advanced biosensors having a high throughput screening capability.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge financial supports from the National Science Foundation (DMR Career 0644864). This work was also partly supported by a grant from Animal, Plant and Fisheries Quarantine and Inspection Agency of Korea (I-AD14-201113-11).

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ABBREVIATIONS PDA, polydiacetylene; EG, ethyleneglycol; CA, cyanuric acid; EDEA, 2,2-(ethylenedioxy)bis(ethylamine); FITC, fluorescein isothiocyanate

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REFERENCES

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dx.doi.org/10.1021/cm3015012 | Chem. Mater. 2012, 24, 2817−2822