Research Article www.acsami.org
Fabrication of High-Performance Magnetic Lysozyme-Imprinted Microsphere and Its NIR-Responsive Controlled Release Property Jinxing Chen,† Shan Lei,† Yunyun Xie,† Mozhen Wang,*,† Jun Yang,*,‡ and Xuewu Ge† †
The USTC-Anhui Tobacco Joint Laboratory of Tobacco Chemistry, CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, and ‡The USTC-Anhui Tobacco Joint Laboratory of Tobacco Chemistry, Research Center of Tobacco and Health, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China S Supporting Information *
ABSTRACT: The preparation of efficient and practical biomacromolecules imprinted polymer materials is still a challenging task because of the spatial hindrance caused by the large size of template and target molecules in the imprinting and recognition process. Herein, we provided a novel pathway to coat a NIR-light responsive lysozyme-imprinted polydopamine (PDA) layer on a fibrous SiO2 (F-SiO2) microsphere grown up from a magnetic Fe3O4 core nanoparticle. The magnetic core−shell structured lysozyme-imprinted Fe3O4@ F-SiO2@PDA microspheres (MIP-lysozyme) can be easily separated by a magnet and have a high saturation adsorption capacity of lysozyme of 700 mg/ g within 30 min because of the high surface area of 570 m2/g and the mesopore size of 12 nm of the Fe3O4@F-SiO2 support. The MIP-lysozyme microspheres also show an excellent selective adsorption of lysozyme (IF > 4). The binding thermodynamic parameters studied by ITC proves that the lysozyme should be restricted by the well-defined 3D structure of MIP-lysozyme microspheres. The MIP-lysozyme can extract lysozyme efficiently from real egg white. Owing to the efficient NIR light photothermal effect of PDA layer, the MIP-lysozyme microspheres show the controlled release property triggered by NIR laser. The released lysozyme molecules still maintain good bioactivity, which can efficiently decompose E. coli. Therefore, this work provides a novel strategy to build practical NIR-light-responsive MIPs for the extraction and application of biomacromolecules. KEYWORDS: protein imprinting, polydopamine, fibrous SiO2, Fe3O4 nanoparticles, lysozyme, NIR-light, controlled release
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INTRODUCTION In nature, antibodies can specifically recognize target molecules in a complicated system by the combination of multiple weak interactions and complementary three-dimensional surfaces,1,2 which has inspired intense efforts to synthesize artificial acceptors by mimicking these specific recognition principles.3−7 Molecular imprinted polymer (MIP) is one of artificial acceptors based on the “lock and key” model, having high chemical stability, low synthesis cost, and outstanding selectivity recognition for small molecules.8,9 However, MIP always show a low recognition performance for large biomolecules, such as protein,10 virus,11 microorganism.12 One big reason is that most of the large template molecules used to create the recognition sites for biomacromolecules in the deeper inside of the MIP matrix can hardly be extracted out by the eluant because of strong diffusion hindrance so that they become the “dead” recognition sites. As a result, the recognition efficiency of MIP will be largely weakened.13−18 Recently, surface imprinting technique has been employed to solve the problem of low gaining rate of the active sites for large molecules.19−22 In this technique, a surface imprinting layer with a nanoscaled thickness is supported on a suitable carrier so that the recognition sites mainly distribute on the surface layer, which is beneficial to the fast mass diffusion. For example, Shi © XXXX American Chemical Society
et al. used radio frequency glow-discharge plasma deposition to form several protein-imprinted polymeric layers with a thickness of about 10 nm on mica sheet.23 These surface imprinting layers exhibited highly selective recognition and rapid binding dynamics for the template proteins. However, along with the high utilization of imprinting sites and recognition rate of the surface imprinting system, a new problem emerges, i.e., how to get enough surface area to reach a high total amount of the recognized target biomacromolecules. Nanomaterials with mesoporous structure (2−50 nm in pore size according to the classification of IUPAC24) maybe a feasible choice because mesopores not only possess fairly high specific surface area to increase the number of recognition sites, but also have enough space for the diffusion of biomacromolecules. Unfortunately, the pore size of some existing classical mesoporous support materials is still less than the mean size of biomacromolecules. For example, the famous mesoporous silica MCM-41 has mesopores with a size below 2.5 nm,25 not suitable for the loading of the proteins generally with a size ≥3.2 nm.26 Therefore, the design and fabrication of the Received: October 22, 2015 Accepted: December 7, 2015
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DOI: 10.1021/acsami.5b10126 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
for 15 min. The reaction system was heated to 75 °C and kept for 16 h. The products, i.e., fibrous SiO2-coated Fe3O4 microspheres (Fe3O4@F-SiO2), were separated and collected from the solution with a magnet, and washed with ethanol three times. The residual surfactant CTAB in Fe3O4@F-SiO2 was removed according to a former report.32 Briefly, the as-prepared Fe3O4@F-SiO2 microspheres were dispersed into 100 mL of ethanol containing 1 g of NH4NO3. The dispersion was stirred at 65 °C for 24 h. After that, the products were separated with a magnet, washed with water twice, and dried in a vacuum oven at 50 °C for 16 h. Synthesis of Lysozyme-Imprinted PDA Layer on Fe3O4@fibrous SiO2 Microspheres. 1. Double Bond Modification on the Fe3O4@FSiO2 Microspheres. 3-(Trimethoxysilyl)propyl methacrylate (5 mL) was dropwise added into 240 mL of ethanol containing 1 g of Fe3O4@ F-SiO2 microspheres. The dispersion was stirred for 24 h at room temperature. The treated Fe3O4@F-SiO2 microspheres were separated by a magnet, washed with ethanol three times, and dried in a vacuum oven at 50 °C for 10 h. 2. Introduction of Carboxyl Groups on Fe3O4@F-SiO2 Microspheres. The above obtained double bonds modified Fe3O4@F-SiO2 microspheres were redispersed in 200 mL of ethanol solution containing 5 mL of mercaptoacetic acid and 100 mg of AIBN. After being bubbled with nitrogen for 30 min, the dispersion was heated to 85 °C within 30 min, and kept for 24 h. The products were separated by a magnet, and washed with water for four times, and dried in a vacuum oven at 45 °C for 12 h. 3. Coating Lysozyme-Imprinted PDA Layer on Fe3O4@F-SiO2 Microspheres. Twenty milligrams of lysozyme and 100 mg of carboxyl group functionalized microspheres were mixed with 40 mL of 10 mM Tris-HCl. The mixture was incubated at 0 °C for 10 h, followed by adding 30 mg of dopamine. The polymerization of dopamine was induced by purging air into the system, and carried out at room temperature for 12 h under mechanical stirring. The product was separated by a magnet, washed with a 5% (v/v) solution of acetic acid containing 10% (w/v) of SDS for five times, and further extracted with water for 3 times to remove the entrapped lysozyme molecules. Finally, the obtained magnetic lysozyme-imprinted Fe3O4@F-SiO2@ PDA microspheres, termed as MIP-lysozyme in this work, were dried in a vacuum oven at 35 °C for 12 h. As a control, the magnetic nonimprinted Fe3O4@F-SiO2@PDA microspheres, termed as NIP, were also prepared at the same procedures except the absence of lysozyme. Imprinting Performance of MIP-Lysozyme Microspheres. Equilibrium Adsorption. MIP-lysozyme or NIP microspheres (2 mg) was dispersed into 3 mL of the aqueous solution of lysozyme with different concentration (from 0.1 to 1.5 mg/mL) in a 5 mL centrifuge tube. The system was incubated at 25 °C for 4 h. After that, the microspheres were separated with a magnet. The concentration of lysozyme in the remaining solution was determined from the UV−vis absorbance at the wavelength of 280 nm for lysozyme. Imprinting Performance and Selective Recognition Ability of MIP-Lysozyme. The imprinting performance of MIP-lysozyme can be evaluated by the imprinting factor (IF), which is defined as33
substrate materials with suitable sizes of mesopores and channels for the diffusion of biomacromolecules have great significance on the development of biological macromolecule imprinting technique. Recently, intense studies have also focused on the applications of the rebinding molecules in MIP.27 However, owing to the strong specific interaction between rebinding molecules and imprinting matrix, the rebinding molecules can be hardly released from the matrix. Therefore, the fabrication of MIPs having stimuli-responsive release ability of the rebinding molecules have received widespread attention. NIR-lightresponsive materials have been widely studied and applied in drug control release system lately,28 since NIR light shows its specific advances in deep penetration distance and fine human security. It is worth noting that polydopamine (PDA), a widely used MIP matrix, has recently been found to exhibit efficient NIR light photothermal effect.29,30 But so far, NIR-lightresponsive MIP has hardly been involved. In this work, we presented a facile route to synthesize core− shell lysozyme-imprinted magnetic Fe3O4@fibrous SiO2@PDA microspheres, which can release the rebinding lysozyme triggered by the irradiation of NIR light. Mesopores with a size of 12 nm throughout the fibrous SiO2 make the specific surface area of Fe3O4@fibrous SiO2 support as high as 570 m2/ g. The lysozyme-imprinted PDA layer was carefully casted onto the surface of Fe3O4@fibrous SiO2 support. The prepared lysozyme-imprinted microspheres manifest enhanced bind capacity, ultrarapid binding rate, and fine selective recognition for lysozyme. They can extract the lysozyme from the egg white, and rapidly release the rebinding lysozyme under the irradiation of NIR laser. The released lysozyme still exhibits high bioactivity that can easily hydrolyze the peptidoglycan in the Escherichia coli (E. coli.) cell walls. Therefore, this work provides a novel strategy to build practical NIR-light-responsive MIPs for the extraction and application of biomacromolecules.
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EXPERIMENTAL SECTION
Materials. Analytical agents including FeCl3, sodium acetate, trisodium citrate dehydrate, ethylene glycol, cetyltrimethylammonium bromide (CTAB), NH4NO3, tetraethylorthosilicate (TEOS), sodium dodecyl sulfate (SDS), and Tris-HCl buffer solution (pH 8.0) were all obtained from Sinopharm Chemical Reagent Co., Ltd., and used as received. 3-(Trimethoxysilyl) propyl acrylate (98%), mercaptoacetic acid (95%), and dopamine hydrochloride (AR) were supplied by Aladdin Industrial Corporation. Lysozyme (Lyz, Mw 14.3 kDa), bovine serum albumin (BSA, Mw 67 kDa), bovine hemoglobin (BHb, Mw 64.5 kDa), trypsin (Mw 23.3 kDa), and cytochrome c (Cyt c; Mw 12.4 kDa) were purchased from Sigma-Aldrich Co. LLC. Deionized water was utilized in all experiments. Preparation of Magnetic Lysozyme-Imprinted Fe3O4@FSiO2@PDA Microspheres. Synthesis of Fe3O4 Particles. The magnetic Fe3O4 particles were synthesized according to a previous report.31 FeCl3 (2.16 g), sodium acetate (2.4 g), and trisodium citrate dihydrate (0.4 g) were dissolved in 80 mL of ethylene glycol under vigorous stirring at 50 °C for 4 h. The obtained solution was transferred into a Teflon-lined stainless-steel autoclave (200 mL in capacity). The autoclave was heated to 200 °C, and kept for 10 h, then cooled to room temperature in the air. The black products were separated by a magnet, washed with ethanol, and deionized water for three times, respectively, and dried in a vacuum oven for 12 h. Synthesis of Fibrous SiO2 (F-SiO2) on Fe3O4 Particles. Typically, CTAB (4 g) and urea (2.4 g) were dissolved in 120 mL of water first. The as-prepared Fe3O4 particles (0.4 g) were then dispersed into the above solution ultrasonically. After the addition of cyclohexane (120 mL), isopropanol (IPA, 4 mL), and TEOS (6 mL) into the above dispersion, the mixture was stirred mechanically at a rate of 250 rpm
IF = Q MIP/Q NIP Where QMIP and QNIP are the binding capacity of the specific protein of MIP-lysozyme and NIP microspheres respectively, which were measured according to the following process: 2 mg of MIP-lysozyme or NIP microspheres was dispersed in 2 mL of an aqueous solution of a protein (0.5 mg/mL). Here, five kinds of proteins, i.e., lysozyme, BSA, BHb, Cyt c, and trypsin, had been investigated. The dispersion was kept for 12 h, and the microspheres were separated by a magnet. The concentration of the protein in the remaining solution was determined from the UV−vis absorbance at the wavelength of 280 nm for lysozyme, 278 nm for BSA, 406 nm for BHB, 410 nm for Cyt c, and 260 nm for trypsin, respectively. At the same time, the selectivity coefficient (α) were used to evaluate the selective recognition ability of MIP-lysozyme, which is defined as the ratio of the IF for lysozyme (IFlys) with respect to that for the competitive protein (IFcomp. protein)34 B
DOI: 10.1021/acsami.5b10126 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces Scheme 1. Schematic Illustration of Preparation Processes of the Lysozyme-Imprinted Microspheres
Figure 1. (a, b) TEM image and SAED pattern of Fe3O4 nanoparticles; (c, d) SEM and TEM images of Fe3O4@F-SiO2 (the insets in d are the elemental mappings of Fe and Si); (e, f) SEM and TEM images of lysozyme-imprinted Fe3O4@F-SiO2@PDA (MIP-lysozyme) microspheres.
α = IFlys /IFcomp.protein
eluted with acetic acid solution (5 vol %). The elution was collected to be analyzed by SDS-PAGE. NIR-Laser Triggered Release of the Rebinding Lysozyme on MIPLysozyme Microspheres. The above obtained MIP-lysozyme microspheres rebound lysozyme in egg white solution were redispersed in 2 mL of Tris-HCl. In each NIR-laser irradiation cycle, the dispersion was first irradiated for 5 min with NIR laser (808 nm, 3.3 W/cm2), and then the laser was turned off. At the moment, the microspheres were separated with a magnet. The clear solution was collected and measured by a UV−vis spectroscopy. Then the above microspheres were redispersed into 2 mL of fresh Tris-HCl solution and standing for 30 min. The above collection and separation process were cycled for 5 times. As a control, the release curve of MIP-lysozyme microspheres without laser irradiation was also recorded. Characterization. The morphology of the prepared particles was studied by transmission electron microscopy (TEM, Hitachi H-7650, 100 kV) and scanning electron microscopy (SEM, JSM6700F, 5.0 kV). Selected area electron diffraction (SAED) patterns were obtained using a high revolution transmission electron microscopy (HRTEM, JEM-2010, 200 kV). The elemental mapping was investigated with
Adsorption Kinetics of Lysozyme. Six milligrams of lysozyme and 15 mg of MIP-lysozyme or NIP microspheres were mixed with 15 mL of 10 mM Tris-HCl buffer in a vortex mixer. At every certain time interval, 1 mL of the mixture was sampled out to measure the concentration of lysozyme with UV−vis spectroscopy method after the microspheres were rapidly separated with a magnet. Application of MIP-Lysozyme in the Extraction and Controlled-Release of Lysozyme. Extraction of Lysozyme from Egg White Solution. Typically, 2 mL of fresh egg white was diluted to 40 mL with 10 mM Tris-HCl buffer. The diluted solution was centrifuged for 10 min at 12 000 rpm. The supernatant was collected, and stored at 4 °C as the stock solution. Two mL of the stock solution was sampled out to be vortex mixed with 2 mg of MIP-lysozyme or NIP microspheres for 1 min. The obtained mixture was mildly shaken at 25 °C for 12 h. Then, the microspheres were separated with a magnet, and rapidly washed with Tris-HCl buffer for three times to remove physical adsorbed protein on the surface. The remaining solution were used for SDS polyacrylamide gel electrophoresis (SDSPAGE) analysis. The collected MIP-lysozyme microspheres were then C
DOI: 10.1021/acsami.5b10126 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 2. (a) N2 adsorption−desorption isotherms of Fe3O4@F-SiO2, MIP-lysozyme, and NIP microspheres, and (b) the corresponding pore size distributions calculated by BJH method from the adsorption branch of the isotherms. energy dispersive spectrometer detector (EDS) by using JEMARM200F TEM at an accelerating voltage of 200 kV. Tristar II 3020 M was used to measure the nitrogen adsorption−desorption isotherms at 77.3 K after the samples were outgassed at 60 °C for 6 h. The specific surface area and pore size distribution were analyzed by Brunauer−Emmett−Teller (BET) and Barret−Joyner−Halenda (BJH) methods, respectively. The total pore volume of the samples was calculated at P/P0 = 0.97. The magnetic properties of the samples were investigated at 300 K using a vibrating sample magnetometer (VSM). The isothermal titration calorimeter (ITC) experiments were performed using an ITC-200 instrument (Microcal). The samples were stirred and degassed for 5 min at 297 K before the measurement. The mass spectrometry analyses were performed on a Bruker Ultrafle Extreme Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometer. α-Cyano-4-hydroxycinnamic acid was used as the matrix in the MALDI method. A laser intensity of 40 Hz was used for the mass spectrometry experiments over a mass range of 10,000−100,000 atomic mass units. The photothermal performance of MIP-lysozyme microspheres in Tris-HCl buffer was measured on an 808 nm semiconductor laser device (ADR-1860, China) with externally adjustable power. The irradiation time was set to 5 min in all the experiments. An online thermocouple (TP-01, Taiwan) with an accuracy of 0.1 °C was immersed into the solution to record the temperature once a second. Thermographs of MIP microspheres dispersion were obtained with an infrared thermal camera (ICI7320, Infrared Camera Inc.) to reflect the temperatures at quartz cell.
and a mean diameter of 400 nm had been obtained, as observed by SEM (Figure 1c). The corresponding TEM image of the microsphere in Figure 1c is shown in Figure 1d, which clearly exhibits a core−shell structure, i.e., a solid core particle with a diameter of ∼130 nm and a shell composed of radial fibers with a thickness of ∼140 nm. The elemental mappings of the microsphere measured by the EDS detector are also shown in Figure 1d. They prove that element Fe only concentrates on the core region, while element Si uniformly distributes in the shell region. Evidently, the microspheres are constituted by a fibrous SiO2 layer (F-SiO2) coated on Fe3O4 nanoparticle core (Fe3O4@F-SiO2). The N2 adsorption−desorption isotherms of the prepared Fe3O4@F-SiO2 microspheres are shown in Figure 2a, which shows typical type IV curves according to BDDT classification with a type H3 hysteresis loops.24,36 A rapid increase of adsorption of nitrogen can be observed when P/P0 is above 0.7, indicating the presence of mesopores. The corresponding pore size distribution calculated from adsorption branch of isotherms by the BJH method is given in Figure 2b. It reveals that the most probable size of mesopores is 12 nm. The BET surface area of Fe3O4@F-SiO2 microspheres is 570 m2/g. The results indicate Fe3O4@F-SiO2 microspheres can provide enough surface area and inner space to support macromoleculeimprinted MIP layer. However, the template lysozyme (PI = 11.0−11.2) is positively charged in Tris-HCl buffer solution (pH 8.0). The residual surfactant CTAB also makes the surface of Fe3O4@FSiO2 microspheres positively charged. The electrostatic repulsion hinders the adsorption of lysozyme on the Fe3O4@ F-SiO2 microspheres. Hence, carboxylation modification on the surface of the Fe3O4@F-SiO2 microspheres had been done to form a lysozyme-imprinted PDA layer on the Fe3O4@F-SiO2 microspheres. The FTIR spectra of the products in each step were given in Figure S1, indicating the functional groups have been successfully modified on Fe3O4@F-SiO2 microspheres. The zeta potential of Fe3O4@F-SiO2 microspheres decreases sharply from +41.5 mV to −33.9 mV after the carboxyl group has been introduced on the surface of Fe 3 O 4 @F-SiO2 microspheres (see Figure S2). Correspondingly, the adsorption capacity toward lysozyme increases greatly from 17.6 mg/g for Fe3O4@F-SiO2 microspheres to 184 mg/g carboxyl functionalized Fe3O4@F-SiO2 microspheres (see Figure S3). Thus, after the adsorption of lysozyme on carboxyl functionalized Fe3O4@ F-SiO2 microspheres, the polymerization of dopamine (acted as both functional monomer and cross-linker) was carried out at
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RESULTS AND DISCUSSION Preparation and Characterization of Magnetic Lysozyme-Imprinted Fe3O4@F-SiO2@PDA Microspheres. To obtain an easily separated mesoporous SiO2 matrix with a suitable space for the imprinting and diffusion of biomacromolecules, a core−shell structured magnetic mesoporous silica microspheres with a pore size of 12 nm were designed as illustrated in Scheme 1. First, hydrophilic and stable Fe3O4 nanoparticles with a mean diameter of 130 nm were synthesized according to the classical solvothermal method (Figure 1a). Selected area electron diffraction (SAED) (Figure 1b) shows polycrystalline-like diffraction, suggesting Fe3O4 consists of many magnetite nanocrystals. The Fe3O4 nanoparticles were then dispersed into a precursor system for silica particles with a fibrous morphology referring to our previous work.35 The following reaction process can be briefly summarized as the hydrolysis and condensation of TEOS on the surface Fe3O4 nanoparticles catalyzed with urea. The silica shell will grow into a fibrous porous morphology directed by CTAB. Thus, after the hydrolysis of TEOS, uniform microspheres with porous surface D
DOI: 10.1021/acsami.5b10126 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 3. (a) Temperature variations of the Tris-HCl buffer containing MIP-lysozyme microspheres with different concentrations with the increase of the irradiation time of NIR laser (808 nm, at a power density of 3.3 W/cm2). The Inset is the photothermal image of Tris-HCl buffer containing 200 μg/mL of MIP-lysozyme under the NIR laser irradiation. (b) Temperature variation of the Tris-HCl buffer containing 120 μg/mL of MIPlysozyme over five laser irradiation ON/OFF cycles.
consistent with the results obtained from the curves in Figure 3a. Furthermore, the photothermal effect has nearly no loss after at least five cycles as given in Figure 3b. The results also can also confirm that PDA layer has been successfully coated on Fe3O4@F-SiO2 microspheres. The excellent photothermal effect of MIP-lysozyme microspheres provides the prerequisite for NIR-light-responsive release of the rebinding lysozyme of the MIP-lysozyme microspheres. The magnetism of the prepared microsphere was evaluated by vibrating sample magnetometer (VSM) to get the saturation magnetization (Ms) values, as shown in Figure 4. The
room temperature, which is beneficial to protect the quaternary structure of lysozyme. In this work, the pH of the reaction system was set as 8.0. The reaction rate was controlled slowly by continuously purging air into the system, resulting in the preparation of a PDA layer on Fe3O4@F-SiO2 microspheres (Fe3O4@F-SiO2@PDA), i.e. MIP-lysozyme, as shown in Figure 1e, f, which show that the formed PDA layer was uniformly coated along the fibrous SiO2 because the pores inside and on the surface of the microspheres are still obvious. The size of MIP-lysozyme microspheres is a little larger than that of primary Fe3O4@F-SiO2 microspheres (Figure 1c). The difference should be the thickness of PDA layer. It ranges several to hundreds nanometers. The average diameter of MIP-lysozyme microsphere is 445 nm, which means that the average thickness of PDA imprinting layer is 45 nm. This indicates that the protein can easily access to the imprinting sites anywhere in the thin PDA layer. The surface area of MIP-lysozyme measured by BET method is 309 m2/g (Figure 2a), smaller than that of Fe3O4@F-SiO2. As another comparison, PDA coated Fe3O4@ F-SiO2 microspheres without imprinted by lysozyme, i.e., NIP microspheres, have a smaller BET specific surface area of 244 m2/g than MIP-lysozyme. At the same time, the intensity of the pores with diameter of 12 nm in MIP-lysozyme and NIP also decreases gradually (see Figure 2b), which indicates PDA only attached to the surface of the pores of Fe3O4@F-SiO2 microspheres to form a thin imprinting layer, rather than completely blocked those pores. The photothermal effect of MIP-lysozyme microsphere was also investigated.29,30 Figure 3a shows the temperature changes of the Tris-HCl buffer containing different content of MIPlysozyme microspheres after being irradiated by NIR laser (808 nm, 3.3 W/cm2) for 5 min. The Tris-HCl buffer containing Fe3O4@F-SiO2 microspheres (200 μg/mL) shows the weak temperature changes during the irradiation of NIR laser, while those containing MIP-lysozyme microspheres exhibited the remarkable photothermal effect. Moreover, the photothermal effect will be enhanced with the increase of the content of MIPlysozyme microspheres. For example, the temperature of the solution containing 200 μg/mL of MIP-lysozyme increased from 26 to 40.5 °C within 5 min’s irradiation of NIR laser. Meanwhile, the photothermal effect can also be recorded by the photothermal images of the aqueous solutions containing 200 μg/mL of MIP-lysozyme under the same NIR laser irradiation (Figure 3a Insets and Figure S4). The red-pink area near the facula indicates the temperature is around 40 °C, which is
Figure 4. VSM curves of (a) Fe3O4, (b) Fe3O4@F-SiO2, and (c) MIPlysozyme microspheres. Inset: the digital photo of the appearance of Tris-HCl buffer containing MIP solution with and without magnet.
hysteresis loops show that there is almost no magnetic hysteresis, indicating that Fe3O4 nanoparticles, Fe3O4@FSiO2, and MIP-lysozyme microspheres have superparamagnetic behavior. The Fe3O4 nanoparticles exhibit the highest magnetization of 79 emu/g. After the coating of F-SiO2 and PDA layer, the magnetization decreases to 58 and 36 emu/g respectively due to the presence of the nonmagnetic parts.37 But still, the MIP-lysozyme microspheres can be separated rapidly within 30 s by a magnet (Figure 4 inset). Once the external magnetic field E
DOI: 10.1021/acsami.5b10126 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 5. (a) Adsorption isotherms and (b) adsorption kinetics of lysozyme on MIP-lysozyme and NIP microspheres.
Figure 6. ITC data of lysozyme binding onto (a) MIP-lysozyme and (b) NIP microspheres at 298 K in Tris-HCl buffer solution.
is removed, the MIP-lysozyme microspheres can be quickly redispersed into homogeneous dispersion upon a slight shake. Imprinting Performance of MIP-Lysozyme Microspheres. The binding isotherms of lysozyme onto MIPlysozyme and NIP microspheres were investigated under various concentration of lysozyme (0.1−1.5 mg/mL) at 25 °C. As shown in Figure 5a, MIP microspheres have a balanced adsorption as high as 710 mg/g when the lysozyme concentration is 1.4 mg/mL, indicating that the imprinted sites have been occupied totally by lysozyme at this condition. At the moment, the adsorption capacity of NIP microspheres is only 128 mg/g, much lower than that of MIP-lysozyme microspheres, indicating the MIP-lysozyme microspheres exhibit a better specific adsorption toward lysozyme. The binding kinetics of MIP-lysozyme and NIP microspheres were studied, and the results were shown in Figure 5b. It can be seen that the adsorption capacity of both MIP-lysozyme and NIP microspheres increase greatly within 10 min. The adsorption process will be completed within 30 min. The rapid binding kinetics of MIP-lysozyme and NIP microspheres implies that most of the affinity sites are located on the surface of PDA layer, where target lysozyme molecules can easily access to.
Thermodynamic parameters of binding process, such as knowledge of rates, affinities, and stoichiometries of protein association with nanoparticles,38,39 play an important role to understand the most basic features of the interaction between nanoparticles and template proteins. Hence, ITC was developed to systematically study the interaction between the lysozyme and MIP-lysozyme microspheres in this work. Figure 6 shows the ITC curves when the lysozyme solution was titrated into the dispersion containing MIP-lysozyme or NIP microspheres. From the raw ITC data, a single set of independent binding sites model was sufficient to achieve a good fit of the calorimetric data. It can be seen that the MIPlysozyme microsphere shows more binding sites (N) than that of NIP microsphere because the more recognition sites were reserved for MIP-lysozyme microsphere after extracted with elution. At the same time, the affinity constant (K) of MIPlysozyme microsphere is 100 times higher than that of NIP microsphere, which is attributed to the specific interaction between the lysozyme and complementary cavity. The heat signals in Figure 6 for MIP-lysozyme and NIP are negative, which indicates that the binding behaviors between lysozyme and microspheres is an exothermal process. Furthermore, the absolute values of ΔH are far higher than F
DOI: 10.1021/acsami.5b10126 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces TΔS in both MIP-lysozyme and NIP groups according to the data in Figure 6, indicating hydrogen bond and electrostatic interaction dominate the recognition process.40 Second, compared with the NIP group, the entropy change in MIPlysozyme group is much less than zero (ΔS ≪ 0), which means the conformation of lysozyme is greatly limited by the complementary cavities when embedded onto the MIPlysozyme microspheres. Therefore, we can conclude that the adsorption process between lysozyme and NIP microsphere simply dominated by physical interaction, i.e., hydrogen bond and electrostatic interaction. In addition to the above physical interaction, the lysozyme was restricted by the well-defined 3D structure of MIP-lysozyme microspheres. Lastly, the ΔG < 0 for both MIP-lysozyme and NIP group shows the binding between protein and microsphere is a spontaneous process. Several proteins including trypsin, BSA, BHb, and Cyt c were chosen as the controls to confirm the selectivity adsorption of MIP-lysozyme microspheres. Figure 7 shows the adsorption
microspheres show a low binding capacity toward each reference protein, i.e., 90 and 74 mg/g for trypsin, 14 and 21 mg/g for BSA, 34 mg/g, 30 mg/g for BHb, 99 and 93 mg/g for Cyt c. The corresponding IFs for lysozyme, trypsin, BSA, BHb, and Cyt c are 4.12, 1.22, 0.67, 1.12, and 1.06 respectively. Hence, the selectivity coefficient (α) of MIP-lysozyme toward trypsin, BSA, BHb, Cyt c can be calculated to be 3.38, 6.15, 3.68, and 3.89, respectively. The high selective adsorption performance of MIP-lysozyme toward lysozyme can be attributed to two major reasons. On one hand, the adsorption capacity of the specific protein is dependent on the strength of the physical interactions between dopamine units and the protein, which is affected by the conformation of the protein.13 It can be induced from the adsorption capacity of each protein on NIP (Figure 7) that lysozyme has a slightly stronger interaction with dopamine than trypsin and Cyt c which have much stronger interaction with dopamine than BSA and BHb. On the other hand, the volume effect of the complementary cavities on MIP microspheres will also make a great contribution to the adsorption capacity of proteins. It is seen that the adsorption capacity of BSA on MIP-lysozyme is even lower than that on NIP because the existence of complementary cavities reduces the contact area of BSA to MIP microspheres, but BSA cannot enter into the complementary cavities due to its much larger size (ca. 7 nm) than lysozyme (ca. 4.3 nm). Trypsin, BHb, and Cyt c have a comparable size (ca. 3.8 nm, ca. 5.9 nm, and ca. 3.3 nm, respectively) with lysozyme so that their adsorption capacities on MIP microspheres are a little higher than those on NIP. The lysozyme has the exactly matched size to the complementary cavities so that its adsorption capacity on MIP microspheres is far above all the other four proteins’. This excellent selective adsorption ability makes the prepared MIP-lysozyme have the potential application on the extraction of lysozyme from biological environments. Application of MIP-Lysozyme in the Extraction and Controlled Release of Lysozyme. Before applying the MIPlysozyme microspheres in a biological environment, its biosecurity should be evaluated first. Thus, the MTT assay of MIP-lysozyme microspheres on HeLa cells was devoted (see Figure S5). It shows that MIP-lysozyme microspheres have a good biosecurity at a concentration ranging from 50 to 500 μg/ mL. Thus, the extraction of lysozyme using MIP-lysozyme from egg white was investigated, followed by the NIR-light triggered release of the rebinding lysozyme, as illustrated in Scheme 2. The MIP-lysozyme microspheres were first incubated in egg white solution. The SDS-PAGE was used here to visualize the
Figure 7. Selective adsorption of MIP-lysozyme and NIP toward different proteins.
performance of MIP-lysozyme and NIP microspheres on different proteins. The MIP-lysozyme microspheres show the highest binding capacity for lysozyme (460 mg/g), but only 112 mg/g on NIP. It is noted that both MIP-lysozyme and NIP
Scheme 2. Schematic Illustration of the Rebinding and Release Processes of Imprinted Microspheres
G
DOI: 10.1021/acsami.5b10126 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 8. (a) SDS-PAGE analysis of egg-white. Lane 1: Protein molecular weight marker. Lane 2:20-fold dilution of egg white before treatment. Lane 3: Remaining 20-fold dilution of egg white extracted by MIP-lysozyme microspheres. Lane 4: Remaining 20-fold dilution of egg white extracted by NIP microspheres. Lane 5: eluate from the rebound MIP-lysozyme microspheres in egg white dilution. (b) MALDI-TOF MS spectra of pure lysozyme, diluted egg white, and releasers from MIP-lysozyme microspheres.
purity of the extracted protein, as shown in Figure 8a. The band of lysozyme is clear in diluted egg white solution (lane 2, 14.4 kDa, according to the MALDI-TOF MS spectra of pure lysozyme in Figure 8b). When the diluted egg white solution was extracted with MIP-lysozyme microspheres, the band of lysozyme disappeared in lane 3, implying the lysozyme in egg white solution had been extracted clearly by MIP-lysozyme microspheres. As a comparison, the band of lysozyme is still obvious in lane 4 using NIP microspheres as the extractant. The above results confirm that the MIP-lysozyme microspheres show high adsorption capacity to lysozyme even in a real sample of egg white solution. In addition, the brightness of the bands for other proteins in egg white hardly changed after the extraction of MIP-lysozyme microspheres, indicating an excellent selective adsorption property of MIP-lysozyme microspheres. At the same time, the rebound MIP-lysozyme microspheres were eluted with the Tris-HCl buffer containing 5 vol % acetic acid. The elution was also analyzed with SDSPAGE (Line 5). Clearly, only lysozyme band appeared in lane 5, thereby suggesting the high selective adsorption ability of MIP-lysozyme microspheres on lysozyme. To recycle the rebinding proteins, the desorption of the rebinding lysozyme from the MIP-lysozyme microspheres had also been investigated. Generally, the rebinding protein can hardly be released at a normal condition since the rebinding molecules have strong interaction with the imprinted matrix by specific adsorption. In this work, the prepared MIP-lysozyme microspheres show a remarkable photothermal conversion effect under the irradiation of NIR light so that the NIR-laser triggered release of lysozyme from the rebound MIP-lysozyme microspheres were investigated and recorded in Figure 9. The NIR laser trigger was operated by laser ON for 5 min, and then laser OFF. When the laser ON, the temperature of the release system increased rapidly from 26 to 38 °C, resulting in a burst of release of lysozyme, i.e., an abrupt rise in the cumulative release percentage. However, the cumulative release percentage increased very slowly when the laser powered OFF. At last, the total release ratio within 8 h reached 60%. As a comparison, the total release ratio without irradiation of NIR laser is less than 35%. The bioactivity of the released lysozyme was also estimated via its decomposition ability of E. coli because lysozyme is often
Figure 9. Lysozyme release curves of MIP under NIR laser irradiation (808 nm, 3.3 W cm−2) for (a) different on/off cycles and (b) the control group released at the same condition without NIR irradiation.
used to lyse bacteria by hydrolyzing the peptidoglycan presents in the cell walls. As a control group, the E. coli was first incubated in the dispersion containing MIP-lysozyme microspheres. The system was irradiated with NIR light for 5 min, and stood for further 60 min to observe the effect of heat on the structure of E. coli (Figure 10b). At this condition, E. coli. nearly has the same morphology with its original status shown in Figure 10a, indicating the mild heat has few influence on E. coli. On the contrary, Figure 10c shows a slightly destroyed shape of E. coli at the presence of the rebound MIP-lysozyme microspheres standing for 65 min without the irradiation of NIR laser. However, the severely destroyed morphology of E. coli is observed at the presence of rebound MIP-lysozyme microspheres under the irradiation of NIR light for 5 min (Figure 10d), caused by the burst release of lysozyme. This means the bioactivity of the released lysozyme has no loss during the adsorption and the release processes in the MIPlysozyme microspheres. H
DOI: 10.1021/acsami.5b10126 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 10. TEM images of E. coli under different environments. (a) original E. coli; (b) MIP-lysozyme microspheres without the irradiation of NIR light; (c) rebound MIP-lysozyme microspheres without the irradiation of NIR light; (d) rebound MIP-lysozyme microspheres under the irradiation of NIR light.
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Notes
CONCLUSION The magnetic core−shell structured lysozyme-imprinted Fe3O4@F-SiO2@PDA microspheres (MIP-lysozyme) have been successfully fabricated. According to the N2 adsorption− desorption isotherms analysis, the Fe3O4@F-SiO2 microspheres possess a high surface area of 570 m2/g and the mesopore size of 12 nm. A lysozyme-imprinted PDA layer was coated on Fe3O4@F-SiO2 microspheres through the polymerization of dopamine on the surface of Fe3O4@F-SiO2 microspheres at room temperature. The prepared MIP-lysozyme microspheres also exhibit a good magnetism of 40 emu/g so that they can be easily separated by a magnet. Further investigations indicate the saturation adsorption capacity of lysozyme on MIP-lysozyme microspheres can attain to 700 mg/g within 30 min. The MIPlysozyme microspheres also show an excellent selective adsorption of lysozyme (IF = 4.12). The binding thermodynamic parameters studied by ITC prove that the lysozyme should be restricted by the well-defined 3D structure of MIPlysozyme microspheres. The MIP-lysozyme can extract lysozyme efficiently from real egg white. Owing to the enhanced photothermal effect of PDA layer, the MIP-lysozyme microspheres show the controlled release property triggered by NIR laser. The released lysozyme molecules still maintain good bioactivity, which can efficiently decompose the E. coli. Therefore, this work provides a novel strategy to build practical NIR-light-responsive MIPs for the extraction and application of biomacromolecules.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51103143, 51173175, 51473152, and 51573174), the Fundamental Research Funds for the Central Universities (WK2060200012, WK3450000001), and Foundation of Anhui Key Laboratory of Tobacco Chemistry (China Tobacco Anhui Industrial CO., LTD) (2014126).
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b10126. FTIR spectra of the products in each step; zeta-potential of Fe 3O 4 @F-SiO2 microspheres before and after carboxylation functionalization; adsorption capacity of Fe3O4@F-SiO2 microspheres for lysozyme before and after carboxylation functionalization; photothermal images of Tris-HCl buffer containing MIP-lysozyme under the NIR laser irradiation; cell toxicity of MIP-lysozyme microspheres.(PDF)
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REFERENCES
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DOI: 10.1021/acsami.5b10126 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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