polypyrrole microspheres with near-infrared-light-controlled release property

Journal of Colloid and Interface Science 548 (2019) 37–47

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Journal of Colloid and Interface Science journal homepage: www.elsevier.com/locate/jcis

Regular Article

Fabrication of inverse-opal lysozyme-imprinted polydopamine/ polypyrrole microspheres with near-infrared-light-controlled release property Wenxiu Yang a, Kun Zeng a, Jiaxin Liu a, Lechen Chen a, Mozhen Wang a,⇑, Shengchi Zhuo b, Xuewu Ge a,⇑ a b

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, PR China Eternal Specialty Materials (Zhuhai) Co., Ltd., Zhuhai, Guangdong 519050, PR China

g r a p h i c a l a b s t r a c t

a r t i c l e

i n f o

Article history: Received 30 January 2019 Revised 4 April 2019 Accepted 6 April 2019 Available online 8 April 2019 Keywords: Molecular imprinting Lysozyme Polydopamine Polypyrrole Inverse-opal microspheres NIR-light response release

a b s t r a c t The combination of the molecular imprinting technology and porous materials is a promising way to obtain high-efficient selective adsorption and separation materials for bioactive macromolecules. In this work, we developed a novel approach to prepare near-infrared (NIR)-light-response inverse-opal lysozyme (Lyz)-imprinted polydopamine/polypyrrole (IO-PDA/PPy-MIP) composite microspheres using micron-sized SiO2 colloidal crystal microspheres as the sacrificed template. The pore size of the IOPDA/PPy-MIP microspheres can be tuned from 200 to 800 nm by the size of silica nanoparticles which self-assemble to form the template SiO2 colloidal crystal microspheres. The IO-PDA/PPy-MIP microspheres show a rapid selective adsorption ability for Lyz due to the inverse-opal macroporous structure. The adsorption capacity exceeds 800 mg/g within 20 min, and the imprinting factor is as high as 24. The bound Lyz molecules can be released rapidly from IO-PDA/PPy-MIP microspheres triggered by the irradiation of NIR laser and remain enough bioactivity to decompose Escherichia coli efficiently. The prepared IO-PDA/PPy-MIP microspheres also exhibit excellent structure stability and good recyclability. The adsorption capacity can remain up to 90% of the initial value after 5 times recycle. This work provides not only a method to prepare novel NIR-light-response inverse-opal macroporous molecularly imprinted microspheres, but also a new perspective on the design of selectively separation materials for the fast, high-efficient recognition and separation of biomacromolecules. Ó 2019 Elsevier Inc. All rights reserved.

1. Introduction Biomacromolecules have tremendous application values due to their important and unique physiological functions [1]. Therefore, ⇑ Corresponding authors. E-mail addresses: [email protected] (M. Wang), [email protected] (X. Ge). https://doi.org/10.1016/j.jcis.2019.04.021 0021-9797/Ó 2019 Elsevier Inc. All rights reserved.

the research in the extraction and purification of biomacromolecules has always been a basic and challengeable subject in life sciences [2–4]. Many methods can be applied to separate biomolecules, such as ion-exchange chromatography [5], electrophoresis method [6], salt fractionation [7], high performance liquid chromatography [8], and gel chromatography [9]. But the selectivity and high cost remain the top concern in this field [10,11]. The

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development of high-efficient adsorption/separation materials towards biomacromolecules has been a pursuing goal for the materials scientists [12–14]. In recent years, molecularly imprinted polymers (MIP) have been widely studied for their expected excellent selectivity and accessibility [15–17]. MIP can be used as smart reagents with the function of molecular recognition [18], separation [19], adsorption [20], electrochemical sensing [21], enzyme mimics [22] and solid phase extraction [23]. Since large biomolecules is more difficult to diffuse compared to small molecules, the capacity of the conventional MIP for biomacromolecules is very limited because plenty of the recognition sites in the deeper inside of MIP matrix can be hardly utilized effectively due to the strong diffusion hindrance [24]. The application of MIP for the adsorption/separation to biomacromolecules is a hot issue in studies [25]. To improve the efficiency of MIP, surface imprinting method has been proposed [26–28]. For example, Li et al. prepared cross-linked polyacrylamide nanowires with bovine Hb-imprinted surface layer [29]. These surface imprinted sites of the nanowires have a good accessibility toward the target protein molecules. The amount of bovine Hb bound on the imprinted nanowires is seven times more than those bound on the control nanowires. Recently, Chen et al. developed a catalase-imprinted polydopamine layer supported on a core-shell structured magnetic Fe3O4/Fe@mesopours fibrous silica composite nanoparticles [30]. The adsorption capacity for the catalase can reach 399 mg g
1. The key to improve the efficiency of surface imprinting is to create a specific surface area as large as possible so as to obtain a maximum adsorption quantity. Inverse opal structural materials (IOM), with advantages of controllable and ordered macropore structures (pore size > 50 nm), large surface areas and capacity, are widely used for adsorption and separation [31–34]. On account of the interconnected ordered macroporous structure, target molecules, even large biomolecules such as protein, can diffuse freely and rapidly through the matrix so that they can easily access the huge surface area inside the IOM. The molecularly imprinted IOM have been referred in some literatures [35,36]. For instance, Wang et al. proposed a horseradish peroxidase imprinted inverse opal structural polyethylene glycol diacrylate and 4-vinylphenylboronic acid composite hydrogel [37]. The composite hydrogel can specifically adsorb and detect horseradish peroxidase as low as the concentration of 1 ng/mL with the adsorption capacity of 24.62 nmol/g. For the imprinting and recognition of biomacromolecules, the biocompatibility of the IOM matrix is also a crucial factor besides the selectivity because the bound biomolecules need to maintain their biological activity in some cases [38]. However, few researches on the preparation of IOM using biocompatible raw materials have been reported. On the other hand, biomacromolecules-imprinted IOM with excellent controlled-release and recycling properties are also necessary so as to achieve the precise separation and utilization of the target biomolecules. But in general, the excellent adsorption and recognition performance of MIP in turn make the adsorbates difficult to be washed off. External forces such as heat, light, change of pH value, can be employed to trigger the release process, which requires the matrix material of MIP to have the ability of responding to those external environmental changes. Based on the above considerations, efficient MIP feature for biomacromolecules should involve large specific surface area, smooth molecular diffusion channels, biosafety, and the function of environmental response to achieve the release of the identified biomacromolecules efficiently. Polydopamine (PDA) and polypyrrole (PPy) exhibit remarkable near-infrared (NIR)-light photothermal effect and have good biocompatibility [39,40], which can be expected to be used as the skeleton materials for the adsorption/ separation of protein with a NIR-light-controlled release property [30,41,42]. But there is no work on the molecularly imprinted

PDA- or PPy-based IOM till now. Herein, we developed a sacrificed hard template method to fabricate novel inverse-opal lysozyme (Lyz)-imprinted polydopamine and polypyrrole (PDA/PPy) composite microspheres. The inverse opal PDA/PPy composite skeleton is formed by the in situ oxidative polymerization of dopamine (DA) and pyrrole (Py) in the gap of the sacrificed SiO2 colloidal crystal microspheres template. The prepared inverse-opal lysozymeimprinted polydopamine and polypyrrole (IO-PDA/PPy-MIP) microspheres have a specific surface area of 59.75 m2/g and an imprinting factor to Lyz up to 24, resulting in a fairly high adsorption capacity for Lyz of more than 800 mg/g. The adsorbed Lyz molecules can be released controllably from IO-PDA/PPy-MIP microspheres under the irradiation of NIR laser and remain enough bioactivity to decompose Escherichia coli efficiently. The prepared IO-PDA/PPy-MIP microspheres also exhibit excellent stability and recyclability. The adsorption capacity can remain up to 90% of the initial value after the IO-PDA/PPy-MIP microspheres were reused for 5 cycles. This work indicates that the IO-PDA/PPy-MIP microspheres are promising high-efficient adsorbents in the field of the extraction and recycling of biomacromolecules. 2. Experimental section 2.1. Materials Analytical reagents including hydrofluoric acid (HF  40%), ethanol, tetraethyl orthosilicate (TEOS), sodium dodecyl sulfate (SDS), trismetyl aminomethane (Tris), hydrochloric acid (HCl, 36– 38%), ammonia (25–28%), hexane, pyrrole, ferric chloride hexahydrate (FeCl36H2O), ethylenediaminetetraacetic acid (EDTA), and acetic acid were all obtained from Sinopharm Chemical Reagent Co., Ltd., and used as received. Tris-HCl buffer solution (50 mM) with pH 7.4 was prepared by mixing 42 mL of 0.1 M hydrochloric acid and 50 mL of 0.1 M Tris aqueous solution, followed by diluting with deionized water to 100 mL. Lysozyme (Lyz, Mw 14.3 kDa, 5000 U/mg), kerosene (reagent-grade), horse radish peroxidase (HRP, Mw 44 kDa, 160 U/mg), cytochrome c (Cyt c; Mw 12.4 kDa, 95%), and dopamine hydrochloride (98%) were supplied by Aladdin Industrial Corporation. Hypermer 2296 (industrial premium grade) was bought from Croda. Bovine serum albumin (98%, BSA, Mw 67 kDa) was purchased from J&K Scientific Co., Ltd. Trypsin (Trs, Mw 23.3 kDa, 1500 U/mg) was purchased from SigmaAldrich Co., LLC. The above reagents were used as received. The protein lotion was prepared by dissolving acetic acid and SDS in water at concentrations of 0.833 M and 0.347 M, respectively. Escherichia coli (E. coli) was provided by Anhui Medical University, Hefei, China. Deionized water was utilized in all experiments. 2.2. Synthesis of SiO2 nanoparticles Monodisperse silica nanoparticles were prepared by a modified Stöber process [43,44]. Typically, a mixture containing 1.6 mL of TEOS, 40 mL of ethanol, and 4 mL of ammonia was magnetically stirred at room temperature for 24 h. Then, monodispersed SiO2 nanoparticles with a size of 200 nm were separated centrifugally from the reaction system, washed with ethanol and water for three times and dried at 50 °C in oven. 2.3. Preparation of SiO2 colloidal crystal microspheres Hypermer 2296 (0.18 g) was dissolved in 6 mL of kerosene. Then 0.6 mL of an aqueous dispersion of the as-prepared SiO2 nanoparticles (0.18 g) was added in dropwise. The mixture was sealed and shaken by hand for 10 min to form an inverse emulsion. The emulsion was poured into a culture dish, and stood at 50 °C for

W. Yang et al. / Journal of Colloid and Interface Science 548 (2019) 37–47

24 h to evaporate water and most of kerosene. SiO2 colloidal crystal microspheres were finally collected from the bottom of the culture dish, and rinsed with hexane to remove the remaining kerosene [45]. 2.4. Preparation of IO-PDA/PPy-MIP microspheres SiO2 colloidal crystal microspheres (200 mg) were immersed in Tris-HCl buffer solution (3 mL, 10 mM, pH 7.4) containing 15 mg of Lyz and 20 mg of dopamine hydrochloride. The system was magnetically stirred gently under vacuum (
0.1 MPa) for 15 min to let Lyz and DA molecules permeate into the voids of silica colloidal crystal microspheres. Then the treated silica colloidal crystal microspheres were separated centrifugally, washed with ethanol, and then dispersed in 2 mL of Py. The system was magnetically stirred gently under vacuum (
0.1 MPa) for 15 min again. The treated silica colloidal crystal microspheres were separated centrifugally. The 13C nuclear magnetic resonance (NMR) spectra and extreme matrix-assisted laser desorption/Ionization time of flight mass spectra (MALDI-TOF-MS) of the residual liquid in Fig. S1 indicated that no dopamine and no lysozyme leaked out from the silica colloidal crystal microspheres during the perfusion of Py. The silica colloidal crystal microspheres containing Lyz, DA and Py were washed with deionized water, and then dispersed in 8 mL of FeCl3 aqueous solution (0.694 M) in an ice-water bath for 12 h. The produced silica colloidal crystal microspheres filled with Lyz, PDA and PPy (SiO2/Lyz/PDA/PPy) were separated centrifugally, washed with deionized water, and then dispersed into 8 mL of hydrofluoric acid. The dispersion was heated to 40 °C in a water bath, and kept for 10 h to remove the silica templates to obtain inverse opal structural Lyz, PDA and PPy (IO-Lyz/PDA/PPy) microspheres. Then the IO-Lyz/PDA/PPy microspheres were separated centrifugally, washed with deionized water and the protein lotion to remove the entrapped Lyz molecules [17]. Finally, the obtained IO-PDA/ PPy-MIP microspheres were centrifuged, washed with deionized water, and freeze-dried by a freeze dryer (BY-FD-1A-50). As a control, non-imprinted inverse-opal PDA/PPy microspheres (IO-PDA/PPy-NIP) were also prepared according to the above same procedure in the absence of Lyz. 2.5. Characterization The morphology of the prepared microspheres was observed by transmission electron microscope (TEM, Hitachi H-7650, 100 kV) and scanning electron microscope (SEM, JSM6700F, 5.0 kV). Samples were dispersed in ethanol, and then the dispersion was dropped onto copper grids for TEM characterization. Ultrathin sections for TEM observation were prepared by Ultramicrotome (Leica UC7FC7). The samples for SEM observation were treated by spraygold. The UV–vis absorption spectra were measured by Shimadzu spectrophotometer (UV-3600). The secondary structures of proteins were determined in Tris-HCl buffer solution using circular dichroism (CD) spectrometer (Jasco-810, Japan). Fourier transform infrared (FTIR) spectra were obtained on a Nicolet 8700 FT-IR spectrometer in the range from 4000 to 400 cm
1 using KBr pellets. Solid 13C NMR spectra were obtained using a Bruker AVANCE AV III 400WB NMR spectrometer with tetramethylsilane as internal standard. The NMR signal was received with a spin rate of 14 kHz to determine the spinning side bands. The cross-polarized time was 2 ms and the delay time was 2 s. 2.6. Adsorption kinetics of Lyz on IO-PDA/PPy-MIP and IO-PDA/PPyNIP microspheres Lyz (12 mg) and IO-PDA/PPy-MIP (or IO-PDA/PPy-NIP) microspheres (12 mg) were mixed in 12 mL of Tris-HCl buffer in a cen-

39

trifuge tube on a shaker table. One milliliter of the mixture was sampled out at every certain time interval to measure the concentration of Lyz by UV–vis spectroscopy method according to the absorbance at 280 nm for Lyz after the microspheres were rapidly separated by centrifugation. The working curve is listed in Fig. S2a. This experiment was repeated 5 times to estimate underlying error shown by error bars. 2.7. Adsorption thermodynamics of Lyz on IO-PDA/PPy-MIP and IOPDA/PPy-NIP microspheres IO-PDA/PPy-MIP (or IO-PDA/PPy-NIP) microspheres (2 mg) were firstly dispersed into 2 mL of Tris-HCl buffer of Lyz at different concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 mg/mL). The systems were kept on a shaker table at 25 °C for 4 h. After that, the microspheres were separated by centrifugation. The concentrations of Lyz in the supernatants were determined according to the UV–vis absorbance at 280 nm for Lyz according to the working curve (Fig. S2a). This experiment was repeated 5 times to estimate underlying error shown by error bars. 2.8. Cycling performance of IO-PDA/PPy-MIP microspheres IO-PDA/PPy-MIP microspheres (20 mg) were added in 20 mL of Tris-HCl buffer of Lyz (1 mg/mL) in a centrifuge tube on a shaker table. After the dispersion was gently shaken at room temperature for 2 h, the microspheres were separated by centrifugation. The concentration of Lyz in the supernatant was determined according to the absorbance at 280 nm for Lyz according to the working curve (Fig. S2a). Then the microspheres were washed with the protein lotion to remove all the adsorbed Lyz [17]. The washed IO-PDA/ PPy-MIP microspheres were centrifuged, rinsed with deionized water, and then re-dispersed in 20 mL of Tris-HCl buffer of Lyz (1 mg/mL) again to investigate their reuse performance. 2.9. Imprinting performance of IO-PDA/PPy-MIP microspheres The IO-PDA/PPy-MIP (or IO-PDA/PPy-NIP) microspheres (2 mg) were dispersed in 2 mL of Tris-HCl buffer containing 1 mg/mL of each single kind of protein (Lyz, BSA, or Cyt c). After the dispersion was gently shaken at room temperature for 2 h, the microspheres were centrifuged. The protein concentrations in the supernatants were determined by the absorbance at 280 nm for Lyz, 278 nm for BSA, and 410 nm for Cyt c on the UV–vis spectra according to the corresponding working curves (Fig. S2). This experiment was repeated 5 times to estimate underlying error shown by error bars. The imprinting performance of IO-PDA/PPy-MIP can be evaluated by the imprinting factor (IF), which is defined as:

IF ¼ Q MIP =Q NIP

ð1Þ

where QMIP and QNIP are the adsorption capacities of Lyz on IO-PDA/ PPy-MIP and IO-PDA/PPy-NIP microspheres, respectively [25]. The selectivity coefficient (a) is used to evaluate the selective recognition ability of IO-PDA/PPy-MIP, which is defined as the ratio of the IF for Lyz (IFlyz) with respect to that for the competitive protein (IFcomp. protein) [25]:

a ¼ IFlyz =IFcomp: protein

ð2Þ

The selective adsorption ability of IO-PDA/PPy-MIP was also investigated by SDS polyacrylamide gel electrophoresis (SDSPAGE). IO-PDA/PPy-MIP (or IO-PDA/PPy-NIP) microspheres (3 mg) were vortex mixed with 3 mL of Tris-HCl buffer solution containing three kinds of proteins, Lyz, HRP, and Trs (0.1 mmol/L for each protein). Then, the mixture was mildly shaken at 25 °C for 2 h. After that, the microspheres were centrifuged. The supernatant was used for SDS-PAGE analysis.

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2.10. NIR photothermal conversion performance of IO-PDA/PPy-MIP microspheres The temperature of the Tris-HCl buffer dispersion of IO-PDA/ PPy-MIP microspheres was measured by an online thermocouple (TP-01, Taiwan) with an accuracy of 0.1 °C immersed in the solution every 10 s under the irradiation of an 808 nm laser produced by a semiconductor laser device (ADR-1860, China) with externally adjustable power. 2.11. NIR-Light triggered release of Lyz bound on IO-PDA/PPy-MIP microspheres The IO-PDA/PPy-MIP microspheres (2 mg) were dispersed in 2 mL of Tris-HCl buffer containing 1 mg/mL of Lyz. After the dispersion was gently shaken by shaking table at room temperature for 2 h to reach equilibrium adsorption, the microspheres were centrifuged and re-dispersed in 2 mL of Tris-HCl buffer. After shaking by shaking table for 30 min, the microspheres were centrifuged and re-dispersed in 2 mL of Tris-HCl buffer. The Lyz concentration of the supernatant was measured by UV–vis spectrophotometry to determine the release amount of Lyz from the IO-PDA/PPy-MIP microspheres at room temperature. The microspheres dispersion was irradiated with NIR laser (808 nm, 3.3 W/cm2) for 5 min, then the microspheres were centrifuged. The Lyz concentration of the supernatant was measured by UV–vis spectrophotometry to determine the release amount of Lyz from the IO-PDA/PPy-MIP microspheres under the exposure of NIR light. The above release processes of Lyz at room temperature and under the exposure of NIR light were repeated for another 4 times. 3. Results and discussion 3.1. Preparation and characterization of IO-PDA/PPy-MIP microspheres In order to obtain IO-PDA/PPy-MIP microspheres with high efficiency and capacity for biomacromolecules, micron-sized SiO2 colloidal crystal microspheres were first prepared to be used as hard templates by water-evaporation-induced self-assembly of SiO2 nanoparticles in an inverse emulsion, as shown in Scheme 1 and Fig. 1. Uniform SiO2 nanoparticles (Fig. 1a) were synthesized by Stöber method with a diameter of around 200 nm and good hydrophilicity due to the abundant hydroxyl groups on the surface (Fig. S3). They tend to locate in water droplets in an inverse emulsion stabilized with Hypermer 2296. During the evaporation of water, the SiO2 nanoparticles distributed in a single water droplet assemble gradually driven by van der Waals forces and hydrogen-bond interaction, forming self-standing colloidal crystal microspheres with the diameters of 4–15 lm, as presented in Fig. 1b. After the polymerization of DA and Py infiltrated in the voids of the SiO2 colloidal

crystal microspheres in the presence of Lyz and the following etching processes in HF solution and the protein lotion in sequence (Scheme 1), self-standing IO-PDA/PPy-MIP microspheres were obtained, as exhibited in Fig. 1c. The IO-PDA/PPy-MIP microspheres were 4–15 lm in diameter, just in accordance with the template colloidal crystal microspheres. The SEM image of the cross section of IO-PDA/PPy-MIP microspheres (Fig. 1d) and TEM image of the corresponding ultrathin sections (Fig. 1e) show the inner inverse opal structure clearly. The pore diameter of the IOPDA/PPy-MIP microspheres is about 200 nm, approximately in line with the size of the original silica nanoparticles, indicating that the polymeric wall material formed in the gap of the colloidal crystal microspheres has enough strength to maintain the porous structure after the solid silica nanoparticles are removed. The N2 adsorption-desorption isotherms and BJH pore size distribution of IO-PDA/PPy-MIP microspheres shown in Fig. 1f also show a type IV curve with nearly no hysteresis loop, which is an indicative of non-porous or macroporous structure according to BDDT classification [46]. The specific surface area of IO-PDA/PPy-MIP microspheres is 59.75 m2/g. FTIR spectrum of the IO-PDA/PPy-MIP microspheres is shown in Fig. 2a, compared with those of pure PDA and PPy synthesized by the oxidative polymerization initiated by FeCl3 in water. The peaks at 1552 cm
1 and 1043 cm
1 are assigned to the symmetrical stretching vibration of pyrrole ring and the bending vibration of CAH on pyrrole ring [41,42,47], which can be observed on the spectra of both PPy and IO-PDA/PPy-MIP. The peaks at 1400 cm
1 and 1090 cm
1 on the spectrum of PDA are attributed to the inplane bending vibration and stretching vibration of phenolic hydroxyl group [48,49], which also appear on the spectrum of IO-PDA/PPy-MIP microspheres. The solid 13C NMR spectra of PPy, PDA, and IO-PDA/PPy-MIP microspheres are shown in Fig. 2b. The characteristic chemical shifts (d) at 30 (Peak 1) and 40 (Peak 2) ppm correspond to the carbons from saturated five-membered ring and amino ethyl group of PDA [48,50], which are also clearly observed in the spectrum of IO-PDA/PPy-MIP microspheres. In addition, the peak at 117 (Peak 3) ppm, corresponds to carbons located in the aromatic core. The quaternary bridgehead carbon is assigned to the signal observed at 128 (Peak 4) ppm and the peak at 143 (Peak 5) ppm is attributed to the bridgehead carbon atom that is adjacent to the electronegative nitrogen atom. The 13 C NMR spectrum of PPy shows a broad peak at about 125 ppm (Peak 6) assigned to the overlap of the signals of the quaternary aromatic carbons and protonated chromatic carbons [47]. The spectrum of IO-PDA/PPy-MIP microspheres also shows an obvious broad peak from 100 to 150 ppm, which can be fitted with Peak 3– 6, as shown in the inset of Fig. 2b. The above results confirm that the prepared inverse opal microspheres do consist of both PDA and PPy components. The pore size of IO-PDA/PPy-MIP microspheres can be regulated easily by changing the size of SiO2 nanoparticles. Fig. 3 shows the morphology of the template silica colloidal crystal microspheres and the corresponding IO-PDA/PPy-MIP microspheres prepared

Scheme 1. Schematic illustration of the preparation process of IO-PDA/PPy-MIP microspheres.

W. Yang et al. / Journal of Colloid and Interface Science 548 (2019) 37–47

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Fig. 1. TEM image of SiO2 nanoparticles (a); SEM images of SiO2 colloidal crystal microspheres (b), IO-PDA/PPy-MIP microspheres and their cross section (c, d); TEM image of the ultrathin section of IO-PDA/PPy-MIP microspheres (e); N2 adsorption–desorption isotherms of IO-PDA/PPy-MIP microspheres (f) (the inset is the corresponding pore size distribution calculated by BJH method from the adsorption isotherms).

Fig. 2. FTIR (a) and solid

13

C NMR spectra (b) of PPy, PDA, and IO-PDA/PPy-MIP microspheres.

using the SiO2 nanoparticles with diameters of 360 nm (Fig. 3a1 and a2) and 780 nm (Fig. 3b1 and b2), respectively. It is seen from Figs. 1 and 3 that the colloidal crystal microspheres basically have a similar size no matter what diameter of the SiO2 nanoparticles is. However, the pore size of IO-PDA/PPy-MIP microspheres is in accord with the diameter of SiO2 nanoparticles, changing from 200 to 780 nm. It is noted that the stability and spherical morphology of IO-PDA/PPy-MIP microspheres become worse with the increase of the size of SiO2 nanoparticles because of the less ordered stacking of larger SiO2 nanoparticles in the colloidal crystals. As a result, the colloidal crystal microspheres consisting of SiO2 nanoparticles larger than 200 nm become unstable during the following monomer infiltration and polymerization processes, as shown in Fig. 3(a2 and b2). Hence, the properties of the IO-

PDA/PPy-MIP microspheres prepared from SiO2 nanoparticles with a size of 200 nm have been further investigated in detail. 3.2. NIR photothermal conversion performance of IO-PDA/PPy-MIP microspheres The NIR photothermal effect of IO-PDA/PPy-MIP microspheres in Tris-HCl buffer solution is shown in Fig. 4a. It shows that the temperature of the Tris-HCl buffer increases with the irradiation time of NIR laser (808 nm, 3.3 W/cm2) in the presence of IO-PDA/ PPy-MIP microspheres. The NIR photothermal effect can be enhanced with the increase of the content of IO-PDA/PPy-MIP microspheres. For example, the temperature of the Tris-HCl solution containing 1 mg/mL of IO-PDA/PPy-MIP microspheres can

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a1

a2

1 μm

2 μm

b1

b2

2 μm

1 μm

Fig. 3. SEM images of the template colloidal crystal microspheres produced by SiO2 nanoparticles with a size of 360 nm (a1) and 780 nm (b1); and the IO-PDA/PPy-MIP microspheres prepared by the corresponding template microspheres (a2, b2).

Fig. 4. The temperature changes of the Tris-HCl buffer dispersion containing different contents of IO-PDA/PPy-MIP microspheres under the irradiation of NIR laser (808 nm, 3.3 W/cm2) (a); Temperature changes of the Tris-HCl buffer containing 1 mg/mL of IO-PDA/PPy-MIP microspheres during six laser irradiation ON/OFF cycles (808 nm, 3.3 W/ cm2) (b).

increase from 25 to 55 °C after 10-min’s irradiation of NIR laser. Furthermore, the NIR photothermal conversion efficiency has nearly no loss after six cycles as given in Fig. 4b, indicating the IO-PDA/PPy-MIP microspheres possess excellent structural stability and provide the prerequisite for NIR-light-responsive release of the bound Lyz. 3.3. Imprinting performance of IO-PDA/PPy-MIP microspheres The adsorption kinetics and thermodynamics of Lyz on the IOPDA/PPy-MIP microspheres at 25 °C are shown in Figs. 5 and 6, respectively, compared with those on the IO-PDA/PPy-NIP microspheres. It can be seen from Fig. 5a that the adsorption amount

of Lyz on the IO-PDA/PPy-MIP microspheres increases rapidly to 720 mg/g within 5 min, and achieves a constant of about 800 mg/ g, i.e. the equilibrium adsorption capacity at this condition, after 20 min. The equilibrium adsorption capacity is almost the same with the quantity of the template Lyz molecules eluted from the IO-Lyz/PDA/PPy with the protein lotion (Fig. S4), indicating a high adsorption efficiency of the IO-PDA/PPy-MIP microspheres. The adsorption rate is so fast that Lyz molecules in a buffer solution (1 mg/mL) can be extracted completely by quickly injecting the solution through a disc filter filled with IO-PDA/PPy-MIP microspheres by four times (Fig. S5). On the contrary, IO-PDA/PPy-NIP microspheres show a weak adsorption ability to Lyz. The adsorption amount rises slowly to 40 mg/g (about 1/20 of that on

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Fig. 5. The adsorption kinetics of IO-PDA/PPy-MIP and IO-PDA/PPy-NIP microspheres in Tris-HCl buffer solution of Lyz (1 mg/mL) (the concentration of microspheres:1 mg/ mL) (a); The plots of ln (qe
qt) versus t (b) and t/qt versus t (c) derived from (a).

Fig. 6. The adsorption isotherms of IO-PDA/PPy-MIP and IO-PDA/PPy-NIP microspheres in Tris-HCl buffer solution of Lyz (the concentration of microspheres:1 mg/mL) (a); The plots of ce/qe versus ce (b) and lnqe versus lnce (c) derived from (a).

IO-PDA/PPy-MIP microspheres) within 30 min, and then levels off. The rapid adsorption kinetics and large adsorption capacity of the IO-PDA/PPy-MIP microspheres confirm that the inverse-opal macroporous structure and large number of molecularly imprinted sites can endow MIP materials with the high adsorption rate and capacity on biomacromolecules. The adsorption kinetics is analyzed by two common models, respectively, i.e. the pseudo-firstorder (Fig. 5b) and the pseudo-second-order equations (Fig. 5c):

pseudo
first
order equation : lnðqe
qt Þ ¼ lnqe
k1 t

ð3Þ

pseudo
second
order equation : t=qt ¼ 1=ðk2  q2e Þ þ t=qe

ð4Þ

where qt and qe are the adsorption amounts (mg/g) of Lyz at the time t and the equilibrium, respectively. k1 (min
1) and k2 (min
1mg
1g) are the corresponding adsorption rate constants. The kinetic parameters in Eqs. (3) and (4) obtained from the slopes and intercepts of the fitted curves and the corresponding linear regression correlation coefficients (R2) are listed in Table 1. According to R2, the adsorption behavior of IO-PDA/PPy-MIP microspheres towards Lyz matches the pseudo-second-order model perfectly. The calculated saturated adsorption capacity based on the pseudosecond-order equation is 833.33 mg/g. This reflects the adsorption

process won’t be controlled by the diffusion of biomolecules, but should be determined by the number of the recognition sites in the IO-PDA/PPy-MIP microspheres. The adsorption isotherms of Lyz on IO-PDA/PPy-MIP and IOPDA/PPy-NIP microspheres at 25 °C are shown in Fig. 6a. IOPDA/PPy-MIP microspheres (1 mg/mL) have an adsorption amount of 811 mg/g (Fig. S6) when the Lyz concentration is 1.2 mg/mL at 25 °C. At the same condition, the adsorption amount of IO-PDA/PPy-NIP microspheres is only 34 mg/g, much lower than that of IO-PDA/PPy-MIP microspheres, which makes the imprinting factor as high as 24, demonstrating that the IO-PDA/ PPy-MIP microspheres exhibit much better specific adsorption toward Lyz, compared to many other non-inverse-opal structural MIP, such as the surface imprinted crygels with saturated adsorption capacity of 10.73 mg/g and IF value of 5 [51] and solid nanoparticles with saturated adsorption capacity of 700 mg/g and IF value of 4 [52]. The experimental adsorption data is fitted using Langmuir model and Freundlich model, respectively, as displayed in Fig. 6b and c:

Langmuir model equation : ce =qe ¼ 1=ðK L  qmax Þ þ ce =qmax

ð5Þ

Freundlich model equation : lnqe ¼ lnK F þ lnce =n

ð6Þ

Table 1 The adsorption dynamic parameters of IO-PDA/PPy-MIP microspheres. Adsorbent

IO-PDA/PPy-MIP

Pseudo-first-order

Pseudo-second-order

k1 (min
1)

qe,cal (mg/g)

R2

k2 (gmg
1min
1)

qe,cal (mg/g)

R2

0.0753

192.27

0.56


2.08E + 11

833.33

1

44

W. Yang et al. / Journal of Colloid and Interface Science 548 (2019) 37–47

where qe and ce are the adsorption amounts (mg/g) and the concentration (mg/L) of Lyz in Tris-HCl buffer solutions at the adsorption equilibrium, respectively. qmax (mg/g) is the maximum adsorption capacity. KL (L/mg) is the Langmuir constant related to the binding energy between adsorbents and Lyz. KF (mg/g) is the Freundlich constant associated with adsorption capacity of adsorbents for Lyz. n is the Freundlich linearity index [53]. All the above fitting parameters are shown in Table 2. According to R2, the adsorption behavior of IO-PDA/PPy-MIP microspheres is more inclined to Freundlich model, which means the adsorption process occurs on heterogeneous surface. The recycled IO-PDA/PPy-MIP microspheres still have excellent adsorption performance. As shown in Fig. 7, the adsorption amounts decrease slightly during 5 cycles, and remain 90% of the value in the first use, indicating that the prepared IO-PDA/PPy-MIP microspheres have excellent structural stability and recyclability. The SDS-PAGE analysis is used to visualize the selective adsorption property of IO-PDA/PPy-MIP microspheres towards different proteins (Lyz, HRP, and Trs) in Tris-HCl buffer solution, as shown in Fig. 8a. The band of Lyz (14.3 kDa), HRP (44 kDa), and Trs (23.3 kDa) is clear in the original mixed proteins solution (Lane 1, 0.1 mmol/L for each protein), according to the marker. When the mixed proteins solution is extracted with IO-PDA/PPy-MIP microspheres, the band of Lyz disappears (Lane 2), implying Lyz molecules have been extracted entirely by IO-PDA/PPy-MIP microspheres. As a comparison, the SDS-PAGE band of the mixed proteins solution extracted with IO-PDA/PPy-NIP microspheres is shown in Lane 3, which has the similar protein bands to the original mixed proteins solution (Lane 1). The above results confirm that the IO-PDA/PPy-MIP microspheres have an excellent selective adsorption ability to Lyz in a complex protein solution. The selectivity coefficients (a) of IO-PDA/PPy-MIP microspheres towards BSA or Cyt c have been measured to be 51 and 10.3, respectively, as shown in Fig. 8b. It is noted that both IO-PDA/ PPy-MIP and IO-PDA/PPy-NIP microspheres show a low adsorption capacity toward each reference protein, i.e., 96 and 206 mg/g for BSA, 162 and 70 mg/g for Cyt c. The corresponding IFs for BSA and Cyt c are 0.47 and 2.32, respectively. Both of them are much lower than the IF for Lyz. The high selective adsorption performance of IO-PDA/PPy-MIP toward Lyz should be attributed to physical interaction between the matrix and Lyz, as well as the volume effect of the complementary cavities on IO-PDA/PPy-MIP microspheres. In our previous work about Lyz-imprinted PDA layer [52], isothermal titration calorimeter (ITC) experiment was developed to systematically study the interaction between lysozyme and PDA matrix. The results prove that the adsorption process between lysozyme and NIP microspheres is simply dominated by physical interaction, i.e., hydrogen bond and electrostatic interaction. However, for MIP microspheres, in addition to the above physical interaction, the volume effect of the complementary cavities, i.e., Lyz will be restricted by the well-defined 3D complementary cavities, also dominates the recognition process of Lyz. As for BSA, it has a much larger size (ca. 7 nm) [52] than Lyz (ca. 4.3 nm). Thus, BSA cannot diffuse into the inner part of the MIP microspheres through the cavities on the outside surface imprinted by Lyz molecules. At the same time, the cavities on the outside surface have no matter in them to produce any interaction with BSA, which means compared with NIP, the effective outside surface area of MIP

Fig. 7. The adsorption amounts of Lyz on IO-PDA/PPy-MIP microspheres after consecutive cycles.

microspheres for the adsorption of BSA is also reduced by the existence of the cavities. As a result, the adsorption capacity of BSA on IO-PDA/PPy-MIP is even much lower than that on IO-PDA/PPy-NIP. While Cyt c (ca. 3.3 nm) [52], Trs (ca. 3.8 nm) [52] and HRP (ca. 3.0 nm) [54] have a smaller size than Lyz, they can diffuse freely through the IO-PDA/PPy-MIP microspheres and the complementary cavities so that it is difficult to be trapped by the complementary cavities, making their adsorption capacity on IO-PDA/PPy-MIP microspheres much less than that of Lyz. This excellent selective adsorption ability makes the prepared IO-PDA/PPy-MIP microspheres have potential application on the extraction of Lyz from biological environments. 3.4. NIR-Light triggered release of Lyz bound on IO-PDA/PPy-MIP microspheres Generally, the bound proteins can hardly be released at a normal condition since the bound molecules have strong interaction with the imprinted matrix by specific adsorption. To recycle the bound Lyz molecules, NIR laser triggered release of Lyz from the rebound IO-PDA/PPy-MIP microspheres has been investigated because of the remarkable NIR photothermal conversion effect of the prepared IO-PDA/PPy-MIP microspheres. It can be seen from Fig. 9a that the released amount of Lyz from IO-PDA/PPy-MIP microspheres at room temperature (26 °C) will be no more than 0.05 mg in 30 min. However, once the NIR laser irradiates the solution, a burst release of Lyz (about 0.3 mg, six times more than that released without NIR irradiation) will occur within 5 min. At the same time, the temperature of the solution increased rapidly from 26 °C to 50 °C. When the laser irradiation is OFF, the release quantity drops down quickly. In order to keep the bioactivity of the released Lyz, we fixed the irradiation time to be 5 min. After five NIR-laser-triggered release, the accumulative release percentage of the bound Lyz can reach 70%, as shown in Fig. 9b. In addition, the cycle performance of the release/adsorption under the irradiation of NIR light is shown in Fig. 9c, which shows that the

Table 2 The Langmuir and Freundlich constants of IO-PDA/PPy-MIP microspheres for Lyz derived from adsorption isotherms. Adsorbent

IO-PDA/PPy-MIP

Langmuir model

Freundlich model

qmax (mg/g)

KL (L/mg)

R2

KF (mg/g)

1/n

R2

2132.15

5.46E-04

0.68

2.7

0.82

0.94

W. Yang et al. / Journal of Colloid and Interface Science 548 (2019) 37–47

45

Fig. 8. The SDS-PAGE analysis of different protein solutions. Lane 1: mixture solution of Lyz, HRP and Trs (0.1 mmol/L for each protein). Lane 2: mixture solution of Lyz, HRP and Trs after being extracted with IO-PDA/PPy-MIP microspheres. Lane 3: mixture solution of Lyz, HRP and Trs after being extracted with IO-PDA/PPy-NIP microspheres (a); the selective adsorption performance of IO-PDA/PPy MIP and IO-PDA/PPy NIP microspheres on different proteins (b).

Fig. 9. The release curves of Lyz from IO-PDA/PPy-MIP microspheres under different NIR laser (808 nm, 3.3 W/cm2) irradiation cycles (a); the cumulative release percentage of Lyz from IO-PDA/PPy-MIP microspheres after five NIR-lase NIR-laser-triggered release (b); the adsorption/release amount of Lyz from IO-PDA/PPy-MIP microspheres under NIR-light-triggered release/adsorption cycles (c).

adsorption/release amount of Lyz had no obvious change after 4 cycles. The result indicates that the IO-PDA/PPy-MIP microspheres have good structural stability and recyclability under the irradiation of NIR light. Fig. 10 shows the CD spectra of Lyz in Tris-HCl buffer solution and Lyz released from the IO-PDA/PPy-MIP microspheres triggered by NIR laser. The raw and released Lyz molecules both show the characteristic peak (207 nm) of a-helical structure on the CD spectrum [55,56]. This result indicates that the secondary structure of Lyz molecules won’t be affected during the adsorption and release processes from the IO-PDA/PPy-MIP microspheres. The lysing behavior of E. coli cells in the presence of EDTA and the Lyz molecules released from IO-PDA/PPy-MIP microspheres triggered by NIR light, was investigated, as shown in Fig. 11. It is apparent that serious shape damage can be observed by treating E. coli with both raw Lyz and Lyz released from IO-PDA/PPy-MIP microspheres triggered by NIR light, confirming that the Lyz molecules maintain their bioactivity during the adsorption and the release processes. It means the prepared IO-PDA/PPy-MIP microspheres can be promisingly applied to separate proteins without any loss of their biologic activity.

Fig. 10. The CD spectra of Tris-HCl buffer solution of raw Lyz (solid line) and Lyz released from IO-PDA/PPy-MIP microspheres triggered by NIR light (dashed line).

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W. Yang et al. / Journal of Colloid and Interface Science 548 (2019) 37–47

Fig. 11. TEM images of E. coli after different treatments: E. coli in Tris-HCl buffer solution (a); E. coli in Tris-HCl buffer solution containing EDTA and Lyz (b); E. coli in Tris-HCl buffer solution containing EDTA and Lyz released from IO-PDA/PPy-MIP microspheres triggered by NIR light (c).

4. Conclusions In summary, we successfully fabricated a novel inverse-opal Lyzimprinted PDA/PPy composite microspheres with NIR-lightresponsive release property for the high-efficient selectively adsorption and separation of Lyz molecules. The inverse-opal macroporous structural feature of the MIP microspheres not only promotes the diffusion of the Lyz molecules in the whole microspheres, but also creates much more adsorption sites on the inner pore interfaces. The IO-PDA/PPy-MIP microspheres possess a high adsorption capacity of more than 800 mg/g and an imprinting factor up to 24, which are better than those reported non-inverse-opal structural MIP [17,20,51,52]. The IO-PDA/PPy-MIP microspheres also have a rapid adsorption rate that Lyz molecules in a buffer solution (1 mg/mL) can be extracted completely by quickly injecting the solution through a disc filter filled with IO-PDA/PPy-MIP microspheres by four times. In addition, the NIR-light responsive matrix materials of PDA and PPy endow the IO-PDA/PPy-MIP microspheres with NIR-light controlled release property. The bound Lyz molecules can be released rapidly from IO-PDA/PPy-MIP microspheres triggered by the irradiation of NIR laser and remain enough bioactivity to decompose E. coli efficiently. The IO-PDA/PPy-MIP microspheres also exhibit excellent structural stability and recyclability under the irradiation of NIR light. Based on extremely high adsorption performance and separation rate of the composite microspheres, further study on the extraction and purification of anti-cancer proteins in the biological systems is under way. Acknowledgements We sincerely thank Prof. Zhishen Ge and Prof. Lihua Yang of the Department of Polymer Science and Engineering of USTC for their kind help in providing the 808 nm semiconductor laser device and guiding the bacteriological test, respectively. We also thank Prof. Yu Zhao of Department of Plastic Surgery, The First Affiliated Hospital of Anhui Medical University, for his kind help in providing E. coli and the valuable instructions of SDS-PAGE analysis. This work was supported by the National Natural Science Foundation of China (Nos. 51573174 and 51773189), and the Fundamental Research Funds for the Central Universities (WK3450000001 and WK3450000004). Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcis.2019.04.017. References [1] G.L. Johnson, R. Lapadat, Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases, Science 298 (5600) (2002) 1911–1912, https://doi.org/10.1126/science.1072682.

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