Protein-Based 3D Microstructures with Controllable Morphology and

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Protein-Based 3D Microstructures with Controllable Morphology and pH-Responsive Properties Shuxin Wei, Jie Liu, Yuanyuan Zhao, Tingbin Zhang, Mei-Ling Zheng, Feng Jin, Xianzi Dong, Jinfeng Xing, and Xuanming Duan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14915 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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ACS Applied Materials & Interfaces

Protein-Based 3D Microstructures with Controllable Morphology and pH-Responsive Properties Shuxin Wei, a Jie Liu,b Yuanyuan Zhao,c Tingbin Zhang, a Meiling Zheng,*,b,d Feng Jin, b Xianzi Dong, b Jinfeng Xing,*,a Xuanming Duan*,c a

School of Chemical Engineering and Technology, Tianjin University, No. 135 Yaguan

Road, Haihe Education Park, Jinnan District, Tianjin 300350, P. R. China. E-mail: [email protected] b

Laboratory of Organic NanoPhotonics and Key Laboratory of Bio-Inspired Materials

and Interfacial Science, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, No. 29 Zhongguancun East Road, Beijing 100190, P. R. China. E-mail: [email protected] c

Chongqing Institute of Green and Intelligent Technology, Chinese Academy of

Sciences, No. 266 Fangzheng Ave, Shuitu Technology Development Zone, Beibei District, Chongqing 400714, P. R. China. E-mail: [email protected] d

School of Future Technologies, University of Chinese Academy of Sciences,Yanqihu

Campus, Huaibei Town, Huaibei Zhang, Huairou District, Beijing, 101407, P. R. China

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KEYWORDS: two-photon polymerization, bovine serum albumin, 3D microstructure, morphology, pH response

ABSTRACT: The microtechnology of controlling stimuli-responsive biomaterials at micrometer scale is crucial for biomedical applications. Here, we report bovine serum albumin (BSA)-based three-dimensional (3D) microstructures with tunable surface morphology and pH-responsive properties via two-photon polymerization (TPP) microfabrication technology. The laser processing parameters including laser power, scanning speed and layer distance are optimized for the fabrication of well-defined 3D BSA microstructures. The tunable morphology of BSA microstructures and a wide range pH response corresponding to the swelling ratio from 1.08 to 2.71 have been achieved. The swelling behavior of the microstructures can be strongly influenced by the concentration of BSA precursor, which has been illustrated by a reasonable mechanism. A panda face-shaped BSA microrelief with reversible pH-responsive properties is fabricated and exhibits unique "facial expression" variations in pH cycle. We further design a mesh sieve-shaped microstructure as a functional device for promising microparticle separation. The pore sizes of microstructures can be tuned by changing the pH values. Therefore, such protein-based microstructures with controllable morphology and pH-responsive properties have potential applications especially in biomedicine and biosensors.


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1. INTRODUCTION Native proteins have congenital advantages such as nontoxicity, biodegradability and biocompatibility1-3, which prompt the diverse applications considering the chemical, optical, mechanical, and electromagnetic properties4-7. Bovine serum albumin (BSA) as one of the native proteins has been widely studied due to the low cost, stimuli-responsive property and the similar nature to human serum albumin.8-13 For example, it can be used as a carrier of ions, metabolites, drugs and hormones,7,14-15 as well as stabilizing and immobilizing agent for some enzymes.16,17 Most notably, BSA-based tunable, dynamic and responsive “smart” three-dimensional (3D) microstructures with well-defined geometries and repeatability are crucial for regenerative medicine and tissue engineering. Two-photon polymerization (TPP) microfabrication technology has excellent capability to produce 3D microstructures with high precision,4,18-20 which is promising for achieving the BSA microstructure with precise configuration. The properties of BSA microstructures have been studied and regulated. Various 2D and 3D BSA microstructures with different shapes, patterns and properties have been investigated, indicating the precise microstructures fabricated by TPP have the capability to adapt to complex 3D biological environments as well as to achieve intelligent response.21-24 Morphology is of critical importance for microstructures, which has a profound influence on the cell behavior or structure quality. BSA microstructures with different voxel dimensions, morphology and porosity have been 3 ACS Paragon Plus Environment


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reported as excellent cell niche for biomedical applications.25 In this work, the authors studied the sub-micrometer topological features. High aspect ratio micropillars, special morphology, were fabricated with aqueous-based photoresists of appropriate viscosity. The micropillars had promising function in creating smart wetting/nonwetting surfaces and influence on the cell behavior.26 Notably, the gradient change of morphology from rough to smooth has not been comprehensively studied. On the other hand, pHresponsive properties are one of the major properties of BSA, which have received a great deal of attention in recent years. Shear group designed a functional microchamber for trapping, incubating and releasing Escherichia coli, benefiting from the stimuliresponsive features of BSA structures.27 The 3D BSA microstructures with dynamic, quantitative and responsive shape-changing can be fabricated and controlled by modulating the layer distance in z direction.28 Modulation of laser processing parameters like layer distance, laser power and scanning speed is vital for fabricating BSA microstructures with different cross-linking density, resulting in the different response degree of the BSA microstructures. Furthermore, based on the stimuli response properties, BSA microdevices with practical function like microlens with tunable focus can be fabricated.29,30 The behavior of undergoing a physical change in response to environmental stimuli is significant for BSA as a promising “smart” material. A wide range swelling extent of BSA microstructure has yet been demonstrated, which is helpful to achieve the protein-based microstructures for different applications. However, the ability to control and quantify the properties of


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BSA microstructures including both morphology and pH response more accurately and widely remains unexplored through synthetical consideration. In this paper, we demonstrate the fabrication of BSA microstructures with tunable properties such as morphology and pH response controlled by BSA concentrations via TPP. We unveil the effect of BSA precursor solution and laser power on fabrication of protein lines. The fabrication window of 3D BSA microstructures is confirmed by optimizing the laser processing parameters. The quality of protein lines and fabrication window indicate a suitable condition for fabricating well-defined 3D microstructures. Microstructures like Roma relief are involved to investigate the relationship between the morphology and BSA concentration. The 3D microstructures have not only tunable surface morphology, yet also a wide range swelling extent in response to pH values by changing the BSA concentration. A panda face-shaped BSA microrelief with reversible pH-responsive properties and unique "facial expression" variations is designed and fabricated to further demonstrate the pH stimulation behavior of BSA microstructures. Finally, based on the pH-responsive properties of BSA microstructures, functional devices like 3D microsieves have been fabricated, whose pore sizes can be tuned by changing pH values. The well-defined, controllable and dynamically responsive “smart” microstructures would have wide potential applications in future. 2. EXPERIMENTAL SECTION 2.1. Materials.



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BSA (96%) and rose bengal (RB, 95%) were purchased from Aladdin Chemical Reagent Company. Sodium hydroxide (NaOH), and hydrochloric acid (HCl) were obtained from Beijing Chemical Reagents Company. pH standard buffer solutions were supplied by Beijing leagene biotech co., ltd. 2.2. Preparation of BSA Aqueous Precursor Solution. Certain amounts of BSA were added to a phosphate buffer solution (pH=7.4) which contained 8.5 mM RB. The mixed solution was fully stirred in a dark room. The adequate mixture was incubated at 4 °C for degassing and keeping BSA fresh. In the experiment, three BSA aqueous precursor solutions of different concentrations were prepared and the amount of BSA in these solutions was 20 wt%, 30 wt% and 40 wt% (corresponding to 250.0 g/L, 428.7 g/L and 666.7 g/L), respectively. Three RB concentrations of 4.2 mM, 8.5 mM and 12.0 mM were used to investigate the influence on protein lines. The UV-vis absorption spectrum of RB is shown in Figure S1. 2.3. TPP microfabrication of BSA Microstructures. In our experiment, a home-built system of TPP was used to fabricate BSA microstructures.20 The system is equipped with a mode-locked Ti:Sapphire laser (Tsunami, Spectra-Physics) whose center wavelength, pulse length, and repetition rate are 780 nm, 80 fs, and 80 MHz, respectively. A 60× oil-immersion objective lens with a high numerical aperture (NA = 1.42, Olympus) was used to tightly focus the laser beam into the BSA precursor solution. In the fabrication process, the BSA precursor solution was dropped on a cover glass substrate which was placed above the xyz-step 6 ACS Paragon Plus Environment

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motorized stage (Scheme 1). The average laser power mentioned in this study was measured before the objective lens and the laser power at the focal plane was 16 % of the measured value. BSA microstructures designed by AutoCAD or 3Ds Max software could be written layer by layer through the laser focus movement driven by computer. After fabrication, the cover glass containing the BSA microstructures was washed by deionized water to remove the unpolymerized BSA precursor and finally the BSA microstructures were remained. 2.4. Swelling Performance and Confocal Fluorescence Imaging. The solutions containing NaOH and HCl with different pH values (pH=1, 5, 9, 11 and 13) were measured by the pH meter (METTLER TOLEDO, FiveGO2) at room temperature. The BSA microstructures for the investigation of swelling performance were prepared as described in section 2.3. Here, the precursor solutions with high and low BSA concentrations were utilized to obtain different cross-linking densities inside BSA microstructures, resulting in different surface morphology and swelling behaviors in external pH stimuli (Scheme 1). The BSA microstructures with different concentrations were equilibrated in pH 5 for 5 minutes and then transferred to the corresponding pH solution (pH=1, 9, 11 and 13) for another 5 minutes equilibration. Every microstructure was observed at two pH values (pH 5 and the corresponding pH value) except the pH cycle experiment. Confocal laser scanning microscopy (CLSM) (Nikon, A1R MP) with a 60× oil-immersion objective lens and 488 nm excitation wavelength (Coherent) was used to excite the BSA microstructures. Simultaneously, 7 ACS Paragon Plus Environment


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the differential interference contrast (DIC) microscopy images are recorded as the bright field images. The area swelling ratio (SWR) of the BSA microstructures was calculated according to the formula28,31 A

SWR= A

0

(1)

where A and A0 are the areas of the swollen BSA microstructure in original pH 5 and the corresponding pH value, respectively. 2.5. Characterization. The morphology of the BSA microstructures was characterized by a field-emission scanning electron microscopy (SEM, S-4800, Hitachi) with an accelerating voltage of 5.0 kV after sputter coating with a gold film (at a current of 10 mA for 60 s) using an auto ion sputtering instrument (MC100, Hitachi). Photochemical mechanism of photoinitiator (RB, 8.5 mM) and BSA precursor solutions were characterized by electron paramagnetic resonance (EPR) spectrometer (Bruker E500) with mercury lamp.


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Scheme 1. Schematic illustration of the fabrication of BSA microstructures using TPP microfabrication technology and the swelling state of different BSA concentrations in pH 1 and pH 11. 3. RESULTS AND DISCUSSION 3.1. TPP of BSA Precursor. In order to elucidate the photoinitiating mechanism of RB and the cross-linking mechanism of protein, EPR measurement is carried out to detect the active species generated by RB. 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) is used as a spin-trap reagent in EPR experiment.32,33 The EPR signals of RB almost have no difference in the dark and light irradiation with DMPO (Figure S2), indicating that the free radicals like hydroxyl radicals, alkyl radicals and superoxide radicals are not generated by RB in light irradiation. 2,2,6,6-tetramethyl-4-piperidinol (TEMP) is used as a spin-label reagent for detecting singlet oxygen molecules (1O2).34,35 The EPR signal intensity of RB is high in light irradiation with TEMP (Figure 1A), demonstrating that the 9 ACS Paragon Plus Environment


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photoexcited molecules at the triplet state could transfer the energy to the ground state molecular oxygen to form singlet oxygen, which is consistent with the previously reported results.36,37 Furthermore, the EPR signals of BSA precursor solutions with different BSA concentrations (20 wt%, 30 wt% and 40 wt% BSA) and 8.5 mM RB are also investigated with TEMP (Figure 1B). The EPR signal intensity of three BSA precursor solutions is significantly lower than that of RB in the same conditions, indicating that protein can consume the 1O2 generated by RB in light irradiation and the cross-linking mechanism of protein involves the reaction of 1O2 with an oxidizable amino acid residue of the protein. Then the covalent cross-linking is produced between an electron deficient protein and another protein monomer with oxidizable amino acid residue.37-39

Figure 1. (A) EPR spectra of photoexcited RB (8.5 mM) using TEMP trapping agent for detecting excited singlet oxygen molecules (1O2) with mercury lamp. Dark control means the sample without light irradiation. (B) EPR spectra of three BSA precursor solutions containing different BSA concentrations (20 wt%, 30 wt% and 40 wt% BSA) and 8.5 mM RB using TEMP with mercury lamp. 10 ACS Paragon Plus Environment

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For fabricating well-defined protein microstructures with appropriate condition, the features of protein lines are firstly investigated. Continuous and smooth protein lines with suitable line widths are prerequisites to formulate an ideal 3D BSA microscale structures. Many parameters can affect the quality of protein lines including laser power, the concentration of BSA and RB. Figure 2A shows the SEM images of solidified protein lines of nine samples. Each sample is fabricated at a constant scanning speed of 44 μm/s. The laser power decreases from the inner ring to the outer ring which is marked by an arrow from 27.0 mW to the threshold with 2.0 mW interval. The relationships between the line width and the laser power with different RB and BSA concentrations are plotted in Figure 2B and Figure S3 according to the SEM images in Figure 2A. The widths of protein lines are strongly dependent on the laser power. The protein line width drops when the laser power decreases, which is consistent with the previous reports.40-42 The BSA concentration and RB concentration are undoubtedly crucial factors that affect the features of the protein lines including the width, threshold and smoothness. Figure 2B clearly shows that line widths increase with the increasing of BSA concentrations at each laser power for all concentrations of RB. It is reasonable that high BSA concentration precursor has more monomers to polymerize compared with low concentration in the same condition. It can be also observed the line widths of high RB concentration are greater than that of low RB concentration at each laser power for all concentrations of BSA (Figure S3). The high RB concentration can improve the polymerization capacity of BSA before saturation relative to the low RB 11 ACS Paragon Plus Environment


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concentration.26 When RB concentration is 4.2 mM, the laser threshold values of the sample with 20 wt% BSA are relatively high, and the lines become intermittent and not smooth. When RB concentration reaches 8.5 mM, the quality of protein lines is obviously improved, particularly for the samples of 20 wt% and 30 wt% BSA. For the sample of 20 wt% BSA with 8.5 mM RB, the threshold is 13.0 mW which is improved by 8.0 mW compared with the sample of 20 wt% BSA with 4.2 mM RB. For the samples of 30 wt% and 40 wt% BSA with 8.5 mM RB, both threshold values are 11.0 mW which is improved by 6.0 mW and 2.0 mW, respectively, compared with the samples of 30 wt% and 40 wt% BSA with 4.2 mM RB. However, the threshold and morphology for all of the BSA samples do not show obvious change with 12.0 mM RB comparing with those with 8.5 mM RB because the amount of initiator is close to saturation. Additionally, excessive increasing the amount of initiator may raise the cytotoxicity of the material and further increase the line width, which has an adverse influence on the fabrication of precise 3D microstructures. Therefore, 8.5 mM is chosen as the optimized concentration of initiator for the fabrication of BSA microstructures in the following study. The minimum line widths of 20 wt%, 30 wt% and 40 wt% BSA with 8.5 mM RB are 164 nm, 126 nm and 209 nm with the corresponding laser powers of 13.0 mW, 11.0 mW and 11.0 mW, respectively, which have high resolution comparing with most of the reported results.25,29


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Figure 2. Effect of BSA concentration (20 wt%, 30 wt% and 40 wt%), RB concentration (4.2 mM, 8.5 mM and 12.0 mM) and laser power on the polymer lines of BSA. (A) SEM images of polymer lines of BSA. The minimum line widths of 20 wt%, 30 wt% and 40 wt% BSA with 8.5 mM RB are 164 nm, 126 nm and 209 nm, which are fabricated with the corresponding laser powers of 13.0 mW, 11.0 mW and 11.0 mW, respectively. (B) The relationships between the line width and the laser power with different BSA and RB concentrations according to (A). Scale bars are 5 μm. 3.2. Morphology of BSA Microstructures.



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The morphology and dimension of protein microstructures can be effectively controlled by laser power, scanning speed and layer distance.1 Laser processing parameters are optimized in order to identify suitable processing range for fabricating stable microstructures. Here, 30 wt% BSA as a representative is adopted to investigate optimal laser processing parameters for fabrication of BSA microstructures. Cuboid shaped microstructures are designed with dimensions of 10 μm× 10 μm× 5 μm (l × w × h). We have considered several parameters including laser power (from 9.5 to 23.5 mW), scanning speed (from 44 to 110 μm/s) and layer distance (from 100 to 500 nm). SEM images of BSA microstructures are shown in Figure 3A. Most of the BSA microstructures have stable and integrate morphology and dimension though slight deformation occurs due to shrinkage upon post-fabrication processing.25 The unstable microstructures are mainly located in the upper left part of Figure 3A while the damaged microstructures are situated in lower right part of Figure 3A. As shown in Figure 3B, the microstructures in red rectangular frame represent the unstable microstructures which are obviously incomplete. The representative of damaged microstructures in green rectangular frame are completely collapsed. On the contrary, the microstructures in yellow rectangular frame exhibit integrate and stable morphology. In general, the combinations of low laser power, fast scanning speed and large layer distance always lead to unstable microstructures while high laser power, slow scanning speed and small layer distance will result in the damaged microstructures. In other words, suitable combination of laser manufacturing parameters is critical to attain the


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microstructures with excellent morphology and integrity, indicating that the quality of the microstructure is related to laser power, scanning speed and layer distance. Indeed, excessive laser power will cause heat polymerization in the processing area and result in internal burning and microstructure explosion. However, high laser power will generate more reactive species to promote polymerization and improve cross-linking density of microstructures within the appropriate range.26 Conversely, for the polymerization at high scanning speed, the exposure time in the processing area is so short that low energy can not initiate sufficient polymerization. Slow scanning speed which has sufficient laser dwell time can enable to generate more active species in the focus zone for effective polymerization. As for the parameter of layer distance, considering that all of the BSA cubic microstructures have the same volume, fewer numbers of laser scanning layers per unit volume are formed in the microstructures with larger layer distance, which results in lower cross-linking density in microstructures.28 Herein, each parameter has an important influence on the quality of the BSA microstructure. The desired microstructure can be obtained only when the parameters are optimized in a suitable range.43



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Figure 3. (A) SEM images showing the morphology of BSA microstructures influenced by average laser power (from 9.5 mW to 23.5 mW), the scanning speed (44 μm/s, 77 μm/s and 110 μm/s) and the layer distance (100 nm, 300 nm and 500 nm) with photoresists containing 30 wt% BSA and 8.5 mM RB. (B) Magnified views of the highlighted areas in (A) with the red, yellow and green rectangular frames, respectively. Scale bars are 5 μm. Here, the suitable laser processing parameters with laser power of 18.0 mW, scanning speed of 77 μm/s and layer distance of 200 nm are chosen for the subsequent processing. Cuboid shaped microstructures (10 μm × 10 μm × 5 μm) of different BSA concentrations (20 wt%, 30 wt% and 40 wt%) are fabricated in such appropriate laser 16 ACS Paragon Plus Environment

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manufacturing parameters, resulting in ideal morphology and dimension (Figure S4). As shown in Figure 4, the Roma reliefs of different BSA concentrations (20 wt%, 30 wt% and 40 wt%) are fabricated in suitable fabrication conditions. It can be clearly found that the morphology of Roma relief has obvious gradient change with the increasing of the BSA concentrations. The 3D relief with 20 wt% BSA concentrations has relatively rough and porous surface morphology. When BSA concentration increases to 30 wt%, the Roma relief becomes more integrated than that with 20 wt% BSA concentrations. The surfaces of 20 wt% and 30 wt% protein microstructures are relatively rough which can be used to fabricate 3D cell scaffolds, bio-implantable microstructures and devices for drug delivery and tissue engineering. Furthermore, 40 wt% protein relief has fine and smooth morphology without apparent pores. The smooth surface morphology not only can be used in biological field, but also have application in numerous photonic and optoelectronic devices. As shown in Figure 4A, the average pore size diameters of the Roma reliefs with different BSA concentrations have been counted in the areas of yellow rectangles, which are 0.39 μm with 20 wt% BSA relief and 0.40 μm with 30 wt% BSA relief. 40 wt% BSA relief does not have visible pores in the surface morphology. Although the average pore size diameters of 20 wt% BSA relief and 30 wt% BSA relief have almost no difference, the number of the pores with 20 wt% BSA relief is significantly more than that with 30 wt% BSA relief. This result indicates that the surface morphology of the complex microstructures with patterns can be controlled from rough to smooth obviously with the increasing of BSA concentrations. 17 ACS Paragon Plus Environment


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Figure 4. Fabrication of 3D reliefs of Rome sculpture style with different BSA concentrations (20 wt%, 30 wt% and 40 wt%) in suitable fabrication conditions. (A) SEM images of 3D BSA microstructures (top view). The yellow squared area is used to estimate the average pore size diameters. (B) SEM images of 3D BSA microstructures (side view). Scale bars are 20 μm. 3.3. pH-Responsive Properties of BSA Microstructures. Proteins have the characteristic property to change the shape in response to external pH stimuli due to the existence of a large number of carboxylic acid groups and amino groups.2,27,44 The number of charged pH-sensitive groups can be manipulated by some factors such as pH values, protein concentrations and so on. Therefore, this manipulation in the swelling or shrinking of the protein microstructures plays an important role in studying the basic properties of proteins and investigating the potential applications.


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We have designed cuboid shaped microstructures with dimensions of 15 μm × 5 μm × 8 μm (l × w × h) as a basic model (Figure 5A) to investigate pH-responsive properties of BSA microstructures with different BSA concentrations in different pH solutions. The SEM images of the cuboid shaped microstructures of different BSA concentrations not only show stable and well-defined microstructures, but also indicate that the morphology and dimension of the microstructures can be controlled by BSA concentrations (Figure 5B). The extent of expansion for the BSA microstructures is highly restricted by the attachment to the surface of glass substrate while the top of the BSA microstructures have a relative big degree of freedom. Thus, the degree of expansion in different height becomes different. In this case, the middle of the BSA microstructures is selected to study pH-responsive properties on x-y plane. The general trend for BSA microstructures is that the microstructures possessed a minimum area at pH 5, which is close to the isoelectric points (pI) of BSA.28 For the microstructures prepared with different BSA concentrations, the tendency of swelling is the same within a wide pH variation from 1 to 13. The swelling degree increases with the increasing of the distance between the external pH values and pH 5 (Figure 5C-G). By increasing the distance between external pH values and pH 5 from 4 to 8, the swelling ratios for 20 wt% BSA microstructures increase from 1.08 to 1.52, while the area swelling ratios of 40 wt% BSA microstructures increase more prominently from 1.31 to 2.72. The area swelling ratios of 30 wt% BSA microstructures increase from 1.12 to 1.88 which are between 20 wt% and 40 wt% BSA microstructures. 19 ACS Paragon Plus Environment


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When the pH values are far from pI, the ionization degrees of weak acid or weak base groups located in BSA microstructures increase, resulting in strong electrostatic repulsion between charged groups and a large swelling extent for microstructures. Hence, the swelling capability of BSA microstructures can be effectively manipulated by tuning pH values. It can be also noted that the BSA microstructures of high concentration swell more than that of low concentration at all the pH values especially in the strong alkalis (pH = 13). It is likely that the BSA microstructures of high concentration have strong electrostatic repulsion originated from the more charged polymer chains in response to external pH stimuli.26,45 For 40 wt% BSA, stimuli-responsive microstructures can undergo large volume changes upon different pH values. Therefore, the degree of swelling of BSA microstructures can be controlled by tuning the BSA concentrations.


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Figure 5. pH-Responsive properties of BSA microstructures. (A) Designed model of cuboid shaped BSA microstructures. (B) SEM images of BSA microstructures with different BSA concentrations (20 wt%, 30 wt% and 40 wt%). (C-F) Confocal fluorescence microscopy images of BSA microstructures with different BSA concentrations at different pH and (G) the corresponding area swelling ratios. Scale bars are 5 μm. 21 ACS Paragon Plus Environment


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It is important to propose a reasonable explanation for the swelling results of pH response that BSA microstructures of high concentration swell more than that of low concentration. It can be observed that the EPR signal intensities of different protein concentrations do not have obvious difference in the same exposure time (40 s), implying that the original photoinitiation efficiency of RB is almost identical (Figure 1 B). In this case, it can be proposed that the density of photochemical cross-linking nodes in the polymer network mainly depends on BSA concentrations in the same fabrication conditions. The density of photochemical cross-linking nodes increases with the increasing of BSA concentrations. Consequently, a reasonable mechanism is proposed that the microstructure of high BSA concentration has bigger density of photochemical cross-linking nodes and more polymer chains between two cross-linking nodes (Figure 6). Herein, larger swelling ratio for BSA microstructures with higher BSA concentrations can be explained by the assumption. At external pH stimuli, the microstructures with high BSA concentration have more charged polymer chains to produce stronger repulsive forces than the binding forces of cross-linking, which results in a larger protein unfolding compared to the microstructures with low BSA concentration. Furthermore, the explanation of the swelling can be verified through the following equation46 ̅̅̅̅ 𝑀𝑐 = 𝜌𝑉̃1 𝑄 5/3 /(1 − 1/𝜒1 )

(2)

where ̅̅̅̅ Mc is the average molecular weight between primary valence crosslinkages unmodified by entanglements, ρ is the density of polymer, ̃ V1 is the molar volume of the solvent, Q is swelling ratio, χ1 is Flory-Huggins interaction parameter. 22 ACS Paragon Plus Environment

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Eq. (2) shows a positive relationship between ̅̅̅̅ Mc and Q. In the swelling experiment, the microstructures with high BSA concentration have bigger Q than that with low BSA concentration. According to Eq. (2), the ̅̅̅̅ Mc of high BSA concentration microstructures is bigger than that of low BSA concentration microstructures, which is consistent with the mechanism. From the other aspect, the microstructures with high BSA concentration have bigger ̅̅̅̅ Mc in the assumption. The Q of the microstructures with high BSA concentration is bigger than that with low BSA concentration according to Eq. (2), which is consistent with the result of the swelling experiment.

Figure 6. Possible photochemical cross-linking ways of BSA microstructures with different BSA concentrations. 3.4. Reversible Swelling Properties of BSA Microstructures. For the sake of investigating reversible swelling properties of BSA microstructures, pH 1 and pH 11 solutions are chosen to represent acid and alkaline environments, respectively. Because the swelling degree of 20 wt% BSA microstructures is relatively small, the microstructures of 30 wt% and 40 wt% BSA are used to study BSA reversible swelling properties. For both cuboid shaped microstructures of 30 wt% and 40 wt% BSA, the excellent reversible swelling properties are displayed over two swelling 23 ACS Paragon Plus Environment


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cycles, including a swelling cycle of alkaline environments and a swelling cycle of acid environments (Figure 7 and Figure S5). In addition, the swelling ratios of microstructures in pH cycle experiment are generally consistent with those in pH response experiment mentioned in section 3.3. It is worth noting that the BSA microstructures can withstand acid and alkaline environments without any influence to exhibit the reproducible dynamic swelling behavior, indicating the stability of protein microstructures.

Figure 7. The area swelling ratios of cuboid shaped BSA microstructures with BSA concentrations of 30 wt% and 40 wt% during the reversible pH cycle. Furthermore, the microstructures of 40 wt% BSA which have larger swelling extents are chosen to investigate the reversible swelling properties for complex microstructures. A panda face-shaped 3D relief is designed by 3Ds Max with dimensions of 35 μm × 33 μm × 11 μm. As shown in Figure 8, the panda face swells markedly, especially the facial features when the pH value is increased from pH 5 to pH 11. The panda facial expression can be considered from happiness to surprise. The 24 ACS Paragon Plus Environment

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3D relief then shrinks back to near the original size after the pH value is decreased to pH 5 with facial expression back to happiness. On the other hand, the microstructure starts to swell with facial variation from happiness to excitement when the pH value decreases from pH 5 to a lower value of pH 1. Finally, the original size of panda face can also be shrunk back when the pH value is back to pH 5 again with face variation back to happiness. In the circulation process of environmental pH value, the protein 3D relief undergoes obvious reversible deformations and "facial expression" variations. This unique feature may have potential applications in image identification and biosensors in response to pH variations. Moreover, fluorescence images of panda face pH cycle can be observed which show promising application in tissue engineering (Figure 8B).

Figure 8. Reversible swelling of BSA-based 3D relief. (A) Bright field images and (B) confocal fluorescence images of the 3D panda relief with reversible deformation in changing the pH values characterized by CLSM. (Inset) The designed 3Ds Max model of a panda relief. The 3D panda microstructure is fabricated by 40 wt% BSA. Scale bars are 20 μm. 25 ACS Paragon Plus Environment


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3.5. Fabrication and “Smart” Response of Microsieves. For creating functional devices of pH sensitive response using cross-linking protein with high BSA concentration, microsieves as a kind of mesh sieve-shaped BSA microstructures are fabricated through secondary processing with 40 wt% BSA. The peripheral frames of microsieves are fabricated using a polymeric photoresist (IP-L) with designed dimensions of 30 μm × 30 μm × 6 μm. After fabrication, the unpolymerized photoresist is removed by ethanol and then the BSA precursor solution is dropped on the substrate for secondary processing.47 The inside suspended grids of microsieves connected with the middle of frame are of 3.0 μm height. Four square pores of equal area are made through three columns and three rows cuboid shaped microstructures. According to the pH-responsive properties of microstructures with high BSA concentration, the pore sizes of the BSA microstructures can be facilely manipulated by changing the pH values (Figure 9A). In addition, IP-L microsieves with the same shape are fabricated as a control (Figure S6). It can be observed that the average pore areas of BSA microsieves decrease with the increasing of the distance between the external pH values and pH 5 while IP-L microsieves have no visible response with the pH changing (Figure 9B). The pore area of BSA microsieves can reach the biggest value (approximately 19.7 μm2) at pH 5 which is larger than that of IP-L microsieves for shrinkage effect upon post-fabrication processing. During changing the pH values (Figure 9A), it is found that the pore areas of BSA microsieves can be adjusted in a wide range from approximately 19.7 μm2 to 7.7 μm2, indicating the microsieves have 26 ACS Paragon Plus Environment

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potential application in separation of microparticles with different sizes upon changing the pH values. Therefore, BSA functional devices like microsieves with different sizes and pore shapes can be fabricated through TPP for more potential applications.

Figure 9. Tunable pore sizes of microsieves. (A) Bright field images and confocal fluorescence images of microsieves in different pH values carried out by CLSM. The peripheral frames and the inside grids of microsieves are fabricated by IP-L and 40 wt% BSA, respectively. Scale bars are 5 μm. (B) The average pore areas of BSA and IP-L microsieves in different pH. 4. CONCLUSIONS In summary, we have systematically demonstrated protein-based 3D microscale structures with adjustable surface morphology and pH-responsive properties through photochemical cross-linking induced by femtosecond laser. The quality of protein lines and processing window of 3D microstructures have been studied in detail. A lateral spatial resolution is 126 nm, which can guarantee the processing accuracy of TPP. BSA microstructures fabricated in optimized laser processing parameters exhibit remarkable stability and integrity. Notably, the properties of BSA microstructures including surface morphology and a wide range swelling ratio from 1.08 to 2.71 in different pH can be 27 ACS Paragon Plus Environment


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controlled by changing BSA concentrations. Benefiting from this finding, we design and fabricate special microstructures with environmental response. A 3D BSA relief of a panda face with "facial expression" variations exhibits the reversible deformation properties. Furthermore, microsieves with tunable pore areas of approximately 19.7 μm2 to 7.7 μm2 by changing the pH values have been successfully fabricated, which are expected to be applied in the separation of microparticles with different sizes in future. The BSA microstructures with tunable morphology and pH-responsive properties would provide new prospects for the potential applications in controlled environmental response, biomimicking and tissue engineering. ASSOCIATED CONTENT Supporting Information. The results of UV-vis characterization, EPR Measurement with DMPO, SEM images, and CLSM images. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] Author Contributions 28 ACS Paragon Plus Environment

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The National Natural Science Foundation of China (31771094, 91323301, 31371014 and 61475164), Tianjin Science and Technology Innovation Platform Program (14TX GCCX00017), the National Key Research and Development Program of China (Grant No. 2016YFA0200500 and 2016YFC1100502), Cooperative R&D Projects between Austria, FFG and China, CAS (GJHZ1720). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Authors thank the support of the National Natural Science Foundation of China (31771094, 91323301, 31371014 and 61475164), Tianjin Science and Technology Innovation Platform Program (14TXGCCX00017), the National Key Research and Development Program of China (Grant No. 2016YFA0200500 and 2016YFC1100502), Cooperative R&D Projects between Austria, FFG and China, CAS (GJHZ1720). REFERENCES (1) Sun, Y. L.; Li, Q.; Sun, S. M.; Huang, J. C.; Zheng, B. Y.; Chen, Q. D.; Shao, Z. Z.; Sun, H. B. Aqueous Multiphoton Lithography with Multifunctional Silk-Centred BioResists. Nat. Commun. 2015, 6, 8612. 29 ACS Paragon Plus Environment


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Protein-Based 3D Microstructures with Controllable Morphology and

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