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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Interchain Overlap Affects Formation of Silk Fibroin Secondary Structures on Hydrophobic Polystyrene Surface Detected via Achiral/Chiral SFG Xu Li, Guozhe Deng, Liang Ma, and Xiaolin Lu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01194 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Interchain Overlap Affects Formation of Silk Fibroin Secondary Structures on Hydrophobic Polystyrene Surface Detected via Achiral/Chiral SFG Xu Li, Guozhe Deng, Liang Ma, Xiaolin Lu* State Key Laboratory of Bioelectronics, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 210096, Jiangsu Province, P. R. China


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ABSTRACT: Silk fibroin (SF) has been engineered in the biomedical applications on account of its structural robustness, biocompatibility and biodegradability. However, in situ study is still lacking with respect to the formation of SF secondary structures at the interface. In this paper, by using methanol as an inducing agent, the formation of SF secondary structures at the polystyrene (PS)/SF solution interfaces was detected with achiral and chiral sum frequency generation (SFG) vibrational spectroscopy. SF solutions with two concentrations above and below the critical overlapping concentration (C*) of SF (~1.8 mg/mL) were chosen, i.e. 90 mg/mL and 1 mg/mL. We found that above C*, before adding methanol to the protein solution, no ordered SF secondary structures could be detected at the PS/SF solution interface; oppositely, after adding methanol to the protein solution, ordered SF secondary structure, e.g. antiparallel β-sheet, could be formed at the PS/protein solution interface. Below C*, both before and after adding methanol to the SF solution, ordered SF secondary structure such as antiparallel β-sheet could be formed. Besides, the addition of methanol could induce the formation of an extended helical structure, verified by the achiral and chiral characteristic bands. Since C* represents a critical solution concentration above which the SF chains can interact with each other and below which the SF chains are isolated in the solution, this achiral/chiral SFG study emphasizes the importance of the chain-chain interaction or spatial confinement on the formation of the protein secondary structures, which provides an additional dimension for the future study of interfacial protein folding.


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1. INTRODUCTION Proteins are one of the most important biological molecules. Over the past centuries, a huge number of proteins have been discovered and investigated on account of the biological functions for metabolic catalysis, DNA replication, cytoskeleton construction, and signal and mass transduction, etc. A long protein chain with the nature-formulated amino acid sequence can selforganize into a folded state with desirable secondary structures.1,2 Understanding proteins’ secondary (or hierarchical) structures provides fundamental knowledge about biological events as well as strategies for developing practical applications such as new biomedical materials.2-5As a ubiquitous structural protein, silk fibroin (SF) from Bombyx mori has been extensively studied and engineered owing to its high mechanical strength and excellent biocompatibility.6-12 It has been used for many applications - for example, as highly human cell-compatible scaffolds7-10 and microfluidic devices.11 Concomitantly, its structure and property can further be optimized via genetic engineering to explore the new applications.12 With this regard, it is of fundamental importance to understand its structural characteristics in advance. Researchers have made great efforts to study SF secondary structures by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), electron diffraction (ED), x-ray diffraction (XRD) and transmission electron microscopy (TEM).13-22 Based on the above studies, an SF protein is believed to be composed of a heavy chain (~390 kDa), a light chain (~26 kDa) and a glycoprotein named P25 (~30 kDa).14 Furthermore, it was found that the SF proteins could have three different states, i.e. Silk I, Silk II and Silk III. Silk I contains random coil and β-turn structures although more details are still under debate.13,14,22 Silk II is mainly composed of an antiparallel β-sheet structure, and Silk III mainly consists of an extended helical structure.14-21 It is believed that the antiparallel β-sheet, featured by a highly repetitive amino acid sequence in


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the heavy chain, -GAGAGS- (“G” for glycine, “A” for alanine and “S” for serine),15 is responsible for the SF’s high mechanical strength. From the perspective of engineering, several methods including vortexing, sonication, electric current, and induction by amphiphilic molecules (e.g. methanol and sodium dodecyl sulfate) have been utilized to prepare SF materials rich in the antiparallel β-sheet.23 It should be noted that the states of Silk I, Silk II and Silk III can change under appropriate conditions. For example, Silk I can be transferred into Silk II in an aqueous solution induced by methanol.16 At the air/water interface, Silk III can be converted into Silk II upon increasing the surface pressure.17,19,20 Generally speaking, the interface as an interaction medium could play an important role during the SF structural transition process triggered by external forces. Therefore, it is important to study the formation or change of SF secondary structures at interfaces. Here, using a submonolayer surface/interface sensitive second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy, we detected the formation and transition of SF secondary structures occurring at interface. By contacting SF solutions with concentrations above and below the critical overlapping concentration (C*) with a polystyrene (PS) surface,24 we created two confined interfacial conditions, corresponding to those with and without protein-protein intermolecular interaction respectively. Using the achiral and chiral SFG,25-28 we explicitly probed the formation and transition of the interfacial SF secondary structures induced by methanol. The interchain overlap/spatial confinement was identified as a key factor to affect the formation and transition of the protein secondary structures. We believe that such knowledge is important for fundamentally understanding protein interfacial structures and for engineering proteins with the help of interfaces. 2. EXPERIMENTAL SECTION


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2.1 Sample preparation First, to create a suitable interface to facilitate the investigation of the SF molecular structures, PS was chosen because of its chemical stability and surface hydrophobicity. Second, water is a nonsolvent for PS, therefore a PS/aqueous solution interface is a sharp interface and ideal for the SFG study. Methanol is a polar organic solvent, which has generally been used to induce SF molecules to form ordered secondary structures in solutions.23 Here methanol was adopted as an inducing agent and added into the SF solution in a volume ratio of 1:1. In this study, PS (Mw=48 kDa and Mn=47 kDa) was purchased from Sigma-Aldrich Co. LLC. and used as received without further purification. Right-angle calcium fluoride (CaF2) prisms were purchased from Chengdu YaSi Optoelectronics Co., Ltd. Toluene and methanol were ordered from Shanghai Lingfeng Chemical Reagent Co., Ltd. PS was dissolved in toluene to prepare the solution. PS films with thicknesses in the range from ~150 nm to ~200 nm on the right-angle CaF2 prisms were prepared by spin coating PS solution in toluene with a 4 wt% concentration on the prism surface. The film thickness was controlled by adjusting the spin speed. The as-prepared PS thin films were then annealed in a vacuum oven at 150 °C for ~15 h to remove the residue solvent molecules. Based on the protocol suggested by Kaplan et al.,23 SF was extracted from the silk cocoons of Bombyx mori in a two-step process with slight modification. Briefly, silk cocoons were placed in the boiling sodium carbonate (Na2CO3, 0.02 M) aqueous solution for 30 min. The obtained fibrous SF was then washed with deionized water for three times under stirring. In this step, the sericin molecules were removed from the silk cocoons, leaving only the SF molecules in the fibrous sample. The fibrous SF sample was dried in a vacuum oven overnight. Afterwards, the degummed fibrous SF sample was dissolved in a lithium bromide (LiBr, 9.3 M) aqueous solution


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and incubated at 60 °C for 2 h under stirring. To remove the dissolved LiBr, the SF solution was dialyzed against deionized water for three days and then stored at 4 °C for later SFG experiments. 2.2 SFG setup A commercial SFG system (EKSPLA, Lithuania) was employed in our SFG experiment. Briefly, a fundamental 1064-nm beam with a pulse width of ~20 ps and frequency of 50 Hz was generated based on a Nd:YAG laser. The 1064-nm beam was then guided to the second- and third-harmonic units to generate a 532-nm beam and a 355-nm beam, respectively. Through the optical parametric generation (OPG)/optical parametric amplification (OPA) (pumped by the 355-nm beam) and the difference frequency generation (DFG) process, a frequency tunable infrared beam covering the mid-IR frequency range was generated, which serves as one input beam for SFG experiments. The other input beam was the 532-nm (visible) beam generated by the second-harmonic unit. The incident angles of the input infrared and visible beams were set to be 53° and 64°, respectively, versus the surface normal. Three polarization combinations were applied in our experiment, i.e. ssp (achiral, s-polarized SFG signal beam, s-polarized input visible beam, and p-polarized input infrared beam), ppp (achiral) and psp (chiral). To avoid the laser damage for our sample, pulse energies of the infrared and visible beams were limited to be ~70 µJ and ~20 µJ, respectively. 2.3 Fresnel coefficients Figure 1 shows a schematic for our SFG experimental sample geometry. To probe the PS/SF solution interface, a PS film was deposited onto the bottom of the right-angle CaF2 prism. In this case, the SFG signals generated from the prism/PS interface may interfere with those generated


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from the PS/SF solution interface due to the limited thickness of the PS film. In this case, the SFG signals at the PS/SF solution interface has to be maximized to guarantee the effective detection of the desired interface. As demonstrated in the previous reports,29,30 the relative Fresnel coefficients (or local field coefficients) for the prism/PS and PS/solution interfaces can be adjusted by tuning either the PS film thickness or the incident angles of the input beams or both. In this study, we chose to tune the film thickness to be in the range from ~150 nm to ~200 nm. As shown in Figure 2, such a film thickness range can allow us to enhance the SFG signals from the PS/SF solution interface, leading to the detection the PS/solution interface rather than the prism/PS interface. It needs to state that the protein amide I signal could only be generated from the PS/protein solution interface, not the PS/prism interface, because the latter interface has no protein molecules.

Figure 1. Schematic showing the SFG experimental sample geometry. A thin film of PS was coated onto a CaF2 substrate by spin-coating and then was put in contact with an SF solution. SFG signals were collected from the PS/SF solution interface.


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In this study, methanol was used as an inducing agent for SF to form ordered secondary structures. It does not contribute to SFG signals in the spectral ranges (amide I and N-H stretching frequency ranges) we focused here. The critical overlapping concentration (C*) was calculated based on the reported molecular weight and the radius of gyration (Rg) for a SF chain, which is ~1.8 mg/mL (see Supporting Information).31 The SF solutions with two concentrations, i.e. 90 mg/mL and 1 mg/mL, were chosen to create two solution states above and below C*, respectively. At the high concentration (90 mg/mL) above C*, the space occupied by a SF chain was intruded by the adjacent SF chains, forming an overlapped state with interchain interpenetration in the solution. The SF interchain interaction should strongly affect the formation of the secondary structures of SF. At the low concentration (1 mg/mL) below C*, the SFG chains were in the isolated state and there was no interchain interaction. Thus, the folding, wrapping and translation of the SF chains are relatively free during the formation of the secondary structures. Here we are using SFG to collect achiral (ssp and ppp) and chiral (psp) SFG vibrational signals in the amide I and N-H stretching frequency ranges. We hope that such SFG results will be able to distinguish the SF interfacial behaviors at the above two different solution concentrations. 3.1 SF secondary structures at interface when the SFG solution is above C* As shown in the achiral SFG spectra of Figure 3, for the case of the SF solution concentration of 90 mg/mL, a prominent peak located at ~1650 cm-1 was observed from the PS/SF solution interface in both ssp and ppp SFG spectra. Judged from the chiral SFG spectra of Figure 4, where no chiral vibrational signals were found in the amide I and N-H ranges, the peak at ~1650 cm-1 in the achiral SFG spectra should come from the random coiled protein molecules adsorbed at the interface (also see the following paragraph).32,33 Once methanol was added, the peak


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intensity decreased substantially and the peak width increased significantly. A small shoulder peak located at ~1625 cm-1 was identified by quantitatively fitting the spectrum, which could be assigned to the B2 mode of the antiparallel β-sheet.32,33 The intensity decrease can be explained by the decreased Fresnel coefficients caused by two reasons. First, the nanobubbles, as suggested by Tsukruk et al.,34 would be generated during the formation of the ordered SF secondary structures upon adding methanol, which would decrease the effective solution refractive indices with respect to the input and output light beams; second, adding methanol would further decrease the solution refractive indices, resulting in the decrease of the Fresnel coefficients.

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Figure 3. Normalized achiral (A: ssp; B: ppp) SFG spectra in the amide I range for the PS/SF solution (90 mg/mL) interface before and after adding methanol. Circles are experimental data and solid lines are the fitted curves. Spectra have been offset for clarity. To extract more information related to the protein secondary structures at the PS/SF interface, the chiral SFG spectra were collected by using psp polarization combination. The psp spectra in the amide I (Panel A) and N-H stretching (Panel B) ranges before and after adding methanol


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were shown in Figure 4. No discernable chiral SFG signals were observed in both the amide I and N-H stretching ranges before adding methanol (Panels A and B in Figure 4). This indicates that either no protein helical or β-sheet structures existed at the pristine PS/SF solution interface or the numbers of helical or β-sheet structures were very small. It should be noted that, with the same number densities at the interface, the SFG signal from α-helix is normally much stronger than that of random coil. Hence, the observed achiral SFG signals at ~1650 cm-1 (Panels A and B in Figure 3) should come from the SF random coiled structures rather than α-helix anchoring on the PS surface. Intriguingly, once the methanol was added, the chiral SFG vibrational signals appeared immediately, indicating the rapid formation of the protein ordered secondary structures. As shown in Figure 4, a prominent peak at ~1625 cm-1 assigned to the B2 mode of the antiparallel β-sheet was observed in the amide I range (Panel A in Figure 4) and a prominent peak at ~3280 cm-1 assigned to the N-H stretching of the antiparallel β-sheet was observed in the N-H range (Panel B in Figure 4).32,33 Based on such spectral evidence, we could know that, at the PS/SF solution interface, the methanol is able to induce SF to form ordered protein secondary structure, e.g. antiparallel β-sheet. Therefore, it is reasonable to assert that, the prominent peak, at the PS/SF solution interface after adding methanol shown in Figure 3, should mainly contain contributions from the random coiled and antiparallel β-sheet structures. However, there remains an intriguing question. As we know, protein adsorption on a surface is generally resulted from the hydrophobic interaction.35 This effect would induce the conformational change of the protein molecules to adapt to the surrounding environment. Consequently, we doubt why the hydrophobic PS surface did not induce SF to form ordered secondary structures with detectable chiral signals. Before answering this question, we would like to see what happened at the PS/dilute SF solution interface with/without added methanol in the solution.


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Figure 4. Normalized chiral (psp) SFG spectra in the amide I (Panel A) and N-H (Panel B) ranges for the PS/SF solution (90 mg/mL) interface before and after adding methanol. The dots are experimental data and the solid lines are the fitted curves. Spectra have been offset for clarity. 3.2 SF secondary structures at interface when the SFG solution is below C* When the concentration of the SF solution was 1 mg/mL (below C*), significant spectral difference was observed compared to the high concentration case. Figure 5 shows the achiral (ssp and ppp) spectra in the amide I range collected from the PS/SF solution interface. Before adding methanol to the protein solution, there were three vibrational peaks located at ~1625 cm-1, ~1650 cm-1 and ~1680 cm-1 for both the ssp and ppp polarization combinations (Panels A and B in Figure 5), which can sequentially be assigned to the B2 mode of the antiparallel β-sheet, random coil and B1 mode of the antiparallel β-sheet, respectively.32,33 Upon adding methanol (Panels A and B in Figure 5), besides the peaks at ~1625 cm-1, ~1650 cm-1 and ~1680 cm-1, a distinct peak located at ~1570 cm-1 appeared in the ssp spectrum. To the best of our knowledge,


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this band was not reported in previously SFG studies on the protein secondary structures. According to the previous infrared spectroscopic investigation on model peptides which mimic the polymorphs of the natural SF proteins by Kaplan et. al.,16 the vibrational peak at ~1570 cm-1 should be attributed to the amide II band of an extended helical structure. Here we tentatively assign this peak to the amide II band of the extended helix. Later we will show that this peak is closely associated with a blue-shifted N-H stretching, which confirms this peak assignment.

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Figure 5. Normalized achiral (A: ssp; B: ppp) SFG spectra in the amide I range for the PS/SF solution (1 mg/mL) interface before and after adding methanol. Dots are experimental data and solid lines are the fitted curves (blue). Spectra have been offset for clarity. Figure 6 shows the chiral SFG spectra (psp) for the PS/SF solution (1 mg/mg, below C*) interface. Before adding methanol, appearance of the peaks at ~1625 cm-1 (Panel A in Figure 6) and ~3280 cm-1 (Panel B in Figure 6) indicates that the antiparallel β-sheet has already been formed, which is in stark contrast to that of the high concentration case (Panels A and B in Figure 4). Upon adding methanol, two prominent spectral characteristics can be identified. One


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is the augmentation of the peak intensity in both the amide I and the N-H stretching ranges (Panels A and B in Figure 6). The other is the broadened linewidth and blue shift (~20 cm-1) of the N-H stretching. The former should come from the increased numbers of the ordered secondary structure (antiparallel β-sheet) at the interface. For the latter, the linewidth broadening indicates there should be more than one vibrational mode contributing to the observed chiral signals and the blue shift indicates that the pristine antiparallel β-sheet structure went through certain structural change or some new ordered structure was formed upon adding methanol. To resolve this issue, the linewidth-broadened and blue-shifted peak was fitted by including the one at ~3280 cm-1 and the other at ~3300 cm-1 (Panel B in Figure 6). Reminiscent of the peak at ~1570 cm-1 (Panel A in Figure 5) assigned to the amide II band of the extended helix,16 this chiral band at ~3300 cm-1 should directly be associated with the achiral band at ~1570 cm-1. We thus assign this band to the N-H stretching of the extended helix, where the relatively weak hydrogen bonding leads to a higher-frequency peak center (~3300 cm-1) in comparison to the that of β-sheet (~3280 cm-1). Table 1 lists the fitted results which help to understand the detected secondary structures at the PS/SF solution interfaces.


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Figure 6. Normalized chiral (psp) SFG spectra in the amide I (Panel A) and N-H (Panel B) ranges for the PS/SF solution (1 mg/mL) interface before and after adding methanol. The dots are experimental data and the solid lines are the fitted curves (blue). Spectra have been offset for clarity. If the spectral characteristics for the concentrated (90 mg/mL, above C*) and dilute (1 mg/mL, below C*) SF solutions are compared, we could find, the hydrophobic PS surface cannot induce the formation of significant numbers of ordered SF secondary structures, i.e. antiparallel β-sheet or extended helix in the concentrated solution. As is known, ordered protein secondary structures like helix and β-sheet are pretty rigid. In order to form such rigid structures, enough space is need for the conformational adjustment of protein molecules and the formation of the final secondary structures. We can argue, when the SF solution concentration is high (90 mg/mL, above C*), corresponding to a more confined situation where the SF chains can interpenetrate with each other, there is not enough space for the conformational adjustment targeting to the ordered secondary structures. Only upon adding methanol, which serves as an external force, the


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barrier from the chain-chain interaction or spatial confinement can be overcome so that the ordered secondary structures can be formed. When the SF solution concentration is low (1 mg/mL, below C*), corresponding to a less confined situation with no SF chain interpenetration or interchain interaction, there is enough space for the conformational adjustment of the SF chains. The formation of the ordered secondary structures can be formed on the hydrophobic PS surface. Upon adding methanol, more ordered secondary structures were formed including shaping of the extended helix. Furthermore, according to Kaplan et al.,17-20, the extended helical and antiparallel β-sheet structures come from the same amino acid sequence (-GAGAGS-). The extended helical structure can exist on the water surface in air and transform into the antiparallel β-sheet by increasing the surface pressure. Their experimental results indicate, in order to form the extended helix, more space is needed in comparison to antiparallel β-sheet. Analogously, this can explain why the SFG signals of the extended helix were not observed at the PS/solution interface when the SF solution concentration was 90 mg/mL (above C*). Similarly, even at 1 mg/mL, we can argue that the spatial confinement at the PS/SF solution interface limited the formation of the extended helix. Only induced by methanol, the extended helix could be formed which coexisted with antiparallel β-sheet. This again highlights the effect of the chain-chain interpenetration or confinement on the formation of the ordered secondary structures at the interface. Although this is only an SFG phenomenological study without any quantification, the information extracted from the achiral and chiral SFG vibrational signals presents a clear cue of the confinement effect on the formation of the protein ordered secondary structures at the interface, especially for the structural proteins, which more or less were overlooked in the previous SFG studies. Considering the fast development of the protein engineering, introducing protein molecules into a confined environment and detecting them will not only help to


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understand the formation of the protein secondary structures but also may provide an additional dimension for the surface protein engineering. Table 1. Fitting results of SFG spectra.

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with metha nol

witho ut metha nol

with metha nol

witho ut metha nol

with metha nol

Assp/ Appp

Assp/ Appp

Apsp

Apsp

Assp/ Appp

Assp/ Appp

Apsp

Apsp

17

amide II of extended helical structure

-

-

-

-

-

36/-

-

-

~1625

12

B2 mode of the antiparallel βsheet

-

4/11

-

25

5/19

12/16

40

60

~1650

17

random coil

59/71

28/34

-

-

36/38

16/29

-

-

10

B1 mode of antiparallel βsheet

-

-

-

-

24/8

30/10

-

-

35


-

-

-

106

-

-

41

50

28

N-H stretching of extended helical structure

-

-

-

-

-

-

-

21

ωq

(cm-1)

~1570

~1680

~3280

~3300

Γq

(cm-1)

Assignment

CONCLUSIONS


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In summary, by combining achiral and chiral SFG, we detected the SF ordered secondary structures induced by methanol at the interfaces between PS and SF solutions above and below C*, i.e. 90 mg/mL and 1 mg/mL, corresponding to two confined conditions with and without the SF interchain interaction, respectively. We found, when the concentration was 90 mg/mL (above C*), due to the spatial confinement, no ordered protein secondary structures with detectable chiral SFG signals existed at the PS/SF solution interface. Adding methanol can induce the formation of antiparallel β-sheet. When the concentration was 1 mg/mL (below C*), the SF secondary structure, i.e. antiparallel β-sheet, can directly be detected at the PS/SF solution interface. Adding methanol can induce the formation of an extended helical structure, featured by a blue-shifted N-H stretching band at ~3300 cm-1(chiral, psp) and an achiral band at ~1570 cm-1 (achiral, ssp), as well as more antiparallel β-sheet structure. This achiral/chiral SFG study highlights the importance of the chain--chain interpenetration or spatial confinement on the formation of the protein ordered secondary structures, which can add a dimension for future study and engineering of the SF protein molecules at interfaces. AUTHOR INFORMATION Corresponding Author E-mails: [email protected] (X. L) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT


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This study was supported by the State Key Development Program for Basic Research of China (2016YFA0501604), the National Natural Science Foundation of China (Grant No. 21574020), the Fundamental Research Funds for the Central Universities, the National Demonstration Center for Experimental Biomedical Engineering Education (Southeast University) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). REFERENCES 1.

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