Superhydrophobic Polyhydroxyalkanoates: Preparation and

Sep 5, 2018 - Poly(R-3-hydroxybutyrate-co-R-3-hydroxyhexanoate) (PHBHHx), a family member of microbial polyhydroxyalkanoates (PHA), is a ...
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Superhydrophobic Polyhydroxyalkanoates: Preparation and Applications Xuemei CHE, Dai-xu Wei, and Guo-Qiang Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01176 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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Biomacromolecules

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Original paper

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5 6

Title:

Superhydrophobic Polyhydroxyalkanoates: Preparation and Applications

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Authors:

9

Xue-Mei Chea,b, Dai-Xu Weia, and Guo-Qiang Chen*a,b,c

10 11

Affiliations

12

a

13

Center for Life Sciences, Tsinghua University, Beijing 100084, China

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b

Center for Nano and Micro Mechanics, Tsinghua University, Beijing 100084, China

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c

MOE Key Lab for Industrial Biocatalysis, Tsinghua University, Beijing 100084,

16

China

Center of Synthetic and Systems Biology, School of Life Science, Tsinghua-Peking

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KEYWORDS:

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PHB, Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), Superhydrophobicity, Oil

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absorbents, Anti-bioadhesion

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ABSTRACT

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Poly(R-3-hydroxybutyrate-co-R-3-hydroxyhexanoate) (PHBHHx), a family member

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of microbial polyhydroxyalkanoates (PHA), is a biodegradable and biocompatible

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material with some hydrophobicity and reasonable strength for packaging and tissue

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engineering applications. In this study, superhydrophobic PHBHHx is fabricated via a

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simple non-solvent-assisted process. The material can absorb all tested hydrophobic

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solvents and oil up to six folds of the material weights from water, permitting

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applications for cleaning environmental oil or solvent pollutions with convenience of

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disposal after the usage due to its biodegradability. With an excellent combination of

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biodegradability and biocompatibility, superhydrophobic PHBHHx films are

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evaluated for anti-bioadhesion properities to exploit possible implant usages. Up to

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100% reductions for platelet adhesions on the superhydrophobic PHBHHx surfaces

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are observed compared with that on the control material surfaces. Superhydrophobic

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biodegradable and biocompatible PHBHHx films demonstrate promising low value

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and high-volume or high-value and low volume applications.

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1. INTRODUCTION

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Superhydrophobic surface has received increasing interest due to their tremendous

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potential in areas of batteries, oil–water separation, optical devices, self-cleaning,

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drug

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anti-bioadhesion1-8. Usually, superhydrophobic surface shows an apparent water

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contact angles larger than 150°9. Superhydrophobicity has been inspired by natural

delivery,

anti-icing

coatings,

microfluidic

devices,

sensors

and

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materials such as lotus leaves and butterfly wings9-10.

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Superhydrophobicity requires two essential features including a micro- or

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nanostructured surface or a nonpolar chemical molecular structures, to help trap a thin

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air layer that reduces the interaction between the surface and the water11. Approaches

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have been investigated to prepare superhydrophobic surfaces, such as self-assembly

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procedure, sol–gel process and phase separation et al9. Among these ways, phase

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separation is the simple one for creating superhydrophobic surfaces with rough

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topography, it takes advantage of the instability generated by the multi-component

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mixture, especially for polymers12. There are some methods to form phase separation,

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including thermal induction13, solvent induction14 and non-solvent induction15. Xue et

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al. reported a porous superhydrophobic PLA film fabricated via phase separation,

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utilizing 1, 4-dioxane as a solvent and ethanol as a non-solvent. The porous

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superhydrophobic PLA film showed a rough micro/nano-porous structure with a CA

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value of 153.5 ± 1.7°16. Similarly, superhydrophobic LDPE films with

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micro-/nanoscale hierarchical topography were also fabricated and the as prepared

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LDPE film had a CA value of 173.0 ± 2.5°17.

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Oil water separation and anti-bioadhesion are two of the main applications of

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superhydrophobic materials. Recently, oil–water separation has become an important

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method to solve environmental contamination issues18-19. Superhydrophobic materials

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together with other smart ones have been prepared and employed to separate oils from

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oil-and-water mixtures20-21. However, it remains a challenge to deal with the used

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superhydrophobic materials due to their non-degradable property in nature. The used

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superhydrophobic materials are burnt and abandoned resulting in secondary

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pollution22-23. Therefore, it is of vital importance to investigate biodegradable

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superhydrophobic materials for oil–water separation for sustainable developments.

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Importantly, surface topography is also a property to tissue implants as it influences

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the interaction with surrounding cells exposed to24-26. Some implants require coating

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to minimize cell adhesion and blood coagulation27. Additionally, some implanted

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surfaces offer an ideal environment for adhesion of microbial cells, causing infection

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and implant failure28. To prevent these medical complications, numerous ideas have

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been proposed29-30, including coatings and changing material compositions.

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Superhydrophobic surfaces with extremely low wettability (CA > 150°) have emerged

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as a potential alternative, which could resist adhesions of microbial cells and

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platelets9. However, the studies are limited to the antifouling properties of a

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superhydrophobic biopolymer surface.

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Negative environmental issues of petroleum-based materials have promoted more

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interests for renewable biomaterials derived from natural resources31-33. The

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utilization of renewable materials can make a great contribution to sustainable

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development34. Polyhydroxylalkanoates (PHA) are a family of polyesters synthesized

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by many kinds of bacteria and some haloarchaea species33, 35-37. More than 150 kinds

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of PHA have been known till now. Because of their excellent biocompatibility,

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biodegradability and thermal-mechano-properties, PHA have attracted a lot of

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attentions38-42. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is one of

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a random copolymer consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate

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(3HHx) monomers. Compared with homopolymer P3HB, PHBHHx has a lower

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melting point and more favorable properties43. Biodegradable PHBHHx has been

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investigated for industrial packaging and biomedical applications44. Sudesh et al.

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found that natural hydrophobic PHA has a significant oil-absorbing ability, which

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may have applications in the cosmetics and skin cares45. Recently, Tsujimoto et al.

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prepared porous PHBHHx monolith exhibiting strong plant oil absorption

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capability46. Dou et al made a micro-ball composed of P3HB for the absorption of

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spilled crude oil from water with adsorption capability of 1.8 g/g47. However, no

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study was found regarding superhydrophobic PHA for oil-water separation.

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This study aimed to develop technology for making superhydrophobic PHBHHx films

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and investigated their applications as environmentally friendly oil removal agent or

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blood clotting prevention implant.

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2. MATERIALS AND METHODS

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2.1. Materials

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Bacterial copolymer PHBHHx was kindly provided by Lukang Pharmaceutical Co.

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Ltd (Shandong, China). It showed a polydispersity of 2.14 and a number-average

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molecular weight (Mn) of 1.14×105 g/mol as measured by gel permeation

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chromatography

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Chromotography. Ethanol, octane, hexane, vegetable oil, silicon oil and chloroform

(GPC),

3HHx

content

of

28

mol-%

detected

via

Gas

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were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd, China. Diesel and

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gasoline were purchased from PetroChina Petrochemical Research Institute.

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Phosphate

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(Sigma-Aldrich), fetal bovine serum (Sigma-Aldrich), cell counting kit-8 (CCK-8,

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Boya Biotech) and the above chemicals mentioned were used as received. The

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platelet-rich plasma (PRP) was obtained from fresh rabbit blood. Human bone

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marrow mesenchymal stem cells (hBMSCs) were supplied by Cyagen, China.

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2.2.

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PHBHHx was dissolved in chloroform and then the solution was poured onto a clean

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glass dish to allow evaporation of solvent at room temperature. Smooth PHBHHx

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films were obtained after storage of one week under vacuum. Superhydrophobic

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PHBHHx

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PHBHHx/chloroform solution with PHA concentrations of 5%, 7.5%, 10% and 12.5%

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were poured onto clean glass dishes, respectively. The dishes containing PHBHHx

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solutions were placed in ethanol for 6 hours to allow full phase separations. After a

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vacuum drying process in an oven at 40oC for 6 hours, PHBHHx films with

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hierarchical structures were successfully created.

buffered

saline

(PBS)

(Sigma-Aldrich)

and

sodium

citrate

Sample Preparation

films

were

fabricated

using

a

phase

separation

process.

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2.3. Contact Angle Measurements

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The PHBHHx/water contact angles were determined by Dataphysics OCA 15Pro

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optical contact angle meter (Dataphysics, Germany). A 2.0 µl aliquot of deionized

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water was dropped onto the PHBHHx films and the water contact angle was obtained

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by an average of parallel tests taken at three positions on each PHBHHx films at room

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temperature.

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2.4. Scanning Electron Microscope

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FEI Quanta 200 scanning electron microscopy (FEI, America) was used to observe

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the surface morphology of the PHBHHx films. Before observation, all of the

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PHBHHx films were coated with a layer of gold and the surface images were taken at

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an accelerated voltage of 10 kV.

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2.5. Atomic Force Microscopy

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To observe the surface morphologies of PHBHHx films, AFM (MFP-3D Infinity,

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Oxford Instruments, U.S.A.) scanning was carried out in a tapping mode with a

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standard ACTA AFM probe (AFM probe, U.S.A.). The film was scanned and images

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of scan size (30 µm × 30 µm) were collected for the PHBHHx film. AFM image

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processing software XEI (Park Systems, U.S.A.) was employed to analyze the images.

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2.6. Solvent and Oil Absorption Test

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The PHBHHx film was put on the surface of oil/water mixture in a glass dish. The oil

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was dyed with Oil Red. Various types of oils and organic solvents were used to

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evaluate the absorption ability of the PHA materials, including octane, hexane,

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vegetable oil, silicon oil, gasoline oil and diesel oil. The weights of the PHBHHx

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material before (W1) and after (W2) absorption were measured, then the weight gain 7

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G were evaluated by the formula of G = (W2 – W1)/W1.

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2.7. Cell adhesion Test

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Human bone marrow mesenchymal stem cells (hBMSCs) were provided by Cyagen

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and were cultured in human bone marrow mesenchymal stem cell basal medium

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(Cyagen, China) with 10% fetal bovine serum in an incubator with 5% CO2 at 37°C.

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Smooth PHBHHx films and superhydrophobic PHBHHx films were carefully placed

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on the bottom of the wells of a 96-well culture plate with the rough surfaces up.

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Before seeding, the plates were sterilized with 75% ethanol for 12 h. 5 × 104 of

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hBMSCs were respectively seeded onto the PHBHHx films and then cultured in the

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medium. After incubations lasting 1, 4 and 7 day, respectively, cell count kit-8

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solution was employed to each well to evaluate the cell viability quantitatively.

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Solution absorbance was measured at 450 nm with a Thermo Scientific Varioskan

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Flash microplate reader (Thermo, America) after incubation at 37°C for 1 h. Three

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specimens were prepared for each sample. FEI Quanta 200 scanning electron

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microscopy (SEM) was used to observe the morphology of in vitro cultured cells on

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the PHBHHx films. A confocal laser microscopy (A1; Nikon) was employed to take

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the Fluorescence images. Before cell staining, the films were washed with PBS. Alexa

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Fluor 555 Phalloidin (Invitrogen, CA, USA) was used to stain actin fibers and

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4,6-diamidino-2-phenylindole (DAPI) (Invitrogen, CA, USA) was used to stain cell

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nuclei respectively.

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2.8. Platelet Adhesion Test

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The method for in vitro platelet adhesion test of the PHBHHx films was according to

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a previous method48-49. A plastic syringe with 3.8% (w/v) sodium citrate solution with

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a final dilution ratio of 1:9 was used to collect fresh rabbit blood. The mixture of

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rabbit blood and sodium citrate solution was centrifuged at the speed of 1500 rpm for

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10 min at 4 °C to obtain platelet-rich plasma (PRP) for the platelet adhesion study.

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The PHBHHx films in dishes were sterilized using 75% ethanol and then washed with

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PBS three times. The films were heated to 37°C, 1 mL of PRP was added to the tested

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films followed by incubation at 37°C for 3 h. The films were fixed with

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glutaraldehyde (2.5% v/v) after being washed with PBS three times. Then after being

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dehydrated in an ethanol–water solution at different ethanol concentrations for 15

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min, including concentrations of 30%, 50%, 60%, 70%, 80%, 90%, 95% and 100%,

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respectively, the platelets on the PHBHHx film were dried and visualized under the

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SEM at a voltage of 10 kV.

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2.9. Bacterial Adhesion Test

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Escherichia coli expressing green-fluorescent-protein (GFP) was cultured in LB

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medium for 8 h at 37°C. Smooth PHBHHx films and superhydrophobic PHBHHx

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films were placed on the bottom of the wells of a 48-well culture plate with the rough

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surface up. Before seeding, the plates were sterilized with 75% ethanol for 12 h and a

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bacterial suspension (1 mL) was seeded onto the films. After incubation at 37°C for 3

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h, the PHA based films were taken out and washed with PBS three times to remove

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the non-adhered bacteria, followed by fixation in 2.5% glutaraldehyde and step by 9

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step dehydration using 30%, 50%, 60%, 70%, 80%, 90%, 95% v/v water/ethanol

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solution and 100% ethanol at last. The samples were analyzed for adhered bacteria on

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the surface. Fluorescence images were then taken with a confocal laser microscopy

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(A1; Nikon). FEI Quanta 200 scanning electron microscopy (SEM) was used to

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observe the morphology of in vitro cultured cells on the PHBHHx films. The number

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of E. coli adhered on the PHBHHx film were obtained from the SEM images taken at

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6000x magnifications. Five images were used to get an average number for each

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sample.

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3. RESULTS AND DISCUSSION

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3.1 Fabrication and Characterization of Superhydrophobic PHBHHx Surface

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PHBHHx films with multi-scale topography were fabricated via a facial phase

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separation method as described16. Typical SEM micrographs of PHBHHx films

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prepared by different PHBHHx concentrations were shown (Fig. 1): the surface

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wettability is largely dependent on their topography50.

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Figure 1. Chemical structure of PHBHHx and SEM studies on surface morphologies

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of PHBHHx films.

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(a)

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Superhydrophobic PHBHHx films prepared using solutions of different PHBHHx

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concentrations, respectively (b) 5%; (c) 7.5%; (d) 10%; (e) 12.5%; CA values of

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87.7±1.3 o, 134.25±0.15o, 133.25±2.15o, 149.75±0.25o and 151.25±0.35o were

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obtained from corresponding surfaces, respectively.

Chemical

structure

of

PHBHHx;

(b)

Smooth

PHBHHx

film;

(c-f)

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With an increasing concentration, these samples demonstrated quite different surfaces

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morphologies with contact angle (CA) varying from 134.25±0.15o and 133.25±2.15o

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to 149.75±0.25o and 151.25±0.35o (Fig. 1). The morphology characterized by

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micro-porous structures and the size of the pores varying from a few microns to tens

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of microns (Fig. 1c), led to a higher CA value of the surface (134.25±0.15°) than that

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of the smooth PHBHHx film (87.4±1.3°) reported46. With increasing PHBHHx

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concentrations, structures of leaf-shapes, flowers-like patterns, domains and

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microspheres were formed, respectively (Figs. 1d, 1e and 1f). Micro-porous and leafy

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structures were obtained at low PHBHHx concentrations (Figs 1c and 1d) while at

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high PHBHHx concentration flower-like patterns and spheres were observed (Figs 1e

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and 1f). This structure transition has been found in other polymers attributing to the

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nucleation and increased crystal growth rate with increasing polymer concentration51.

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Flower-like structures were revealed to contain nano-fibrilar-like patterns, while

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microspheres were shown with deep grooves in a nano-scale. The micro-nano binary

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structure has been known to significantly increase the hydrophobicity of a surface

237

compared with the surface of only micro-scaled domains51. When scaling down the

238

domain size of a surface, a significant increase in the CA value was found (Fig. 1f).

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The surface became superhydrophobic with a CA value of 151.25±0.35o when

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micro-nano scale hierarchical structures were observed there (Fig. 1f).

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Figure 2. Roughness of PHBHHx films characterized by AFM.

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(a) Smooth PHBHHx film; (b-e) Superhydrophobic PHBHHx films prepared by

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solutions of different PHBHHx concentrations, respectively (b) 5%; (c) 7.5%; (d)

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10 %; (e) 12.5%; (f) Surface roughness of PHBHHx films in a-e.

246

247

We also carried out the atomic force microscopy (AFM) test and typical AFM surface

248

topography pictures were shown in Fig 2. It could be observed that the

249

superhydrophobic PHBHHx films had a quite roughness which may contributed to

250

increase of water contact angle and anti-adhension ability (Fig. 2f). Superhydrophobic

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materials have been investigated for separating oil-in-water mixtures and some

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medical applications1-3. In this study, a multi-scale PHBHHx film exhibiting a very

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high CA value (Fig. 1f) was successfully prepared and explored for both oil

254

absorption and anti-bioadhesion studies. It is emphasized that the superhydrophobic

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PHBHHx film was not chemically modified.

256 257

3.2 Solvent and/or Oil Absorption by Superhydrophobic PHBHHx

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The oil absorption materials can concentrate oil and then transform them by the

259

removal of the oils containing absorption materials52. To achieve this goal, porous

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superhydrophobic structure is required to gain high absorption capacity and selectivity.

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Several biodegradable polymers are employed to fabricate porous structures for oil

262

absorption53. Cervin et al. prepared the superhydrophobic nanocellulose aerogels and

263

used the aerogels as a separation medium for separating oil from water54. As

264

mentioned above (Fig 1), a superhydrophobic PHBHHx film was prepared via a facile

265

phase separation.

266

267

Figure 3. Oil absorption studies.

268

Absorption of a colored oil (dyed with Oil Red O) in the mixture of oil/water using a

269

smooth PHBHHx films (a) and superhydrophobic PHBHHx films (b), respectively.

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Coming into contact with a layer of colored oil on the water layer, the

272

superhydrophobic PHBHHx film floated on the surface and absorbed all oils from the

273

mixture of oil/water in a very short period of time (Fig. 3b), while the smooth

274

PHBHHx film could only absorb small amount of oil (Fig. 3a). It shows absorption

275

ability of up to 4~6 times its own weight for absorption of oil and other solvents

276

including octane, hexane, vegetable oil, silicon oil, gasoline oil and diesel oil (Fig. 4).

277

278

Figure 4. Absorption capacity of smooth PHBHHx films and superhydrophobic

279

PHBHHx films investigated using six types of oils and organic solvents.

280

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Because of the rough structures and superhydrophobic property, the PHBHHx films

282

have high oil-absorption capacity, which are 3~6 times higher than that of smooth

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PHBHHx films. PHBHHx films can be directly buried then biodegraded in soil when

284

completing their usages, this promises the superhydrophobic PHBHHx films as an

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environmentally friendly candidate for cleaning oil-contaminating water.

286 287

3.3 Adhesion of Cells on Superhydrophobic PHBHHx

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Superhydrophobic surfaces are actively studied also in biomedical areas to control

289

cellular interaction1, 55. Adhesion of cells on a material surface is usually related to the

290

surface wettability1. To investigate cell adhesions on smooth PHBHHx film and

291

superhydrophobic PHBHHx films, hBMSCs were used as model cells. Morphology

292

of hBMSCs were visible after 1 day of culture on the smooth PHBHHx film and

293

superhydrophobic PHBHHx surfaces, respectively.

294

295

Figure 5. Comparison of hBMSCs (Human bone marrow mesenchymal stem cells)

296

adhesion on the smooth and superhydrophobic PHBHHx surfaces after 1 day (24 h) of

297

incubation. SEM images of cells attached on (a) smooth PHBHHx surface and (b)

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superhydrophobic PHBHHx surface. Insets: Fluorescent microscopy images of cells

299

attached to the surfaces. Inset scale bar: 50 µm.

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On the smooth PHBHHx surface, a significant amount of cells were clearly observed,

301

indicating that cells could adhere and spread well on the surface (Figs. 5a and 5b),

302

with normal morphology (Fig. 5a), indicating good compatibility of PHBHHx. This

303

agrees with previous study related to significant cell growth on a smooth PHBHHx

304

film55. On the superhydrophobic PHBHHx surface, the amount of cells adhered is

305

considerably lower than that on the smooth PHBHHx surface. Moreover, shapes of

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the cells adhering to the superhydrophobic PHBHHx surface were abnormal with only

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a few hBMSCs grown individually (Fig. 5b).

308

309

Figure 6. Viability of hBMSCs on the smooth and superhydrophobic PHBHHx

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surfaces at different culture times assayed via CCK-8.

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The spread of hBMSCs on the superhydrophobic PHBHHx surface is limited due to

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the fact that cells were not in full contact with the surfaces in the growth medium, as

314

indicated by the Cassie−Baxter model for the superhydrophobic surfaces56. Song et al

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also observed that very few cells were able to adhere to the superhydrophobic PLLA

316

surfaces in comparison with the smooth ones57. SEM results are consistent with

317

fluorescent microscopy images, both demonstrates that fewer cells adhered onto the

318

superhydrophobic PHBHHx surface than that on the smooth PHBHHx surface.

319

Quantitative results of cell culture for different days are displayed (Fig. 6). The cell

320

count kit-8 solution was used to measure the cell adhesion on different substrates. It

321

was obvious that the viability of hBMSCs on the superhydrophobic PHBHHx surface

322

was much lower than that on smooth PHBHHx surfaces, only a few cells were

323

observed capable of adhering onto the superhydrophobic PHBHHx surfaces.

324 325

3.4 Adhesion of Platelets on Superhydrophobic PHBHHx

326

Surface properties of a material are important for platelet adhesion35. Platelet adhesion

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on superhydrophobic PHBHHx was conducted using the platelet-rich plasma (PRP)

328

method27. SEM was employed to visualize the morphology and calculate the number

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of the platelets adhered on the PHBHHx surface (Fig. 7). After 3 h of inoculation, the

330

platelets spread over the entire smooth PHBHHx surface, yet too few platelets were

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observed on the superhydrophobic PHBHHx surface (Fig. 7).

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333

Figure 7. Comparison of platelet adhesion on the smooth PHBHHx and

334

superhydrophobic PHBHHx. (a) SEM image of the platelet adhesion on the smooth

335

PHBHHx surface (scale bar 10 µm). (b) SEM image of the platelet adhesion on the

336

superhydrophobic PHBHHx surface (scale bar 10 µm).

337

338

Results in this study agree with other studies demonstrating that a hydrophobic

339

surface prohibits platelet adhesion while a smooth one tends to promote platelet

340

adhesion48.

341 342 343

3.5 Adhesion of Bacteria on Superhydrophobic PHBHHx

344

Due to the potential harm of intensive usage of biocides and antibacterial Ag+,

345

superhydrophobicity has attracted increasing interests especially for reduction on

346

bacterial adhesion.55 In current study, the ability of superhydrophobic PHBHHx film

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to prevent bacterial adhesion was studied by culturing E. coli on the surface.

348

Comparison of bacterial adhesion on the smooth and superhydrophobic

349

Figure 8.

350

PHBHHx surfaces after 3h of incubation.

351

SEM images of bacteria attached on (a) smooth PHBHHx surface and (b)

352

superhydrophobic PHBHHx surface; Insets: Fluorescent microscopy images of

353

bacteria attached to the surfaces. Inset scale bar: 100 µm. (c) Corresponding bacterial

354

numbers on different PHBHHx film, averaged from 5 images for each film.

355

356

Fluorescence microscopy images of bacterial adhered onto PHBHHx surface (Fig. 8)

357

shows a sharp reduction on bacterial adhesion on the superhydrophobic PHBHHx

358

compared with the smooth PHBHHx film, as also evidenced by morphology changes

359

and bacterial number (Fig. 8). The E. coli were clearly observed in good shapes and

360

number on the smooth PHBHHx film, while only few bacteria were found on the

361

superhydrophobic PHBHHx film. Quantitative estimation of adhered bacteria was

362

conducted using SEM to show a clear reduction of adhered E. coli (Fig. 8c). The

363

combination of micro-nanostructure and superhydrophobicity may lead to the

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improved anti-adhesion ability. This outcome is of great significance to further

365

promote the use of superhydrophobic PHBHHx.

366

CONCLUSION

367

4

368

For the first time, the superhydrophobic surfaces of PHBHHx containing a

369

micro-nano binary rough structure have been successfully prepared. The

370

superhydrophobic PHBHHx materials exhibit high selectivity and high separation

371

efficiency for oils-in-water and some solvents-in-water. Because of their

372

biodegradability, the used PHBHHx can be easily disposed, promising the

373

superhydrophobic PHBHHx as an environmentally friendly material for separation of

374

oil from water. A sharp reduction of adhered hBMSCs, platelets and bacteria on the

375

superhydrophobic PHBHHx surface was observed compared with the smooth

376

PHBHHx surface. A superhydrophobic PHBHHx may be an excellent candidate for

377

implant applications.

378 379

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AUTHOR INFORMATION

381 382

Corresponding Author

383

* Guo-Qiang CHEN (Chen GQ)

384

* E-mail: [email protected]

385

Author Contributions

386

X.M.C. conducted the studies and prepared the draft paper. D.X. W. assisted the biological study. G.Q.C.

387

supervised the study.

388

Notes

389

The authors declare no competing financial interest.

390

Funding sources

391

This research was financially supported by a grant from Ministry of Sciences and Technology (Grant No.

392

2016YFB0302504), and grants from National Natural Science Foundation of China (Grant No. 31430003 and No.

393

31600072). Tsinghua President Fund also supported this project (Grant No. 2015THZ10).

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Superhydrophobic Polyhydroxyalkanoates: Preparation and Applications

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Authors:

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Xue-Mei Chea,b, Dai-Xu Weia, and Guo-Qiang Chen*a,b,c

576 577

Affiliations

578

a

579

Center for Life Sciences, Tsinghua University, Beijing 100084, China

580

b

Center for Nano and Micro Mechanics, Tsinghua University, Beijing 100084, China

581

c

MOE Key Lab for Industrial Biocatalysis, Tsinghua University, Beijing 100084,

582

China

Center of Synthetic and Systems Biology, School of Life Science, Tsinghua-Peking

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