Highly tunable and scalable fabrication of 3D flexible graphene

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Biological and Medical Applications of Materials and Interfaces

Highly tunable and scalable fabrication of 3D flexible graphene micropatterns for directing cell alignment Jiao Yang Lu, Xin Xing Zhang, Qiu Yan Zhu, Fu Rui Zhang, Wei Tao Huang, Xue Zhi Ding, Li Qiu Xia, Hong Qun Luo, and Nian Bing Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04416 • Publication Date (Web): 27 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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ACS Applied Materials & Interfaces

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Highly Tunable and Scalable Fabrication of 3D Flexible Graphene Micropatterns

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for Directing Cell Alignment

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Jiao Yang Lua, Xin Xing Zhanga, Qiu Yan Zhua, Fu Rui Zhanga, Wei Tao Huanga,*, Xue Zhi Dinga, Li

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Qiu Xiaa, Hong Qun Luob & Nian Bing Lib,*

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a

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Hunan Normal University, Changsha 410081, P. R. China

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b

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School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R.

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China

State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science,

Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education),

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*Corresponding author:

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E-mail: [email protected]. Fax: (+86)731-8887-2905; Tel: (+86)731-8887-2905

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E-mail: [email protected]. Fax: (+86)23-6825-3237; Tel: (+86)23-6825-3237

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Abstract

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Patterning graphene allows to precisely tune its properties to manufacture flexible functional materials or

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miniaturized devices for electronic and biomedical applications. However, conventional lithographic techniques are

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cumbersome for scalable producing time- and cost-effective graphene patterns, thus greatly impeding their practical

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applications. Here we present a simple scalable fabrication of wafer-scale 3D graphene micropatterns by direct

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laser tuning graphene oxide reduction and expansion using a LightScribe DVD writer. This one-step laser-scribed

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process can produce custom-made 3D graphene patterns on the surface of a disc with dimensions ranging from

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microscale up to decimeter-scale in about 20 minutes. Via control over laser-scribing parameters, the resulting

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various 3D graphene patterns are exploited as scaffolds for controlling cell alignment. The 3D graphene patterns

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demonstrate their potential to biomedical applications, beyond fields of electronics and photonics, which will allow

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to incorporate the flexible graphene patterns for 3D cells or tissues culture to promote tissue engineering and drug

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testing applications.

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Keywords: 3D graphene micropatterns, cell alignment, laser-scribing, graphene oxide, scalable fabrication

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ACS Paragon Plus Environment

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Introduction

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The graphene-family materials like graphene, its oxidized and reduced form (graphene oxide and reduced

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graphene oxide), have attracted tremendous imagination of loads of researchers in many areas of research,

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including electronics, biomedicine1-2, sensors, photonics, and energy,3 owing to their remarkably high surface area,

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distinct electrical, chemical, and optical properties.4 Scalable and controlled patterning of graphene and its

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derivatives is predicted to lead to interesting properties5 and allows to precisely tune properties of graphene-based

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materials6 for potentially practical applications in tailor-made and sophisticated flexible materials or devices.7

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Recently, lithographically defined graphene-based patterns8 (such as graphene-based micro/nanoribbons,9

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micro/nanomeshes) have already been developed in pursuit of this goal. Although the graphene-based patterns

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generated by several of these methods exhibit positioning accuracy and patterning uniformity, the conventional

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lithographic techniques (including photolithography,10 soft lithography, electron-beam lithography, interferometric

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lithography, and ion beam lithography), which are time-consuming, labor-intensive, complex and expensive in

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commercial applications, require several steps, expensive masks10 or templates, post-processing, and so on.8

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Among various innovative techniques, because wavelength- and power-tunable lasers have been extensively used

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for modern manufacturing. Thus, laser-based techniques for processing graphene hold promise for commercial

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applications. Recently, laser irradiation techniques with advantages of rapid processing speed, no physical contact,

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and large scan area have been widely studied and applied in the graphene synthesis, reduction, and patterning for

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fabrication of various flexible electronic devices.11-12 However, one main challenge, that is, the need for the heavy,

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bulky and expensive equipments7, 13-14 working in special conditions (like a vacuum or airtight with inert gas15),

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still exists.

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Laser-scribed graphene oxide (GO)-reduced graphene oxide (rGO) conversion and patterning technology

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creatively invented by Kaner et al. using a LightScribe DVD writer,16 is an attractive candidate, because the method

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is simple, cost-and time-efficient. Since this development, laser scribing technology has been utilized for achieving

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band-gap modulation,17 conductivity enhancement,16 functionalities elimination, fabricating porous structure,18 and

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doping graphene19 or phase reversion of graphene-like materials (such as MoS2),20 which reveals its potential

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applications in electronic, optoelectronic and electroacoustic devices,18, 21 such as capacitors,22-23 sensors,16, 18, 24-25

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and memory chips.17 However, there are two barriers to laser-scribed graphene’s application and competing with

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other techniques. On one hand, although many macroscopic graphene-based patterns with various sizes and shapes

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can be created by laser-scribed technology, the challenge is to fabricate tailor-made and sophisticated 2- or 2


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3-dimensional (2D/3D) graphene-based patterns with dimensions ranging from microscale (even nanoscale) to

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decimeter-scale and with excellent control over patterning parameters (tailored shapes, pattern widths, length,

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spacing, height, densities) on arbitrary substrates. On the other hand, very few study takes exploration of other

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applications of graphene-based patterns produced by various techniques, including laser-scribed technology,

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beyond the fields of electronics,11, 26 and photonics,27-30 (such as sensors and devices) for biomedical purposes,8 like

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tissue engineers, cell differentiation1-2, or drug screening.

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Some interesting attempts have been made to utilize graphene-based micropatterns (such as fluorinated31-32

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graphene lines,33-34 rGO lines,35-36 GO grooves,37 lines38 and grids,39) with dimensions ranging from several tens to

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several hundred micrometers fabricated via conventional lithographic and laser irradiation techniques33 as a

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promising platform and cell-guiding physical cues for controlling cell adhesion, alignment,31, 35, 37 differentiation,39

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and gene delivery,38 which has great potential in biomedical applications (such as tissue engineering).40 Regrettably,

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these techniques are very difficult for producing low-cost, scalable graphene-based patterns, which greatly impedes

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the translation of interesting findings relating control of cell behavior into practical applications. Herein, we

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demonstrate on highly tunable and scalable fabrication of tailor-made and sophisticated 3D graphene micropatterns

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over large areas on arbitrary substrates using a LightScribe DVD writer for guiding neuronal growth (Scheme 1).

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This single-step laser scribing process is simple, fast, readily scalable, inexpensive. A commercially available

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LightScribe DVD burner and a standard size DVD costing about 30 USD can produce wafer-scale (~100 cm2 area)

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custom-made 3D graphene patterns with dimensions ranging from microscale (even nanoscale) up to

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decimeter-scale in about 20 minutes. Via control over laser-scribing parameters, the resulting 3D graphene-based

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micropatterns with various tailored shapes, line widths, heights, densities on arbitrary substrates can be exploited as

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cell culture substrates to directly support cell attachment, growth, and alignment.

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Scheme 1. Schematic demonstration of tunable and scalable manufacturing of 3D flexible graphene mircopatterns

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via laser scribing technology for directing cell alignment.

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Results and discussions

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Fabrication and characterization of 3D laser-scribed graphene scaffolds with different pattern

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sizes. After launched for 12 years, LightScribe technology has been successfully used to fabricate rGO, MoS2,20

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copper nanoparticle41 macroscopic patterns for some potential applications.16-18,

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between the laser-scribed parameters and the resulting patterns are still rarely known. In the laser scribing process,

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the labeling parameters play important roles for the formation of the scribed patterns (see the details about

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hardware, software, and working principle of LightScribe technology in SI, Fig. S1-S5). Here we bypass the

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specialized coating by covering the disk with a layer of GO film on arbitrary substrates, which can then be fast

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patterned in bulk on directly to form tailored arbitrary shapes. Fig. 1 shows a photograph, their optical microscope,

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SEM, and AFM images of the 18 circles with a diameter of 0.6 cm patterned on the GO film by using different

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print modes, grayscale values, and contrast. As expected, the 788 nm infrared laser makes the golden-brown GO

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layer become black and convert to reduced graphene oxide (called laser-scribed graphene, LSG). The Best mode,

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gray value 0, and Enhanced contrast can result in the blackest pattern (Fig. 1a, first circle on the first line).

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Switching the print mode (Normal or Draft) or changing higher gray values can produce the lighter patterns (Fig. 1a,

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other circles). In comparison with Enhanced contrast, Default contrast further makes the patterns paler (Fig. 1a, the

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9 circles below). Thus, using the Best mode, Enhanced contrast, the lowest gray value can produce the best image 4


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However, the relations

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quality, because the 788 nm infrared laser can burn more durably and frequently with higher energy and precision

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(see the following detailed characterization). Using Draft or Normal imaging modes, Default contrast, the higher

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gray value provides a shorter burn time, but also results in a lighter label, because of the laser pulses with lower

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precision, shorter time, and lower frequency.

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For better understanding the laser-scribing graphene process, the macroscopic LSG patterns were characterized

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by optical and SEM microscope (Fig. 1b,c). Interestingly, as shown in Fig. 1b, the macroscopic LSG patterns with

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different shades consist of microscale lines and/or dots. Fig. 1c exhibits their typical SEM images. The pristine GO

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film displays a comparatively flat morphology. Whereas, the LSG regions possess an expansive 3D morphology

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consisting of ordered lines and/or dots with different length, width, height, and spacing due to rapid degassing of

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gases generated and released during laser treatment.18 The darker region indicates the larger scribed area, the

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smaller spacing, the larger width, and the higher density of microscale lines and/or dots, while the lighter regions

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correspond to the smaller patterned area, the wider spacing, the smaller width, and lower density of microscale

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lines and/or dots (Fig. 1b,c, S6). Thus, it is concluded that time cost and image quality mainly depend on the laser

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energy density and duration which directly affect the scribed area, spacing, density, width, height, and length of the

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microscale lines and/or dots. Adjusting print mode (Best, Normal, Draft) can control laser energy density from high

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to low and regulate the LSG microscale line and/or dot width (Fig. S6a), and spacing (ranges from 0 to ~50 µm, Fig.

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S6b). And switching the contrast can further control laser energy density and adjust the width, height (Fig. 1d, Fig.

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S6e) and density of lines and/or dots (Fig. 1b,c). Interestingly, using the Best mode, Enhanced contrast, gray value

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0 can give the maximum laser energy, resulting in the exfoliated reduced graphene sheets with large-area flat

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morphology (Fig. 1a, first circle on the first line, Fig. S7). Changing the gray value from 0 to 200 can control the

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LSG dot or line length, which ranges from ~50 µm to ~11.4 cm. Moreover, repeating the laser-scribe process in the

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same region also can further obtain bold lines, reduce line spacing, increase scribed area (Fig. S8a,b). The images

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not only give a clear definition between laser scribed graphene and un-treated GO regions, but also demonstrate the

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level of precision possible when using this method to pattern GO. Thus, just simply changing or combining

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different laser-scribe parameters (print modes, gray value, contrast, repeat times) can precisely fabricate tailor-made

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and sophisticated graphene patterns with dimensions ranging from microscale to decimeter-scale and with excellent

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control over patterning 3D morphology (lines and/or dots, line and/or dot thickness, height, spacing, densities) on

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arbitrary substrates (Fig. S9).

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Figure 1. Fabrication of tailor-made and sophisticated 3D LSG micropatterns. A digital photograph (a), their

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optical microscope (b), SEM images (c) of the 18 laser-scribed graphene circles with a diameter of 0.6 cm obtained

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by using different print modes, grayscale values, and contrast. The print modes are Best, Normal, Draft from left to

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right. The gray values are 0, 100, 200 from top to bottom. The 9 circles above and below are produced by Enhanced

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and Default contrast, respectively. The golden-brown region represents an untreated GO area; The black color

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represents LSG regions. Scale bar, 0.6 cm and 100 µm. (d) The AFM images and height analysis of 3D LSG

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micropatterns (see Fig. 1c, yellow boxes).

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We further examined morphology and chemical changes of the LSG micropatterns by using SEM, Raman

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spectrometer, X-ray photoelectron spectroscopic (XPS) analysis, and water contact angle (CA). As shown in Fig. 2a,

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untreated GO regions appear flat in contrast to the expanded and exfoliated laser reduced graphene oxide. Porous

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structure which size ranges from ~25 to ~200 nm could be found on the LSG surface (Fig. 2b, inset), mainly

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attributing to laser heating.18 As shown in Fig. 2c, the D peak of LSG Raman spectrum exhibits a little

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enhancement caused by the graphite edges at ~1354 cm─1, which is in agreement with SEM results (Fig. 2b). Its

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G-band peak displays a decrease and shift to smaller wavenumber from 1599 to 1593 cm─1, which indicates

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sp2-bonded carbons re-establishing and a defect decrease within basal planes.42 A prominent 2D peak is seen in

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LSG indicates conversion of GO into a reduced form and generation of few-layer graphene. The diminishment of

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an S3 peak at ~2928 cm─1 points to decreasing disorder for LSG.43 Fig. 2d-f shows the XPS results of GO and LSG.

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Fig. 2d reveals the significant change in carbon-to-oxygen ratios for GO (~2.3) and LSG (~8.3), which

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demonstrates that the LSG micropatterns are only partially reduced. The C1s peaks of GO (Fig. 2e) can be

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decomposed into carboxyl (288.1 eV), epoxy carbons (286.9 eV), hydroxyl carbons (284.6 eV), and π-π* peak

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(290.4 eV). Whereas in the LSG spectrum, sp2 C-C bonds peak obviously increases as well as oxygen-containing

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groups peaks greatly decreases (Fig. 2f). Simultaneously, the resulting LSG film has a better electrical conductivity

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(Fig. S8c), attributing to the declined oxygen content from 31% to 11.2%. Furthermore, after laser treatment, the

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CA of LSG remarkably increases from 49° to 77° (Fig. 2g), indicating enhancement of hydrophobicity,44 which can

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be attributed to removal of oxygen contents45 and enhancement of surface roughness.46

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Figure 2. The morphology and chemical changes of LSG micropatterns. (a) The SEM images of LSG

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micropattern and un-treated GO regions. The arrow indicates the laser scanning direction. (b) A zoomed-in view of

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LSG and GO regions in Fig. 2a, yellow boxes. The inset: The higher magnification SEM images of porous

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structures on the LSG surface and flat structures on the GO surface. Lightscribe parameters: Normal mode, gray

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value = 100, Default contrast. (c) Raman and (d-f) XPS comparisons between GO and LSG. (g) The black laser

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treated area (LSG) and golden-brown untreated area (GO) exhibit obviously different contact angles, respectively.

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Lightscribe parameters: Best mode, gray value = 0, Default contrast.

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The Effect of the LSG patterns on the Behavior of PC12 Cells. To investigate potential biomedical

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applications of LSG patterns, we firstly assessed the biocompatibility of GO and LSG substrates (produced by Best

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mode, gray value 0, Enhanced contrast, repeating 3 times) for neuron-like PC12 cell culture. According to the MTT

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and LDH results (Fig. 3a,b), there wasn’t significant difference in viability and LDH release of PC12 cells growing

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on GO and LSG substrates by comparing with cells seeding on cell culture plate (the control). For further 8


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evaluating the GO and LSG toxicity,47 we used a ROS indicator (DCFH-DA) to measure intracellular ROS changes

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after cultured on three substrates. As shown in Fig. 3c, the difference of ROS levels was not significant (P > 0.05)

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among GO, LSG, and the control group. The Hoechst 33258 staining results in Fig. 3d revealed that PC12 cells on

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the control had normal nuclei morphology. The nucleus on other two substrates did not show any significant

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difference across 48 h of culture, indicating no apoptotic cells present.48 These results suggest that GO and LSG

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substrates are non-cytotoxic.

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Figure 3. The biocompatibility of GO and LSG substrates for PC12 cell culture. Cell viability as assessed by

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MTT assay (a) and LDH concentration in the medium (b) after being cultured on the cell culture plate (control),

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GO and LSG (produced by Best mode, gray value 0, Enhanced contrast, repeating 3 times) for 1, 2, 3, and 4 days (n

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= 7 samples). (c) Effects of the cell culture plate (control), GO, LSG substrates on ROS generation in PC12 cells (n

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= 9 samples). (d) Representative fluorescence images of Hoechst-33258-stained PC12 cells cultured for 48 h on the

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cell culture plate (control), GO, LSG substrates (n = 3 samples). Scale bar, 10 µm. Data in a,b,d are mean ± s.e.m.

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from three experiments.

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3D flexible graphene micropatterns for directing cell alignment. To investigate cell interaction with the

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LSG micropatterns on clear glass coverslips, PC12 cells were differentiated on the different topography of LSG

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patterns for 48 h, and then stained with hematoxylin for easy to be observed by optical microscope. As shown in

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Fig. 4a-i, PC12 cells showed a good spreading and attachment to the GO surface or at the GO-LSG junction (for

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LSG patterns), and revealed strikingly different growth patterns on flat GO film, LSG dots, and LSG lines. Cells

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seeded on GO film showed randomly oriented growth (Fig. 4a, Fig. S10a, left). While cells on the LSG dot patterns

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started to appear more aligned organization with increase in density of the dots (Fig. 4b,c, Fig. S11a,b). Cells on the

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LSG line patterns grown and differentiated along the lines and further shown more elongated in a bipolar manner

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with decrease in spacing of lines (Fig. 4d-i, Fig. S10a, Fig. S11c-h). To further quantify the orientation of PC12

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cells on the LSG patterns, the corresponding optical microscope images (Fig. 4a-i) are analyzed by using the

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two-dimensional fast Fourier transform (2D FFT) approach, which converts spacial signals to mathematically

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defined optical data (Fig. 4a-i, insets). The 2D FFT power spectra for the randomly oriented cells differentiated on

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the flat GO (Fig. S10a, inset) and sparse LSG dot substrates showed a symmetric distribution of spots about the

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center (Fig. 4a,b, insets) which indicated no directionality, whereas the 2D FFT power spectra for aligned cells

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differentiated on the dense LSG dot and LSG line (Fig. S10a, inset) substrates were more oval, indicating cell

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alignment (Fig. 4c-i, insets). The anisotropy of the 2D FFT spectra were further quantified by analyzing

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long-to-short axis ratio. Value equal to or higher than 1 respectively reveal an isotropic distribution and a preferred

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direction of cell alignment.49 Values for GO and sparse LSG dot substrates were not obviously greater than 1.

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However, values for the dense LSG dot and LSG line substrates were more than 1.5 (Fig. S12), further proving the

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effect of GO-LSG-GO groove on aligning cell. Radial intensity plots of the aligned cells on dense LSG dots and

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LSG lines exhibited sharp peaks, while random cells on GO and sparse LSG dots exhibited random spikes (Fig.

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S13). According to the wind-Rose analysis, angle of PC12 cells on the LSG lines mainly distributed between ±30°

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(Fig. 4k,l). In contrast, when seeded on GO and LSG dots, PC12 cells showed less organized growth patterns,

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having a much wider variation of ±75° (for GO, sparse LSG dots) and ±52° (for dense LSG dots), respectively, (Fig.

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4j). These results support that the line topography of LSG may be the major determinant of alignment.

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Figure 4. Comparison of PC12 cell interaction with the different LSG patterns. Optical microscopic

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morphology of PC12 cells differentiated on the GO and different LSG pattern substrates (a-i) for 48 h after staining

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with hematoxylin. Insets represent the corresponding 2D FTT power spectra. Scale bar, 50 µm. (j-l) Angular

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histogram of orientation of PC12 cells from the images in a-i, showing cell elongation in the direction of LSG lines

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(n > 100 cells per group).

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Environmental scanning electron microscope (ESEM), in comparison to a conventional SEM, allows to

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directly visualize biological samples without additional sample drying or coating. To verify the effect of different

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topography of LSG lines on the alignment of PC12 cells, cell growth and differentiation on the five kinds of LSG 11



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line substrates fabricated by different laser-scribe parameters (Draft 1 time, Draft 2 times, Normal 1 time, Normal 1

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time + Draft 1 time, Best 1 time) were further investigated by SEM (Fig. S14) and ESEM (Fig. 5, S15). As

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illustrated in Fig. 5a, i-iv, the PC12 cells with neurites on the LSG line substrates with obvious GO-LSG-GO

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grooves preferred to attach on LSG regions (see statistical analysis in Fig. S16) and exhibited bipolar orientation

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along the line direction. The corresponding 2D FFT power spectra showed a much more oval shape, revealing cell

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alignment (Fig. 5a, insets). By analyzing long-to-short axis ratio, values for those substrates were obviously greater

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than 1 (Fig. S17). Moreover, radial intensity plots of those 2D FFT outputs consisted of sharp peaks, which further

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confirm a considerably high anisotropic orientation (Fig. S18). This finding was in agreement with optical

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microscope analysis of cells differentiated on LSG lines, further proving that cell alignment was attributed to line

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topography. However, as for LSG substrate (produced by the Best 1 time) without obvious GO-LSG-GO grooves

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(Fig. 1a, first circle on the first line, Fig. S7), the PC12 cells revealed a less aligned morphology (Fig. 5a,v).

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Moreover, the corresponding 2D FFT power spectrum was relative circular and its long-to-short axis ratio value is

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remarkably less than that with obvious GO-LSG-GO grooves. According to radial intensity plots, the PC12 cells on

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no GO-LSG-GO groove substrate had loads of low frequency signals, reflecting a low anisotropic orientation. The

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wind-Rose analysis indicated that PC12 cells on the LSG line substrates with obvious GO-LSG-GO grooves

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exhibited a confined alignment between ±15° (Fig. 5b, i-iv). In contrast, when seeded on the LSG substrate without

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GO-LSG-GO grooves, PC12 cells showed a much wider variation of ±30° (Fig. 5b, v). These results indicate that

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the topographical cue of GO-LSG-GO grooves plays a key role in the guidance of neurites growth.

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Figure 5. Typical ESEM images of PC12 cells differentiated on the LSG line patterns for 48 h. (a) Low and

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high magnification ESEM images showing different growth patterns of PC12 cells differentiated on LSG line

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patterns obtained by various print modes and times (i, Draft 1 time; ii, Draft 2 times; iii, Normal 1 time; iv, Normal

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1 time + Draft 1 time; v, Best 1 time). Other lightscribe parameters: gray value = 0, Enhanced contrast. Insets

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represent the corresponding 2D FTT power spectra. Scale bar, 20 µm. (b) Angular histogram of orientation of PC12 13



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cells from the images in Fig. 5a, showing cell elongation in the direction of GO-LSG-GO grooves (n > 100 cells

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per group).

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Conclusions

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Compared to most reported graphene-based patterns on certain substrates via other techniques,31-39 the

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reported laser-scribed process of 3D graphene-based micropatterns can selectively and precisely tune GO to rGO

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conversion and pattern GO-rGO-GO structures, which benefit to utilize the different properties (conductivity,

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hydrophily, adsorptivity) of GO and rGO for potential applications in electronics, biomedicine, energy. The current

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repurposing laser-scribing approach provides a standard, general and robust platform for micro/nano-fabrication in

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a time- and cost-effective manner, and can be easily extended to produce and pattern various graphene hybrids

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(such as polymer/graphene, metal nanostructures/graphene hybrids), other emerging nongraphene 2D materials,

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and their hybrids, which will fulfill the requirements to amend capability of graphene devices and fit them into

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more applications in various fields.

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3D graphene micropatterns with various sizes and shapes can be directly reused as highly anisotropic

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substrates for investigating cell behaviors in vitro, like cell alignment and cell-cell interactions. The demonstrated

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cell biocompatibility, differentiation, and alignment on the 3D graphene micropatterns substrates, which are

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essential in tissue engineering application, would inspire researchers to utilize various graphene hybrids with

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micro/nanostructures for important applications, like nanotechnology, biomedicine, biosensors, and energy.

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Although some methods can create higher resolution nanopatterns, laser-scribed technology has a competitive

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advantage due to its highly tunable and scalable fabrication capability with low cost. In future, the more powerful

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laser-scribed systems with higher precise and depth, can be achieved by simply improving the system of

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LightScribe DVD burner, such as laser wavelength (Blu-ray), power, beam focusing, and so on. Moreover, this will

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allow researchers to incorporate the flexible laser-scribed graphene-based mircopatterns for 3D culture of cells,

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tissues, and even organs to promote tissue engineering applications.

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Experimental Section

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Manufacturing of LSG patterns. LSG was obtained according to the Kaner group reported method.22-23 GO was

28

prepared according to the Hummer’s method.50 Briefly, 15 mL of the GO aqueous solutions (0.34 mg/ml) was

29

dropped onto a disk or a thin substrate glued to the DVD surface such as polyethylene terephthalate or glass 14


Page 15 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

coverslips for drying overnight. Various custom-made patterns can be inscribed at precise locations onto GO layer

2

via LightScribe-supported application software (SureThing Disc Labeler Gold 6), which controls the labeling

3

parameters, such as print modes, grayscale values, and contrast. Changing LightScribing parameters not only

4

tunably transforms yellow GO into different shade of black LSG patterns consisted of micropatterns (such as

5

microscale lines and/or dots), but also adjusts the spacing, density, width, height, and length of graphene

6

micropatterns.

7

Characterization of LSG patterns. The morphology of LSG patterns and untreated GO were observed via a

8

JSM-7100F scanning electron microscope with field emission function (JEOL Ltd., Japan). Contact angle

9

measurement was performed by a TX 500 H spinning drop interfacial tensiometer (KINO Ltd., USA). According to

10

our reported method,51 AFM and XPS characterization were performed on a Bruker Multimode-8 AFM/SPM

11

system and EscaLab 250 photoelectron spectrometer and analyzed with Gwyddion 2.30 and XPSPEAK41 software.

12

Raman spectra were observed via a Raman microspectrometer (Renishaw InVia, 514 nm Ar+ laser) in the spectral

13

range of 100–4000 cm–1. Optical microscope imaging of LSG patterns was performed on an Olympus BX51

14

microscope.

15

Cell culture. The PC12 cells (ATCC CRL-1721) were cultured in RPMI 1640 medium (Invitrogen), containing 10%

16

horse serum (Sigma) and 5% fetal calf serum (Sigma), penicillin (10 units/ml), and streptomycin (10 µg/ml) at

17

37 °C in a water-saturated atmosphere at 5% CO2. Before the cell seeding, the GO films and LSG patterns were cut

18

into proper size pieces, so that they can fit in 6-, 24- or 96-well plates. The GO films or LSG pieces with various

19

patterns were then sterilized by treating with 75% ethanol for 2 h, UV irradiation for another 1 h, lastly rinsed twice

20

in PBS solutions.

21

Cytotoxicity assay. Cells were divided into three groups: cell culture plate (control group), GO film (GO group),

22

and LSG substrate (LSG group, note that the LSG substrate produced by Enhanced, Best mode, gray value 0). The

23

MTT and LDH assay were utilized to compare cell cytotoxicity of control, GO, and LSG groups. PC12 cells (about

24

2×104 cells/well) were seeded on three substrates in the 96-well culture plate and incubated for 1, 2, 3, and 4 days.

25

Subsequently, MTT and LDH assay were performed by referring reported method52 and the Dojindo’s protocols

26

with ELx800 plate reader (Bio-Tek).

27

Fluorescence staining. After being incubated on three substrates for 48 h, PC12 cells were stained by Hoechst

28

33258 (Sigma)53 for 10 min or 2’,7’-dichlorofluorescin diacetate (DCFH-DA, see Beyotime’s protocols) for 30 min, 15



Page 16 of 22

1

and then observed with a Nikon ECLIPSE TE2000-u microscope. Note that for comparing different substrates, the

2

imaging parameters are consistent. The fluorescent intensity analysis of ROS was executed on at least five images

3

of two replicated experiments by using ImageJ.

4

Cell differentiation. After attaching for 10 h onto the different substrates, PC12 cells were stimulated with nerve

5

growth factor (100 ng/ml, Sigma), and its response to the different topographies is analyzed according to cell

6

alignment.

7

Hematoxylin staining. After being differentiated on three substrates for 48 h, the fixed PC12 cells were rinsed

8

three times in PBS, stained with hematoxylin solution (Aladdin Reagent, China) for 10 s and immediately rinsed

9

with double distilled water, finally photographed with a light microscope (Olympus BX51, Japan).

10

SEM imaging of cells. For SEM imaging, cells differentiated on the LSG patterns were fixed, dried, and then

11

observed.54 For ESEM imaging, cells differentiated on the LSG patterns were fixed with 4% paraformaldehyde and

12

2.5% glutaraldehyde, and then directly imaged in hydrated state at 20 kV using ESEM mode (Quanta 200, FEI,

13

USA). The signals were collected using a gaseous secondary electron detector (GSED).

14

Image analysis. The width, spacing, density, and length of microscale LSG lines and/or dots (or lines on label layer

15

of DVD) was directly obtained by using ImageJ software for analyzing their SEM or optical microscope images.

16

These pictures were also converted to grayscale images (8-bit), treated by using threshold function of ImageJ, and

17

then analyzed the laser-scribed area. The optical microscope and ESEM images of cell morphology on different

18

substrates were firstly adjusted by using the ImageJ threshold function, edited for removing the LSG lines or dots

19

by using the PhotoScape v3.7 software (http://www.photoscape.org/), processed by applying 2D FFT functionality

20

in ImageJ. The long to short ratio was analyzed by referring other report.49 The resulting 2D FFT power spectra

21

were analyzed with an oval profile plug-in.55 Cells alignment analysis was examined by measuring the angle

22

between neurites axis and the LSG lines or dots. Then the orientation distribution of each group was indicated by

23

using a Rose plot diagram.

24

Statistical analysis. Results were indicated as mean ± s.e.m. Continuous variables were compared by Student’s t

25

test. Statistical analysis was done using GraphPad Prism 5. P < 0.05 and > 0.05 represent significance or not,

26

respectively.

27 28 16


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1 2

Acknowledgements

3

This work was funded by the National Natural Science Foundation of China (No. 21505042 and 21675131),

4

Scientific and Technological Plan Project from Changsha of China (No. KQ1707010), Municipal Science

5

Foundation from Chongqing City (No. CSTC-2015jcyjB50001), Natural Science Foundation (No. 2016JJ3084),

6

Research Foundation of Education Bureau (No. 15K084), Cooperative Innovation Center about Engineering and

7

New Products of Developmental Biology (No. 20134486), and Open Foundation for Microbial Molecular Biology

8

(No. 2014-03) from Hunan Province, Doctoral Research Foundation (No. 150612) and Youth Foundation from

9

Hunan Normal University (No. 31403).

10 11

ASSOCIATED CONTENT

12

Supplementary Information. The details about hardware, software, and working principle of LightScribe

13

technology, the relations between the labeling parameters and the resulting LSG patterns, scalable fabrication of

14

various graphene macroscopic and microscale patterns, and comparison of PC12 cell interaction with the GO and

15

LSG line pattern (optical microscope images, SEM images, Radial sum intensity plot, long-to-short axis index,

16

Rose plot diagram), is available in Supporting Information. This material is available free of charge via the Internet

17

at http://pubs.acs.org.

18 19

Notes

20

Competing financial interests: The authors declare no competing financial interests.

21 22

Corresponding Author

23

*E-mail: [email protected]. Fax: (+86)731-8887-2905; Tel: (+86)731-8887-2905

24

*E-mail: [email protected]. Fax: (+86)23-6825-3237; Tel: (+86)23-6825-3237

25 26

Author contributions

27

All authors have given approval to the content of the manuscript.

28 17



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Page 18 of 22

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Highly tunable and scalable fabrication of 3D flexible graphene

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