Three-dimensional patterning of solid microstructures through laser reduction of colloidal graphene oxide in liquid-crystalline dispersions

Graphene materials and structures have become an essential part of modern electronics and photovoltaics. However, despite many production methods, applications of graphene-based structures are hindered by high costs, lack of scalability and limitations in spatial patterning. Here we fabricate three-dimensional functional solid microstructures of reduced graphene oxide in a lyotropic nematic liquid crystal of graphene oxide flakes using a pulsed near-infrared laser. This reliable, scalable approach is mask-free, does not require special chemical reduction agents, and can be implemented at ambient conditions starting from aqueous graphene oxide flakes. Orientational ordering of graphene oxide flakes in self-assembled liquid-crystalline phases enables laser patterning of complex, three-dimensional reduced graphene oxide structures and colloidal particles, such as trefoil knots, with"frozen"orientational order of flakes. These structures and particles are mechanically rigid and range from hundreds of nanometres to millimetres in size, as needed for applications in colloids, electronics, photonics and display technology.


Introduction
Owing to unique physical properties 1,2 , graphene-based materials and devices are shaping the future of photonics, electronics, optoelectronics and energy applications [2][3][4][5][6][7][8] . For example, they promise to replace rare and brittle indium tin oxide transparent electrodes in the display industry 1,3 . The most effective, low-cost and scalable approach to prepare graphene based materials is the reduction of graphene oxide (GO) 9,10 , which can be produced in abundance. Graphene oxide can be reduced by multiple methods  , including chemical reduction 10 , photoreduction [6][7][8][10][11][12][13][14] and even reduction mediated by biological microorganisms 15 . Most of these methods require specific precursors and conditions, which make them expensive and unsuitable for large-scale production. Multiple lithographic methods 5,16 have been developed for post-production of reduced graphene oxide (rGO) and its micropatterning into useful structures and assemblies, but they are time and resource consuming and limited by advances in mask production. Recently, reduction of GO and micropatterning was achieved using laser irradiation [6][7][8][10][11][12][13][17][18][19][20][21][22][23]27 . However, its use so far has been limited to reduction and direct writing of patterns in dry two-dimensional (2D) solid films of GO.
Here we produce fully three-dimensional (3D) functional solid microstructures of rGO in an aqueous nematic liquid crystal (LC) of 2D GO flakes using pulsed near-infrared laser scanning and characterize them using nonlinear optical microscopy and other techniques. We show that photoluminescence of laser-reduced rGO is increased compared with pristine GO, which allows for using the same excitation beam for simultaneous reduction of GO into desired microstructures and their subsequent nonlinear photoluminescence imaging. Orientational order of LC phases promotes homogeneous internal structure of complex 3D rGO patterns. Our approach is reliable and scalable, does not require special conditions or chemical reducing agents and can be used in ambient conditions in high concentration aqueous dispersions of GO flakes. Because laser reduction with tightly focused femtosecond light is an intrinsically depth-resolved nonlinear process, it can produce layered, sandwiched, as well as fully 3D microstructures, including nonplanar surfaces such as those with the topology of trefoil torus knots, by programmable spatial steering of a tightly focused laser beam, without lithographic masks. Various 3D topologically nontrivial solid microstructures of rGO obtained with this approach can be used in electronic and optoelectronic devices and in basic research. clean, untreated glass substrates (Fig. 1a). Graphene oxide flakes (Fig. 2a) are hundreds of nanometres wide graphene sheets with a basal plane and edges decorated by oxygen-containing groups including carboxyls, hydroxyls, epoxides and others 10 , which cause screened electrostatic repulsion that stabilize the colloidal flakes against aggregation prompted by interlayer forces between flakes, making them both strongly hydrophilic and stable in colloidal dispersions. They can be dispersed in deionized water to form different lyotropic discotic LC phases at higher concentrations [29][30][31][32] . In this work, we use aqueous GO flakes at 0.25-1 wt% to achieve a stable self-assembled nematic LC phase (Fig. 1b,c and Supplementary Fig. 1). Flat GO flakes align edge-on at the interface with glass substrates 29,31 and spontaneously orient in the bulk so that normals to the surfaces of disk-like planes point roughly along the LC director n, which describes the average local orientation of normals to the flakes 29 Supplementary Fig. 3) at 850 nm, which allows for nonlinear, intrinsically depth-resolved imaging of nematic textures 33 in GO LCs 29 . On the other hand, illumination of a GO nematic with a pulsed laser beam of high laser energy density (fluence) results in a permanent physical change with properties drastically different from the pristine samples. We attribute this permanent change of illuminated area to a local reduction of GO flakes by a laser beam [6][7][8][10][11][12][13]27 . Figure 1b shows a bright-field micrograph of a square area illuminated by a laser beam of high fluence, E  60-70 mJ cm -2 . Its appearance is dark (Fig. 1b) and its transmittance T is lower (Figs 1d, 3a) than through the unaffected bright area. At the same time, the laser-treated area produces significantly higher intensity of photoluminescence, I PL (Figs 1e, 2e, 3c), allowing for the simultaneous nonlinear photoluminescence imaging of a produced pattern (Fig. 1c). Owing to the intrinsic depthresolved nature of this nonlinear optical process 34,35 , the laser reduction of aqueous GO is spatially localized in a small volume of about 300 nm in diameter defined by optical resolution [33][34][35] and called a voxel. This 3D localization allows for voxel-by-voxel micro-patterning of rGO not only in the plane of the sample (Fig. 1b,c,f) but also across the sample thickness (Fig. 1g), which sets our approach apart from previous techniques [6][7][8][10][11][12][13][17][18][19][20][21][22][23]27 of photothermal or photo-induced reduction of GO and 2D micropatterning in thin dried films. Three planes of rGO, whose thickness is determined by optical resolution, were produced at different heights within a thick GO nematic dispersion sample (Fig. 1f) using a tightly focused laser beam. This is achieved without employing multi-layer lithography 5 .
Programmable control of a scanning laser beam allows for precise voxel-by-voxel reduction of GO and micropatterning not only in-plane at different heights (Fig. 1f,e) but also continuous reduction across a thick layer of a GO nematic ( Supplementary Fig. 4).

Characterization of rGO.
To probe how a femtosecond pulsed laser beam at 850 nm modifies aqueous GO samples, we use ultraviolet-visible ( Fig. 2e and Supplementary Fig. 2), Raman ( Fig. 2f and Supplementary Fig. 5) and X-ray photoelectron (XPS; Fig. 2g,h) spectroscopies, as well as optical  (Fig. 2f), which can be attributed to removal of oxygen-containing groups 8,10 , as we additionally verify using XPS. Figure 2g,h shows XPS spectra, which clearly indicate the removal of oxygen-containing groups during the 850-nm laser irradiation of GO in water. XPS survey spectra ( Fig. 2g) show signals of carbon and oxygen from both GO and rGO, however, the intensity of the oxygen peak O1s from an rGO area is significantly decreased, indicating that oxygen was partially removed. The high-resolution C1s XPS spectra (Fig. 2h) were deconvoluted into peaks corresponding to carbon in, respectively, C-C, C-OH, C=O and O=C-OH functional groups, which allows us to estimate the relative content of carbon not bound to oxygen as ~ 45.8% in GO and ~ 58% in the rGO area. The observed levels of reduction (Fig. 2g,h) are similar to those reported in previous studies of 2D laser reduction in solid thin films 11 . In addition, measurements of resistance ( Supplementary Fig. 6) of produced plain rGO stripe microstructures, which were performed using methodology similar to that developed for the study of photoreduction of GO in solid thin films 11 , indicate that rGO microstructures are more conductive as compared with the electrically insulating GO.
Differences in physical properties between pristine and laser-reduced GO are also detected by optical spectroscopy (Fig. 2e) and optical bright-field and nonlinear photoluminescence microscopy ( Fig. 3). The absorbance of rGO areas increases in the visible range and the onset of the absorbance peak is redshifted ( Supplementary Fig. 2b). The transmittance of rGO within the visible range is decreased (Fig. 3a) as they change appearance from transparent yellowish to opaque dark brown ( microscopic observations (Fig. 1b,c) allow concluding that for the used parameters of the irradiation process the onset of reduction is not very sharp. Experiments also show that at high laser powers, E >> 100 mJ cm -2 , the process most probably can become more violent with rapid expulsion of water and oxygen and damaging integrity of a sample. We therefore constrain the irradiation parameters so that the onset of reduction and rGO flake aggregation into microstructures is smooth and continuous and dependent on duration of irradiation (Fig. 3). Importantly, the laser patterning is done through scanning of a tightly focused beam to reduce the structure voxel-by-voxel, without causing bubbles or disrupting orientational ordering of the flakes. Progressive reduction of GO flakes by the scanned beam focused to a volume <1 m 3 and diffusion of flakes around the region of scanning allow us to avoid flows and non-homogeneity of the GO dispersion in the region of reduction. As the GO flakes within the micro-patterned structure collapse atop of each other due to lowering of electrostatic repulsion forces caused by reduction, water is displaced from inter-flake regions continuously during the scanning, so that the effect of these localized flows on GO flake ordering can be neglected. Solid structures of rGO are rigid and preserve their shape even under mechanical stress induced, for example, by the flow of LC during shear of confining glass plates ( Supplementary Fig. 8a,b). The rGO structures produced in the bulk of the LC fluid are permanent colloidal objects and, at normal conditions, do not "dissolve" in an aqueous GO dispersion. For example, such microstructures in the form of square-shaped sheets of rGO could be re-dispersed in water and then transferred onto solid substrates for scanning electron microscopy (SEM) imaging, an example of which is shown in the inset of Fig. 1c. Thus, this approach may allow for facile production of not only surface-attached micro-and nano-structures, but also complex-shaped colloids 34 Fig. 3 and Supplementary Fig. 10). Figure 4 shows different complex multi-level patterns of rGO produced in a bulk and at a surface of aqueous GO nematic and imaged by optical and photoluminescence microscopy. Using transmission-mode bright-field microscopy, one can see all the elements of micropatterns at the same time, but blurred depending on the location of the microscope's focal plane relative to the features of micropatterns (Fig. 4a,d,f): an image is focused at a single level (Fig. 4a) of rGO "nanostairs" going from the top to the bottom substrate, or it is focused on the different parts of the text (Fig. 4d,f) Supplementary Fig. 6), can be envisaged as prototypes of electronic elements for applications in electronics and optoelectronics, such as thin film capacitors 6,18,20 and field transistors 6 . Importantly, our approach is suitable for direct mask-free production of not only in-plane or sandwiched structures and devices 6,7,12,14,22 but also fully 3D interconnecting patterns, structures and nonplanar surfaces, which is one of the key advances as compared with, for example, in-plane structures presented in a pioneering study on photoreduction of GO in solid thin films 11 . To further demonstrate this capability, we produce an array of rGO trefoil knots ( Because the internal orientation of rGO flakes within the colloidal structures matches that of the surrounding GO flakes, the fabricated knotted particles differ from the polymerized ones studied recently 34 in that they do not induce noticeable director distortions or topological defects (Fig. 5). This demonstrates that topological defects can be avoided when the easy axis (boundary conditions) for the director orientation on the surface of complex-shaped particles is such that it mostly matches that of the surrounding LC, even when the colloidal inclusions exhibit nontrivial surface topology of torus knots.

Discussion
We have demonstrated selective reduction of GO flakes in the bulk of an aqueous LC dispersion of GO flakes at ambient conditions using nonlinear excitation by a near-infrared femtosecond laser light, which allows for mask-free micropatterning of 3D solid planar and nonplanar structures and surfaces of reduced GO. Besides its simplicity, this reliable, scalable method does not require special conditions or chemical reduction agents and can be implemented at ambient conditions in aqueous dispersions of GO flakes. Advantages of micropatterning in a discotic nematic LC phase include the possibility of producing patterns having more homogeneous internal structure and the potential to attain "frozen" orientational order of GO flakes, which can also be modified before reduction by applying an external fields 32 or using other stimuli. Our approach can be used for scalable production of graphene-based devices for photonics, electronics 20 , optoelectronics 12 , information 22 and energy storage 7 applications. Furthermore, solid colloidal particles of rGO produced here are enabling new explorations in other research areas such as, for example, for probing the interplay of topologies of surfaces, molecular fields and defects in soft condensed matter 34 . In this regard, the ribbon-like shape of thin rGO colloidal structures may allow for obtaining colloids with surfaces lacking orientability, such as Möbius strips 39 , which would be otherwise difficult to produce using techniques like two-photon polymerization 34 .

Methods
Preparation of aqueous dispersions of graphene oxide flakes. The improved, mostly single-layer GO flakes used in this work were synthesized by methods 28 modified for large-scale production and obtained as a dispersion in deionized (DI) water at a concentration of 2.5 g l -1 (0.25 wt%). The synthesis of GO flakes and their purification procedure was as following: 3 g of xGnP 5-m-graphitenanoplatelets (XG Sciences, Inc.) and 200 ml of 9:1 H 2 SO 4 /H 3 PO 4 solution were added to a 1-l Pyrex beaker. The mixture was set to stir continuously using an IKA Werke RW 16 mechanical stirrer.
Slowly, 9 g of KMnO 4 were stirred into the mixture, which turned a deep dark green colour.
Precautions were taken to avoid exceeding ~ 5 wt% of KMnO 4 per addition and to assure that the change in colour from green to purple is complete before introducing more oxidant (this is important as solutions with more than 7 wt% of KMnO 4 added to H 2 SO 4 could explode upon heating). The mouth of the beaker was covered as much as possible using aluminum foil and the reaction was heated to 40C using a resistance-heated water bath. After 6 h, the mixture had thickened and turned from dark green to purple-pink. At this point, additional 9 g of KMnO 4 were added and allowed to react for another 6 h. When the reaction was completed, the resulting slurry was quenched over 200 ml of icewater containing 5 ml of 30% H 2 O 2 . The resulting bright yellow GO suspension was purified by a series of centrifugations and resuspensions in a pure solvent. All centrifugations were performed at 4,000 r.p.m. for 90 min, and all re-suspensions were performed by shaking the centrifuged GO for To retrieve a produced rGO microstructure for SEM imaging, a sample was disassembled and the GO dispersion, together with the rGO microstructure, was released into a Petri dish with DI water. Then, after "washing" it in DI water, the rGO microstructure with some water was soaked into a pipet tip and placed onto a silicon wafer (Supplementary Fig. 8c). Once the residual water evaporated, we performed SEM imaging.
Laser-induced reduction and optical characterization. The integrated 3D laser induced reduction and nonlinear photoluminescence imaging of GO aqueous samples was performed at room temperature using a multimodal nonlinear optical microscopy setup 33 coupled to an inverted Olympus microscope IX-81 ( Supplementary Fig. 3). A tunable (680-1,080 nm) Ti:sapphire oscillator (140 fs, 80 MHz, Chameleon Ultra II, Coherent) was used as an excitation source. The excitation beam was directed to the sample by a system of mirrors (DMs) and focused into the sample using the Olympus high numerical aperture (NA) oil objective UPLSAPO 100/NA = 1.4 (OL1). The spatial 3D position of the excitation beam in the volume of the sample was controlled with the galvanomirror scanning unit (Fluoview FV300, Olympus). An average laser power for photoluminescence imaging was controlled by a pair of Glan polarizer and half-wave plate to be <1 mW in a sample to prevent the photo-and thermal damage. The excitation of GO flakes was performed at 850 nm ( Supplementary Fig. 2a), and the unpolarized photoluminescence light was detected in a range of ~ 400-700 nm (Fig. 2e) in a backward mode with a Hamamatsu photomultiplier tube H5784-20 (PMT1). The transmission-mode single wavelength bright-field image was collected in a forward mode with PMT2. The polarization of excitation could be varied using a half-or quarter-wave retardation plate mounted immediately before an objective. The same setup was utilized for a 3D reduction of aqueous GO flakes using a pulsed laser beam at 850 nm and laser fluence E < 100 mJ cm -2 . The dwell time, or scanning speed, was also controlled by a scanning unit FV300. Olympus Fluoview software was used for data acquisition and image reconstruction, and ImageJ software was used for data processing and analysis. Polarizing microscopy observations of samples in visible light and measurements of photoluminescence spectra were performed using the same IX-81 equipped with crossed polarizers, CCD camera (Flea, PointGrey) and a spectrometer USB2000 (OceanOptics) mounted onto a microscope. Raman spectra were measured using Renishaw inVia Raman microscope with excitation at 514.5 nm and low power to reduce unwanted heating or optical effects induced by a laser. XPS spectra were collected using a PHI Quantera XPS microprobe (Physical Electronics, Inc.) and SEM imaging was performed using a Zeiss Auriga FIB-SEM instrument.     Supplementary Figure S6 | Current-voltage dependence of an rGO thin film resistor. Currentvoltage characteristics of a single rGO ribbon of dimensions ~600 m  50 m  0.5 m (length  width  thickness) that was micropatterned using laser reduction. The resistance data for the thin film of rGO were measured by a multimeter at the frequency of 1 kHz. Resistance of the rGO-resistor was evaluated from a linear fit as R  61.5 k.