Layer-by-Layer Assembled Bacterial Cellulose/Graphene Oxide Hydrogels with Extremely Enhanced Mechanical Properties
KeywordsBacterial cellulose Nanocomposite Graphene oxide Biosynthesis Nanofiber Hydrogels
A modified in situ static culture method (layer-by-layer assembly, LBLA) was developed.
The LBLA method ensures uniform distribution of graphene oxide (GO) in bacterial cellulose (BC) and makes very thick BC/GO hydrogels with homogeneous structures.
The BC/GO hydrogels show greatly enhanced mechanical properties over bare BC.
Nano-carbon materials, such as one-dimensional (1D) carbon nanotube (CNT) and two-dimensional (2D) graphene (GE) and graphene oxide (GO), are believed to be promising candidate materials for tissue engineering and regenerative medicine applications owing to their large specific surface area, high porosity, and excellent mechanical properties [1, 2, 3, 4, 5, 6, 7]. Among these carbonaceous nanomaterials, GO is considered a promising material for biological applications owing to its excellent biocompatibility, better dispersibility in water than GE, and abundant surface functional groups [8, 9, 10, 11, 12]. Furthermore, GO can support and accelerate adhesion, proliferation, and differentiation of various mammalian cells [12, 13, 14, 15].
Despite its solubility in water, GO tends to aggregate in physiological environments because of nonspecific binding to proteins , which will inevitably lower its reinforcement effectiveness when used as nanofillers in GO-based nanocomposites. To make full use of its good dispersion in water, GO has been used to reinforce a natural polymer, bacterial cellulose (BC), to form BC/GO nanocomposites. For instance, Feng et al.  prepared the BC/GO nanocomposite by mechanically mixing BC fragments with a GO aqueous dispersion. The obtained nanocomposites show a tensile strength of 242 MPa in the dry state (5 wt% GO), which is an improvement of 22% compared to that of bare BC (198 MPa). Liu et al.  prepared a BC/GO nanocomposite (in the dry state) with a tensile strength of 18.48 MPa by a one-step cross-linking method. In both cases, however, the obtained nanocomposites broke the intrinsically three-dimensional (3D) structure of the BC, which is the most precious feature distinguishing it from other natural polymers. Incorporation of GE or CNTs into the inner core of a 3D BC network, by filtration or post-immersion in a solution of GE or CNTs, is challenging because of the lack of large pores (> 20 μm) in pristine BC .
In order to retain the advantageous 3D structure of BC, Yoon et al.  reported a post-processing immersion method to fabricate BC-CNT nanocomposites by immersing BC pellicles in CNT solutions. However, this method cannot work when the BC pellicles are thick. More importantly, in the case of GO, the post-processing immersion method might not be feasible for the preparation of BC/GO nanocomposites, since GO is much larger than CNT and thus cannot enter the inner structure of BC pellicles. In our previous studies, a one-pot in situ biosynthesis approach was developed, and a BC/GO nanocomposite with homogeneous GO nanosheets in a BC matrix was successfully fabricated . The BC/GO nanocomposite (in a dry state) showed high mechanical properties and improved electrical conductivity, compared to those of the pristine BC. Unfortunately, neither post-processing immersion nor in situ biosynthesis could enable GO to penetrate the internal area of the BC network when the BC/GO hydrogel was thicker than 2 mm. Therefore, a great deal of effort is required to improve the uniform dispersion of GO in a 3D integrated BC matrix, particularly when thick BC/GO products are required.
Herein, we report a novel in situ layer-by-layer assembly (LBLA) method for fabricating thick (≥ 5 mm) BC/GO nanocomposite hydrogels with highly dispersed GO nanosheets bundled by 3D interconnected BC nanofibers. The sophisticated porous structures and improved mechanical properties of the as-prepared BC/GO nanocomposites were investigated carefully.
3.1 Materials and Methods
Yeast extract, tryptone, disodium phosphate (Na2HPO4), and acetic acid were used as-received for BC production. A commercially available aqueous dispersion of GO with a concentration of 0.5 mg mL−1 was purchased from Nanjing XFNANO Materials Technology Co. Ltd., China. The bacterial strain, Komagataeibacter xylinus X-2, was kindly provided by Tianjin University of Science and Technology, Tianjin, China.
3.2 Preparation of BC, c-BC/GO, and BC/GO
Prior to inoculation, the culture medium (pH = 4.5) of BC, composed of 2.5% (w/v) glucose, 0.75% (w/v) yeast extract, 1% (w/v) tryptone, and 1% (w/v) Na2HPO4, was sterilized at 121 °C for 30 min. This recipe has been reported in our previous work [22, 23]. To prepare BC/GO nanocomposite hydrogels, the GO suspension was added to the above-mentioned culture medium of BC under intense stirring for 60 min. A pure BC membrane (≈ 3 mm in thickness and denoted as BC0 hereinafter), prepared by a conventional static culture method, was placed in a culture dish. Afterward, the GO-dispersed culture medium was sprayed onto the surface of the as-obtained BC0 membrane, on which new BC grew at the interface of the BC0 membrane and the GO-dispersed culture medium, leading to a BC/GO film. When the medium was consumed, additional GO-dispersed culture medium was sprayed onto the surface of the newly prepared BC/GO film, and the second layer of the BC/GO film was produced. The process continued until the desired hydrogel thickness was reached.
GO content in various BC/GO nanocomposite hydrogels prepared in this work
Vculture medium/VGO dispersion
GO content with respect to BC (wt%)
3.3 Characterization Methods
Thermogravimetric analysis (TGA) was conducted using a TGA instrument (STA449F3) with a heating rate of 10 °C min−1 from room temperature to 800 °C. The Brunauer–Emmett–Teller (BET) surface area, pore size, and volume were measured by the nitrogen adsorption method using a surface area analyzer (NOVA 2200e).
Static tensile tests of BC and BC/GO hydrogels (dimensions: 50 × 10 × 2 mm3) were conducted using a micro-electromagnetic fatigue testing machine (MUF-1050, Tianjin Care Measure & Control Co., Ltd., Tianjin, China) at a strain rate of 0.1 mm s−1. At least five specimens were tested for each sample, and the averages and standard deviations were reported.
4 Results and Discussion
Surface chemistry of the BC/GO nanocomposites was examined by FTIR analysis (Fig. 3b). In the spectrum of BC, characteristic peaks (3348, 2892, 1429, and 1061 cm−1 due to –OH bonds, asymmetric stretching vibration of C–H, asymmetric angular deformation of C–H bonds, and antisymmetric bridge stretching of C–O–C, respectively [18, 32, 33, 34]) are observed. In the spectrum of GO, three peaks, centered at 3361, 1730, and 1621 cm−1, corresponding to the stretching vibrations of O–H, C=O, and C=C bonds [17, 35], respectively, are observed. The major characteristic absorptions of GO and BC are also noted in the BC/GO nanocomposites. It is noted that the peak intensity of the –OH group (3348 cm−1) decreases with increasing GO content in the BC/GO nanocomposites as compared with that observed with pure BC. In addition, an intense peak at 1579 cm−1 is noted, and its intensity increases with the GO content. This may indicate a strong interaction (hydrogen bonding) between BC and GO, which causes downshift of the GO C=O group band. The formation of hydrogen bonding is important for enhancing the mechanical properties of BC/GO composites [36, 37].
To further determine the surface chemistry of the BC/GO nanocomposites, XPS analysis was carried out, and the results are presented in Fig. 3c. Both BC and BC/GO-2 show almost identical wide-scan spectra (Fig. 3c1). Furthermore, it seems that the C 1s spectra of BC and BC/GO-2 are the same, both showing four peaks of C–C or C–H (284.6 eV), C–O (286.5 eV), O–C–O (287.5 eV), and O–C=O (288.5 eV). However, a careful comparison reveals a difference in the sub-peak intensity of C–C or C–H. As expected, the incorporation of GO improves the C–C or C–H peak strength of BC/GO-2.
Figure 3d presents the Raman spectra of GO and BC/GO-2. The two curves are similar, both showing a D band (1344 cm−1, which corresponds to the disordered structure of GO sheets) and a G band (1595 cm−1, which represents the first-order scattering of the E2g vibrational mode) [38, 39]. However, determination of the intensity ratio of D band to G band, ID/IG, reveals a significant difference between GO (0.86) and BC/GO-2 (0.97). The improvement in ID/IG of BC/GO-2 over GO can be ascribed to the removal of some oxygen-containing functional groups on the surface of GO , since GO can be deoxygenated in alkaline solutions  during sample preparation (boiled with a 0.5-M NaOH solution for 15 min and cleaned with 1 wt% NaOH for 2 days), in line with our previous work . As shown in Fig. 3d, besides the D and G bands, two peaks located at 2680 and 2931 cm−1 are also noted in the spectra of GO and BC/GO-2, which can be assigned to the 2D and D + G bands, respectively . The 2D band is highly sensitive to the stacking of graphene, and the intensity ratio of 2D band to G band (I2D/IG) is an important parameter to evaluate the layering of graphene materials [42, 43]. The I2D/IG ratios of single-, double-, triple-, and multi- (> 4) layer sheets are typically > 1.6, ~ 0.8, ~ 0.30, and ~ 0.07, respectively . The calculated I2D/IG is 0.66 and 0.68 for GO and BC/GO-2, respectively, which suggests a few-layered texture of GO in BC/GO-2, in accordance with the specification of the providers of the GO suspension.
BC/GO nanocomposites with a sophisticated nanostructure have been fabricated via a novel in situ LBLA strategy. The LBLA method involves two simultaneous steps: self-assembly of 1D BC nanofibers into a 3D structure and self-bundling of 2D GO nanosheets by 1D BC nanofibers. The BC/GO hydrogels show greatly improved mechanical properties over those of bare BC and other BC/GO counterparts prepared by conventional static culture methods. The intriguing nanostructure with strong hydrogen bonding, close mechanical bundling, and even distribution of 2D GO nanosheets throughout the 3D BC network are the main reasons why the LBLA-derived BC/GO hydrogels are ultra-strong. These results show that such a novel LBLA strategy has great promise in the development of high-performance BC-based hydrogels for various applications.
This work is supported by the National Natural Science Foundation of China (Grant Nos. 51572187, 51563008, 51662009, 31660264), the Provincial Natural Science Foundation of Jiangxi (Grant No. 20161BAB206149), and the Key Project of Natural Science Foundation of Jiangxi Province (Grant No. 20161ACB20018).
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