*Article* **Two-Photon–Near Infrared-II Antimicrobial Graphene-Nanoagent for Ultraviolet–Near Infrared Imaging and Photoinactivation**

**Wen-Shuo Kuo 1,2,3,† , Yen-Sung Lin 4,5,† , Ping-Ching Wu 6 , Chia-Yuan Chang 7 , Jiu-Yao Wang 3 , Pei-Chi Chen 3 , Miao-Hsi Hsieh 3 , Hui-Fang Kao 8 , Sheng-Han Lin 9, \* and Chan-Chi Chang 10, \***


**Abstract:** Nitrogen doping and amino group functionalization through chemical modification lead to strong electron donation. Applying these processes to a large *π*-conjugated system of graphene quantum dot (GQD)-based materials as electron donors increases the charge transfer efficiency of nitrogen-doped amino acid-functionalized GQDs (amino-N-GQDs), resulting in enhanced twophoton absorption, post-two-photon excitation (TPE) stability, TPE cross-sections, and two-photon luminescence through the radiative pathway when the lifetime decreases and the quantum yield increases. Additionally, it leads to the generation of reactive oxygen species through two-photon photodynamic therapy (PDT). The sorted amino-N-GQDs prepared in this study exhibited excitationwavelength-independent two-photon luminescence in the near-infrared region through TPE in the near-infrared-II region. The increase in size resulted in size-dependent photochemical and electrochemical efficacy, increased photoluminescence quantum yield, and efficient two-photon PDT. Therefore, the sorted amino-N-GQDs can be applicable as two-photon contrast probes to track and localize analytes in in-depth two-photon imaging executed in a biological environment along with two-photon PDT to eliminate infectious or multidrug-resistant microbes.

**Keywords:** sorted-graphene quantum dot; excitation-wavelength-independent photoluminescence; two-photon photoinactivation; near-infrared-II two-photon bioimaging; multi-drug resistant microbe

#### **1. Introduction**

Graphene quantum dot (GQD)-based materials with *π*–*π* configurations and surface groups have a high surface area, large diameter, and excellent surface grafting. These materials may cause intrinsic–state and defect-state emission to achieve photoluminescence

**Citation:** Two-Photon–Near Infrared-II Antimicrobial Graphene-Nanoagent for Ultraviolet–Near Infrared Imaging and Photoinactivation. *Int. J. Mol. Sci.* **2022**, *23*, 3230. https://doi.org/ 10.3390/ijms23063230

Academic Editor: Ana María Díez-Pascual

Received: 17 January 2022 Accepted: 1 March 2022 Published: 17 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(PL). Intrinsic–state emission is induced by the quantum size effect, zigzag edge sites, or the recombination of localized electron–hole pairs; by contrast, defect-state emission originates from defect effects (energy traps) [1,2]. The PL emission of a material determines its suitability for imaging and photochemistry [3]. GQDs can be bonded with nitrogen atoms (N-GQDs) to alter their chemical composition and modulate their band gap, enhancing their photochemical properties and facilitating the fabrication of tunable luminescence in bioimaging and photodynamic (or photoinactivation) applications [4,5]. In addition, primary amine molecules (also known as amino group–functionalized molecules) can be chemically modified to cause strong electron donation and significantly alter the electronic properties of nitrogen–doped amino acid–functionalized GQD (amino-N-GQD) materials, increasing electrochemical and photochemical activities [6].

Combining multiphoton and near-infrared (NIR) excitation is an effective approach for investigating photoexcitation. This approach involves less absorption and a shorter photoexcitation period than other types of excitation. Moreover, this approach involves ultra-low energy consumption. These attributes enable the deep penetration of biological specimens and effective observation [7]. This study used a novel inverted optical microscopy system with a femtosecond Ti-sapphire laser with a repetition rate of 80 MHz and optical parametric oscillators (Mai Tai Spectra-Physics, Santa Clara, CA, USA; Scheme S1). The derived amino-N-GQDs with quantum confinement in the sp<sup>2</sup> domain and intrinsic– state and defect–state emissions can cause an excitation wavelength–dependent PL phenomenon. Therefore, the amino-N-GQDs were sieved through membranes with pores of various sizes to ensure that they exhibited homogeneous atom doping functionalities, which enabled the investigation of electronic and intrinsic properties related to optical behavior under quantum confinement effects [8,9]. This phenomenon results in excitation wavelength-independent two-photon luminescence (EWI-TPL) emission at a two-photon excitation (TPE) wavelength of 960 nm in the NIR-II region [7,10]. X-ray photoelectron spectroscopy (XPS) revealed the enlargement of the sorted amino-N-GQDs, which increased the number of C–N groups and pyridinic-, amino-, and pyrrolic-N functionalities. This increase could induce a radiative recombination of localized electron-hole pairs, resulting in significant two-photon properties, including favorable two-photon absorption (TPA), high TPL emission, excellent absolute TPE cross-sections, a short lifetime, a high ratio of radiative to nonradiative decay rates, and high post-TPE stability. In addition, the mean lateral size increased, which resulted in a high PL quantum yield (QY) and high efficiency in photodynamic therapy (PDT, or photoinactivation) under TPE [excitation wavelength (Ex): 960 nm; ultralow energy: 222.7 nJ pixel−<sup>1</sup> ; photoexcitation period: 100–170 scans; total effective exposure time: 0.65–1.11 s]. The results indicated that the sorted amino-N-GQDs are promising as two-photon contrast probes for tracking and localizing analytes in detail in two-photon imaging (TPI) of a biological environment in two-photon PDT; they can be used to eliminate infectious microbes effectively.

#### **2. Results**

Amino-N-GQDs were synthesized from graphene oxide sheets through ultrasonic shearing according to the modified Hummers' method (Figure S1, Table S1, and Scheme S2a) [11]. XPS indicated that the as-prepared amino-N-GQDs with homogeneous O and N distributors exhibiting high crystallinity and uniformity were sieved through membranes with pores of various sizes (Figures S2a–d, S3 and S4). Low-magnification and high-resolution transmission electron microscopy (HR-TEM; inset images in Figure S2a–d) were used to characterize the amino-N-GQDs. The mean lateral sizes of the sorted dots were set to 9.1 ± 0.2 nm (amino-N-GQD 9.1), 9.9 ± 0.2 nm (amino-N-GQD 9.9), 11.1 ± 0.3 nm (amino-N-GQD 11.1), and 12.0 ± 0.3 nm (amino-N-GQD 12.0). Further characterization results indicated that the sorted amino-N-GQDs were successfully prepared (Figures S3–S5).

Zigzag edge sites, localized electron–hole pairs, and quantum effects have been used to induce intrinsic-state emission in GQD-based materials; however, defect effects (energy traps) have been used to trigger defect-state emission [2,6,7]. To demonstrate such

effects, Figure 1a displays the sorted amino-N-GQD dispersions, various levels of PL emission (gray-level images), dots with slight variations in size, and wavelengths at 630 nm encompassing the NIR-I window. The *x*–*y* focal point and *z*-axis resolution (full width at half maximum, FWHM) of the laser system were set to approximately 0.45 and 0.90 µm, respectively (Figure 1b). Satisfactory TPA in the NIR-II window was measured in subsequent experiments using the custom femtosecond Ti-sapphire laser optical system displayed in Scheme S1 (for details on the system, please refer to the Materials and Methods section), with an extension of approximately 960 nm (Figure 1c). Applying the most effective excitation wavelength can significantly enhance the two-photon properties of the materials used for bioimaging with TPEs [12]. Figure 2a shows the TPL spectra of the increase in the size of the sorted amino-N-GQDs, indicating red-shifted peaks of amino-N-GQD 9.1, amino-N-GQD 9.9, amino-N-GQD 11.1, and amino-N-GQD 12.0 at approximately 719, 772, 810, and 862 nm, respectively, in the NIR region under TPE (power: 222.7 nJ pixel−<sup>1</sup> ; scans: 20 or 170; total effective exposure time: approximately 0.13 or 1.11 s; Ex: 960 nm). The emission peaks determined via PL spectrophotometry for amino-N-GQD 9.1, amino-N-GQD 9.9, amino-N-GQD 11.1, and amino-N-GQD 12.0 were observed at approximately 719, 772, 810, and 862 nm, respectively, and they exhibited EWI-PL features (Figure S6). XPS revealed that the electron redistribution increased as the number of carbonyl groups increased (Figure S3), which decreased the energy gaps, resulting in TPL red shifts [13]. The quadratic dependence of the TPL increases with TPE power during this process [14]. Figure 2b demonstrates the existence of a two-photon process with an exponent of 2.00 ± 0.02 for sorted dots and conventional fluorophore (e.g., rhodamine B and fluorescein; Figure 2b). μ −

**Figure 1.** (**a**) Photographs of materials without and with 630 nm (gray-level) light excitation. (**b**) *z*-axis scan of thin gold film for measuring the second harmonic generation signal at various positions. The laser system's *z*-axis resolution (full width at half maximum, FWHM) was 0.90 µm (fit using the Gaussian function). (**c**) Relative TPA spectra of the sorted amino-N-GQDs. TPE signals were obtained at 900–1000 nm and at 127.3 nJ pixel−<sup>1</sup> . Delivered dose: 0.75 µg mL−<sup>1</sup> material.

− − − μ <sup>−</sup> **Figure 2.** (**a**) Relative TPL spectra of materials at a TPE power of 222.7 nJ pixel−<sup>1</sup> [20 and 170 scans (total effective exposure times: ~0.13 and 1.11 s), respectively; cut off = 900 nm, determined using cascading filters]. (**b**) TPL intensity dependence on the excitation power (logarithm) of materials and fluorophores; the slope is approximately 2.00 <sup>±</sup> 0.02. TPE power = 1272.8–5091.2 nJ pixel−<sup>1</sup> ; *R* <sup>2</sup> > 0.999. (**c**) Two-photon stability of the amino-N-GQDs, rhodamine B, and fluorescein at a TPE power of 222.7 nJ pixel−<sup>1</sup> with 20, 100, and 170 scans. The normalized integrated area was calculated by dividing the emission intensities of the integrated area after photoexcitation by those of the newly prepared material without photoexcitation. Delivered dose: 0.75 µg mL−<sup>1</sup> material. Data are presented as means ± standard deviations (*n* = 6).

μ

<sup>−</sup> μ <sup>−</sup>

− *π π* → The sorted amino-N-GQDs with homogeneous O and N functionalities can be used to investigate the intrinsic electronic properties related to optical behavior with quantum confinement, leading to EWI-TPL under TPE. The sorted amino-N-GQDs exhibited two-photon stability, which could be attributed to the limited photobleaching due to the post-TPE TPL intensity of the dots (Figure 2c). Rhodamine B and fluorescein's fluorescence demonstrated low robustness against photobleaching upon TPE exposure (power: 222.7 nJ pixel−<sup>1</sup> ; scans: 20, 100, or 170; total effective exposure time: approximately 0.13, 0.65, or 1.11 s). Ultraviolet photoelectron spectroscopy revealed *n*–state levels that were maintained at approximately the same energetic positions (6.6–6.8 eV; Figure S7), regardless of the size determined through TEM and Raman spectroscopy (Figures S2 and S5), confirming the highest occupied orbital level of the sorted dots. The quantum confinement resulting from the particle size regulates the wavelengths of radiative transitions. The EWI-TPL emissions from the sorted amino-N-GQDs indicate the absence of trap states between the *n*-state and *π*\* energy levels. A change in particle size did not cause any disturbance at the *n*-state level. The EWI-TPL of the sorted dots could be attributed to *π*\*→*n* recombination, which triggers electron transition and phonon scattering. Measurements revealed that the absolute fluorescence QY [15] of the materials ranged from 0.39 (for amino-N-GQD 9.1) to 0.48 (for amino-N-GQD 12.0); these values are higher compared to those documented in other studies [16,17]. Desirable yields were achieved because of the electron-donating species in the sorted amino-N-GQD structure. XPS revealed that many C–N configurations functioned as electron-donating species and increased the material QY through the inhibition of nonradiative transitions (Figure S3). However, the low QY was because of the large number of electron-withdrawing carbonyl functional groups acting as non-radiative trap centers

(Figure S3). Characterization of the sorted amino-N-GQDs revealed that the successfully prepared GQDs exhibit EWI-TPL characteristics. However, a large cross-section is typically preferred for monitoring molecular actions. The sorted amino-N-GQDs exhibited a large absolute TPE cross-section, ranging from 55,946 to 60,728 Goeppert-Mayer (GM) units (1 GM = 10−<sup>50</sup> cm<sup>4</sup> s photon−<sup>1</sup> ), which was more than 2900 times the magnitude of fluorescein (~19.2 GM; Table 1). The absolute TPE cross-sections of the amino-N-GQD 9.1, amino-N-GQD 9.9, amino-N-GQD11.1, and amino-N-GQD12.0 were approximately 55,946, 57,332, 59,051, and 60,728 GM, respectively (Table 2; for detailed calculations, please refer to the Materials and Methods section). This difference indicates a high ratio of energy absorption to energy input in the biospecimens. This phenomenon is highly favorable for TPI [18]. Moreover, the TPI emissions of the sorted dots (Figure 3a–d) occurred on a surface through the two-photon process, as shown in Figure 2b (power: 222.7 nJ pixel−<sup>1</sup> ; scans: 20; total effective illumination: ~0.13 s; Ex: 960 nm; scan rate: 6.53 ms scan−<sup>1</sup> ; scan area: <sup>200</sup> <sup>×</sup> <sup>200</sup> <sup>µ</sup>m<sup>2</sup> ; for details regarding the calculation, see the Materials and Methods).

**Table 1.** TPE cross-section of materials at an excitation wavelength of 960 nm. Delivered dose: 0.75 µg mL−<sup>1</sup> material.


<sup>a</sup> Rhodamine B was selected as a reference to determine the TPE cross-section. The relevant calculations are shown in the Materials and Methods section; <sup>b</sup> Forster, L.S.; Livingston, R. The absolute quantum yields of the fluorescence of chlorophyll solutions. *J. Chem. Phys.* **1952**, *20*, 1315–1320.



Because the inverted optical microscopy system was not suitable for investigating in vivo assay processes, the biological environment was mimicked by embedding an *Escherichia coli* (*E. coli*; 3.98 ± 1.37 µm in length and 0.98 ± 0.34 µm in width, calculated from 400 counts of bacteria) strain in a collagen matrix [19]. The TPI action occurred at a specimen depth of 180 µm under TPE (power: 222.7 nJ pixel−<sup>1</sup> ; scans: 20; total effective illumination: ~0.13 s; Ex: 960 nm; Figure 3e,f). Bacteria were observed under TEM (Figure 4a), but they were undetectable in the TPL images (Figure 3(e-1)), similar to the collagen matrix (Figure 3(e-2)). Lipopolysaccharides (LPSs) are major components of the outer membrane of *E. coli*. The physiologically stable and biocompatible sorted-amino-N-GQDs (Table S2 and Figure S8; a selected concentration of 0.75 µg mL−<sup>1</sup> material was used in sequential experiments conducted in the dark) were coated with anti-LPS antibody (AbLPS) through electrostatic interaction to increase efficiency and specificity (Scheme S2b). This resulted in the absorption of a substantial amount of sorted dot–AbLPS on the surface of the bacteria. No exceptional morphology (Figure 4b) was observed on the surface of the bacteria. In contrast, when the GQD size was increased using amino-N-GQD 9.1, amino-N-GQD

9.9, amino-N-GQD11.1, and amino-N-GQD12.0, a high fluorescence QY and large crosssection was detected in the TPL images (Figure 3f). However, the bacteria treated with the photoexcited material–AbLPS hybrid were severely damaged when the power was increased to 222.7 nJ pixel−<sup>1</sup> with 100 or 170 scans (with a total effective illumination of ~0.65 or 1.11 s), which resulted in abnormal morphology, as observed through TEM (Figure 4c,d). TPL decreased after 100 scans (Figure 3g) and was undetectable after 170 scans (Figure 3h) at a depth of 180 µm. For unlabeled bacteria, two-photon autofluorescence (TPAF) was not observed for the intrinsic fluorophores under TPE at the same power (Figure 3i). By contrast, TEM images revealed limited attachment and nonspecific binding for the surface of the *E. coli* strain (without antibody coating) treated with the sorted dots (power: 222.7 nJ pixel−<sup>1</sup> ; scans: 20; Ex: 960 nm; Figure 4e). Subsequently, TPI revealed almost no TPL emission at 180 µm (Figure 3j). Therefore, the *E. coli* strain treated with the photoexcited sorted dots exhibited a normal morphology even after photoexcitation (power: 222.7 nJ pixel−<sup>1</sup> ; scans: 170; Ex: 960 nm; Figure 4f). Under the same conditions, a clear TPI without TPL emission was observed for bacteria without the antibody-coated materials (Figure 3k). However, the images captured at a depth of >180 µm contained spherical aberrations, which severely degraded the image quality. Such aberrations were caused by the mismatch between the refractive indices of the aqueous sample and the maximum *z*-depth of the optical laser system, as well as the influence of the set objective, detection efficiency, and maximum *z-depth* of the optical laser system [20]. Therefore, TPI was not detected at a depth of 200 µm for all the sorted dots (Figure S9). In this study, the maximum *z-depth* for the detection of TPL emission with the laser optical system was 180 µm. This can be attributed to the detection efficiency and set objective, which was set to the depth to obtain the optimal resolution for examining the amino-N-GQDs used as two-photon contrast probes, particularly the sorted amino-N-GQDs with a large lateral size.

The changes in bacterial cell walls and oxidation were examined. The deterioration of the surrounding biological surface substrates was attributed to the reactive oxygen species (ROS) observed through PDT under TPE. These changes could cause bacterial atrophy, morphological damage, and distortion (Figure 4c,d) because of amino-N-GQD desorption from the bacterial surface (Figure 3g,h). The LIVE/DEAD kit was used to investigate the green fluorescence of the living bacteria and an additional incubation time of 3 h was necessary to induce PDT action effectively, leading to the elimination of bacteria (amino-N-GQD 12.0 was used to conduct this experiment; viability > 99%; Figure S10a). The results indicated that the bacteria were almost completely undamaged by exposure to laser treatment (power: 222.7 nJ pixel−<sup>1</sup> ; scans: 170; total effective illumination: ~1.11 s; Ex: 960 nm). The bacteria treated with the photoexcited amino-N-GQD 12.0–AbLPS hybrid without incubation were also nearly undamaged (viability > 99%; Figure S10b). After 3 h of additional incubation, the same panel was observed, and the results indicated that the dead bacteria were somewhat distinguishable (represented by red fluorescence in Figure S10c). Bacterial viability was then quantified for further antimicrobial testing, which revealed nearly complete elimination of the bacteria treated with the amino-N-GQD 12.0–AbLPS hybrid (elimination > 99%; Figure S10d, corresponding to Figures 3h and 4d) and the strong antibacterial effect of the amino-N-GQDs in PDT. Thus, no other photochemical activity (e.g., photothermal effect) was observed after photoexcitation. In addition, the bacteria not treated with the antibody–coated materials exhibited almost no antimicrobial effect under similar conditions (Figure S10e–h, corresponding to Figures 3k and 4f).

− μ <sup>−</sup> μ <sup>−</sup> **Figure 3.** TPL images (gray-level) of the (**a**) amino-N-GQD 9.1, (**b**) amino-N-GQD 9.9, (**c**) amino-N-GQD 11.1, and (**d**) amino-N-GQD 12.0 at a TPE power of 222.7 nJ pixel−<sup>1</sup> with 20 scans. (**e-1**) *E. coli* alone, (**e-2**) collagen matrix alone and bacteria subjected to sorted amino-N-GQD–AbLPS treatment at a 180 µm depth (222.7 nJ pixel−<sup>1</sup> ) with (**f**) 20 scans and (**g**) 100 scans through TPE. (**h**) Images acquired after an additional 170 scans. (**i**) TPAF image of the unlabeled bacteria. TPL images of bacteria treated without the antibody-coated materials with (**j**) 20 scans and (**k**) 170 scans through photoexcitation under the same conditions. All images were acquired after 3 h of additional incubation to make the PDT action effectively. TPE wavelength: 960 nm. Delivered dose (OD600): approximately 0.05 of *E. coli* or 0.75 µg mL−<sup>1</sup> material–AbLPS.

μ <sup>−</sup>

μ <sup>−</sup>

μ μ

− μ <sup>−</sup> **Figure 4.** TEM images of the (**a**) bare *E. coli* (with 20 scans), (**b**–**d**) sorted amino-N-GQD–AbLPStreated *E. coli* (with 20, 100, and 170 scans), and (**e**,**f**) sorted amino-N-GQD-treated *E. coli* (with 20 and 170 scans) under TPE (222.7 nJ pixel−<sup>1</sup> ). All images were acquired after 3 h of additional incubation. TPE wavelength: 960 nm. Delivered dose (OD600): approximately 0.05 of *E. coli* or 0.75 µg mL−<sup>1</sup> material–AbLPS.

− ROS plays a crucial role in PDT by enabling the detection of superoxide anion radicals (O<sup>2</sup> <sup>−</sup>), hydrogen peroxide (H2O2), and singlet oxygen (1O2). In PDT, ROS are formed when molecular oxygen reacts with a photoexcited photosensitizer (PS) exposed to a suitable wavelength of light and energy. Photosensitized reactions involving oxygen are categorized as type I or II. A light-sensitized (excited) PS can directly react with a suitable substrate (unsaturated lipids, proteins, or nucleic acids) to produce unstable radicals through proton or electron transfer (type I reaction), leading to oxygenated products in the presence of oxygen, such as O<sup>2</sup> −, hydrogen peroxide (H2O2), or hydroxyl radicals (OH). Subsequently, it reacts with molecular oxygen to form <sup>1</sup>O<sup>2</sup> through energy transfer (type II reaction). ROS can induce DNA damage, inactivate enzymes, and oxidize amino acids, causing bacterial injury. However, a considerable amount of <sup>1</sup>O2, O<sup>2</sup> −, and H2O<sup>2</sup> was generated, and falsepositive ROS signals were observed, which could be due to interactions among the sorted amino-N-GQDs, singlet oxygen sensor green (SOSG), trans-1-(2′ -methoxyvinyl)pyrene (*t*-MVP), 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT),

glutathione (GSH), and 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA), which might compromise the results (Tables S3 and S4). The ROS generated by the bacteria treated with the sorted amino-N-GQD–Ab were monitored (Tables S5 and S6), and their signals were consistent with the <sup>1</sup>O<sup>2</sup> phosphorescence signals emitted at 1270 nm from the sorted amino-N-GQDs (Figure 5a). The material without antibody coating (Tables S7 and S8) generated less ROS than the AbLPS–coated material. Furthermore, <sup>1</sup>O<sup>2</sup> QY (Φ∆) values for amino-N-GQD 9.1, amino-N-GQD 9.9, amino-N-GQD 11.1, and amino-N-GQD 12.0 [21] were approximately 0.26, 0.28, 0.31, and 0.34, respectively. This study demonstrates the antimicrobial potential of the developed materials against *E. coli* in PDT. The bactericidal capability of the dots was investigated at a low dose of 0.75 µg mL−<sup>1</sup> in the dark (TPE energy: 222.7 nJ pixel−<sup>1</sup> ; scans: 20; Ex: 960 nm). No significant difference in viability was observed between the panels (Figure S11a,b, corresponding to Figures 3f and 4b, respectively). After 100 scans, TPE still exhibited no bactericidal effect on the bacteria alone, and without TPE, the material in the panel exhibited considerable biocompatibility with the bacteria treated with the sorted dot–Ab hybrid (Figure S11c,d). However, under TPE, the sorted amino-N-GQDs exhibited excellent bactericidal capability (~89%, 93%, 98%, and 100% elimination for amino-N-GQD 9.1–AbLPS, amino-N-GQD 9.9–AbLPS, amino-N-GQD 11.1–AbLPS, and amino-N-GQD 12.0–AbLPS, respectively, amounting to an approximate 0.90–7.82 log<sup>10</sup> reduction; Figure S10c,d, corresponding to Figures 3g and 4c). In contrast, the observed bacterial viability was higher for the materials without antibody coating (>98% viability) compared to the materials with the coating (Figure S11c,d). Although antimicrobial capabilities were still not apparent (~6%, 8%, 9%, and 11% elimination for amino-N-GQD 9.1, amino-N-GQD 9.9, amino-N-GQD 11.1, and amino-N-GQD 12.0 without the coating antibody, respectively), the sorted dots exhibited 100% antimicrobial efficacy against all *E. coli* strains treated with the sorted dot–AbLPS hybrid under TPE at 170 scans (Figure S11e,f, corresponding to Figures 3h and 4d). The surface protein, protein A, on the cell wall of a multidrug-resistant (MDR) strain of gram-positive methicillin-resistant *Staphylococcus aureus* (MRSA) was considered. Thus, the material was coated with Abprotein A to form a material–Abprotein A hybrid that eliminated MRSA (Figures S12 and S13), demonstrating a trend similar to that shown in Figure S11. These results were attributed to the sorted amino-N-GQDs functioning as a two-photon PS to generate ROS involved in PDT. These results also demonstrated the effectiveness of the antibody coating in enhancing the functions of the materials. Additionally, the trend of ROS generation in MRSA treated with the sorted dot–Abprotein A hybrid (Tables S9–S12) under TPE was similar to that of ROS generation in *E. coli* treated with the material–AbLPS hybrid (Tables S3–S8).

μ <sup>−</sup> **Figure 5.** (**a**) Phosphorescence spectra of the sorted amino-N-GQDs (obtained at 1270 nm). (**b**) Decay profiles of the time-resolved room-temperature TPL material. Delivered dose: 0.75 µg mL−<sup>1</sup> material.

*π*

*π*

Amino-N-GQDs exhibited remarkable quantum confinement, and their edge effects could be altered to increase their electrochemical, electrocatalytic, and photochemical activities [6,9]. Strong electron donation and large *π*-conjugated systems increased the charge transfer efficiency of the amino-N-GQDs [22], which resulted in favorable TPA, post-TPE stability, TPE cross-sections, and TPL. Additionally, they increased the ratio of radiative to non-radiative decay rates (amino-N-GQD 9.1: 0.64; amino-N-GQD 9.9: 0.69; amino-N-GQD 11.1: 0.82; and amino-N-GQD 12.0: 0.92; please refer to the Materials and Methods section for the calculation; Table 2). The results indicated that the material passed mainly through the radiative pathway as the fluorescence QY increased (amino-N-GQD 9.1: 0.39; amino-N-GQD 9.9: 0.41; amino-N-GQD 11.1: 0.45; and amino-N-GQD 12.0: 0.48) and the lifetime decreased (from 1.13 to 0.93 ns; Figure 5b, Tables 2 and 3). Radiative electron–hole pair recombination was observed and it was induced by N dopants and amino groups on the surface of the GQD-based material, which increased the intrinsic-state emission. However, for N dopants and amino groups, the presence of edge amine groups can increase the maximum occupied molecular orbital energy of the graphene flakes [23]. Thus, the narrowing of the orbital band gap, which increased the PL QY, could be attributed to the resonance between the delocalized *π* orbitals and the molecular orbital of the primary amine. XPS revealed that the C–O, C=O, and amide groups, which induced the nonradiative recombination of localized electron–hole pairs and prevented intrinsic–state emission [24], were favorable for small materials (Figures S3 and S4). The PL QY increased with increasing particle size. In addition, chemical modifications strongly affect the electronic properties of the amino-N-GQDs, enabling strong electron donation in primary amine molecules, which is also known as amino group functionalization. Singlet-triplet splitting of the amino-N-GQDs resulted in intersystem crossing and a high triplet-state yield. This splitting process was efficient, and it could compete with the process of internal conversion between multiple identical states, resulting in the creation of ROS for involvement in PDT [9,22]. As the number of edge sites increased, the number of C–N, pyridinic-, amino-, and pyrrolic-N groups increased (Figures S3 and S4). Similarly, as the size of the amino-N-GQDs (Figures S3 and S4) increased (Figures S2 and S5), their antibacterial ability and the number of ROS generated increased (Figure 5a and Tables S3–S8), leading to a highly efficient PDT process.

**Table 3.** Lifetime data and parameter generated using a time-correlated single-photon counting technique involving a triple-exponential fitting function while monitoring the emission under TPE. Delivered dose: 0.75 µg mL−<sup>1</sup> material.


#### **3. Materials and Methods**

#### *3.1. TEM Observation of the Negatively Stained Bacteria*

Bacteria were picked from colonies and suspended in a 1% aqueous sodium phosphotungstate solution (Sigma Aldrich Co., St Louis, MO, USA) at pH 7.0. Droplets of the suspensions were allowed to dry on grids coated with the Formvar. Thereafter, the samples were subjected to TEM.

#### *3.2. Molecular Weight of the Sorted-Amino-N-GQDs*

The theoretical diameter of benzene is 0.243 nm with a molecular weight of 72 (ignoring the H atoms). According to Figure S2, the mean lateral sizes of the sorted-amino-N-GQDs were approximately 9.1 ± 0.2 nm (amino-N-GQD 9.1), 9.9 ± 0.2 nm (amino-N- GQD 9.9), 11.1 ± 0.3 nm (amino-N-GQD 11.1), and 12.0 ± 0.3 nm (amino-N-GQD 12.0). For the sorted-amino-N-GQDs, assuming there was no leakage from a layer of material and ignoring the exposed functional groups, the benzene number and molecular weight could be approximately 1027 and 26,217 g mol−<sup>1</sup> , 1261 and 32,027 g mol−<sup>1</sup> , 1519 and 38,418 g mol−<sup>1</sup> , 1801 and 45,390 g mol−<sup>1</sup> (Table S14). The following measurement for the cross-section of TPE was performed using the estimated molecular weights.

#### *3.3. Femtosecond Laser Optical System for the Measurements of TPA and TPL*

A novel inverted optical microscopy system with a femtosecond Ti-sapphire laser [repetition rate: 80 MHz; Mai Tai with optical parametric oscillators, Spectra-Physics, Santa Clara, CA, USA] optical system: an inverted optical microscope (Zeiss, Oberkochen, Germany); an x–y galvanometer scanner (Cambridge, MA, USA); a triple-axis samplepositioning stage (Prior Scientific Instruments Ltd., London, UK); a *z*–axis piezoelectric nano-positioning stage (Mad City Labs, Madison, WI, USA); photomultiplier tubes (Hamamatsu, Shizuoka, Japan); a data acquisition card with a field-programmable gate array module (National Instruments, Austin, TX, USA) (Scheme S1).

All the Materials and Methods used in this study can be found in the Supplementary Materials.

#### **4. Conclusions**

Nitrogen doping and amino group functionalization, which result in strong electron donation, can be achieved through chemical modifications. Large *π*-conjugated systems of GQD-based materials acting as electron donors can be chemically manipulated with a low TPE energy in a short photoexcitation period to increase the charge transfer efficiency of the sorted amino-N-GQDs. This study used a novel femtosecond Ti-sapphire laser optical system (power: 222.7 nJ pixel−<sup>1</sup> ; scans: 100–170; total effective exposure time: ~0.65–1.11 s; excitation wavelength: 960 nm in the NIR-II region) for chemical modification. The sorted amino-N-GQDs exhibited increased TPA, post-TPE stability, TPE cross-sections, and TPL through the radiative pathway. The lifetime and quantum yield of the sorted amino-N-GQDs decreased and increased, respectively. Additionally, the sorted amino-N-GQDs exhibited EWI-PL in the NIR region and generated ROS after the TPE. Increasing the mean lateral size increased the number of C–N, pyridinic–N, amino–N, and pyrrolic– N functionalities, which induced the radiative recombination of localized electron–hole pairs and provided greater PL QY and efficient PDT action through TPE, enabling the sorted amino-N-GQDs to be applied in contrast probes to track and localize analytes in two-photon PDT.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ijms23063230/s1.

**Author Contributions:** Conceptualization, data curation, formal analysis, project administration, investigation, methodology, funding acquisition, resources, software, supervision, validation, visualization, W.-S.K.; data curation, formal analysis, investigation, methodology, funding acquisition, resources, software, validation, visualization, P.-C.W., C.-Y.C. and J.-Y.W.; formal analysis, investigation, methodology, software, validation, visualization, P.-C.C., M.-H.H. and H.-F.K.; conceptualization, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, S.-H.L., Y.-S.L. and C.-C.C. The manuscript was written through contributions of all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Nanjing University of Information Science and Technology, China (2018r047); State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, China (CMEMR2021-B11); An Nan Hospital, China Medical University, Taiwan (ANHRF110-34); Allergy Immunology and Microbiome Center, China Medical University Children's Hospital, China Medical University, Taiwan (1JA8); E-Da Hospital, Taiwan; Ministry of Science and Technology, Taiwan (MOST 110-2221-E-006-013-MY3); Academia Sinica Healthy Longevity Grand Challenge Competition, Taiwan (AS-HLGC-110-07).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Graphene for Antimicrobial and Coating Application**

**Viritpon Srimaneepong 1 , Hans Erling Skallevold 2 , Zohaib Khurshid 3 , Muhammad Sohail Zafar 4,5 , Dinesh Rokaya 6, \* and Janak Sapkota 7, \***


**Abstract:** Graphene is a versatile compound with several outstanding properties, providing a combination of impressive surface area, high strength, thermal and electrical properties, with a wide array of functionalization possibilities. This review aims to present an introduction of graphene and presents a comprehensive up-to-date review of graphene as an antimicrobial and coating application in medicine and dentistry. Available articles on graphene for biomedical applications were reviewed from January 1957 to August 2020) using MEDLINE/PubMed, Web of Science, and ScienceDirect. The selected articles were included in this study. Extensive research on graphene in several fields exists. However, the available literature on graphene-based coatings in dentistry and medical implant technology is limited. Graphene exhibits high biocompatibility, corrosion prevention, antimicrobial properties to prevent the colonization of bacteria. Graphene coatings enhance adhesion of cells, osteogenic differentiation, and promote antibacterial activity to parts of titanium unaffected by the thermal treatment. Furthermore, the graphene layer can improve the surface properties of implants which can be used for biomedical applications. Hence, graphene and its derivatives may hold the key for the next revolution in dental and medical technology.

**Keywords:** graphene; coatings; bioactivity; tissue engineering; bone regeneration

#### **1. Introduction**

Graphene, having a sp 2 configuration, is made from a thin sheet of carbon atoms (Figure 1) [1–3]. The various forms of graphene include pure/pristine graphene, graphene oxide (GO) containing –COC–, –COOH, or –COH, reduced GO (rGO), and animated graphene oxide (AGO). Graphene materials have outstanding various properties with good mechanical strength, high surface area, elasticity, stiffness, excellent biocompatibility, superior electrical and thermal conductivity, and ease of functionalization [4–8]. Therefore, graphene is attractive in different fields including medicine and dentistry [3,9–11]. The review aims to present an introduction of graphene and presents a comprehensive up-to-date review of graphene as an antimicrobial and coating application in medicine and dentistry.

**Citation:** Srimaneepong, V.; Skallevold, H.E.; Khurshid, Z.; Zafar, M.S.; Rokaya, D.; Sapkota, J. Graphene for Antimicrobial and Coating Application. *Int. J. Mol. Sci.* **2022**, *23*, 499. https://doi.org/ 10.3390/ijms23010499

Academic Editor: Ana María Díez-Pascual

Received: 2 December 2021 Accepted: 30 December 2021 Published: 2 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Structure of graphene [12].

#### **2. Production of Graphene**

‐ Different grades of graphene can be prepared by various methods of production depending on the type of application. Such methods of production include mechanical exfoliation of graphite, epitaxial growth of graphene, liquid-phase exfoliation (LPE), chemical vapor deposition, and molecular assembly [3,4,13]. The most common methods are shown in Figure 2. ‐

**Figure 2.** Various methods of production of graphene.

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The simplest method of production of graphene is by mechanical exfoliation in which the graphite is subjected to tape exfoliation followed by transfer of graphene to a substrate [14,15]. Through this method, the greatest quality of graphene is produced, however, to scale-up the process is not possible [16]. The characteristics of graphene produced from various methods differ, as shown in Table 1. Graphene can grow epitaxially on SiC (silicon carbide) at high-temperature (1300–1800 ◦C) [17]. This method produces atomically smooth graphene nanostructures but may contain certain manufacturing defects. Furthermore, molecular assembly induces modulation of graphene using metal phthalocyanines [18] which is effective to improve the electronic properties [19,20] and the molecular ordering is critical to achieving potential shapes [19]. Liquid phase extraction (LPE) is important for the mass manufacture of graphene [21,22]. Common reported techniques of LPE include sonication [23], jet cavitation [24], micro-fluidization [25], and high-shear mixing [22]. Sonication can produce high concentrations of monolayer to few-layer graphene [23,26]. The factors responsible for the graphene exfoliation include the sonication process, shear forces, the dispersion medium, and the centrifugation process [27–29]. Graphene is also grown on non-metallic substrates such as SiO2, h-BN, or quartz, using chemical vapor deposition, which allows direct deposition of high-quality graphene [30,31]. Chemical vapor deposition of graphene can result in 3D structures having low density, high surface area, and fast electron transport [32–34]. These properties are suitable for engineering, nanotechnology, and biomedical applications.

**Table 1.** Various methods of production of graphene and their properties [4].


#### **3. Structure and Properties of Graphene**

A flat, 2D, sheet of graphene is single to multi-layered while graphene 3D structures can be produced to take various forms (flakes, foams, shells, and hierarchical structures) [32,35]. A graphene film may be comprised of a monolayer, bilayer, or multi-layer. Monolayer graphene is very thin (0.35 ± 0.01 nm) [36] and multilayer graphene has <10 layers [6], as reported by Raman scattering, scanning probe microscopy, and optical contrast [37]. The 2D graphene layers can have a pore size of less than a millimeter which can subsequently be incorporated into porous 3D graphene forms [38]. The 3D foam structures have a larger surface area, strength, are stiff, lightweight, and provide excellent electronic and thermal conductivity, and pathways for ionic transport.

The structure of GO and rGO and their process of production is shown in Figure 3 [39]. Generally, GO is manufactured by the oxidation of graphite from Hummers' method [40,41]. By thermal-, chemical-, and electrochemical reduction, GO yields rGO. GO and rGO have functional capabilities and wider applications beyond that of pristine graphene [3,42,43].

The AGO can be produced from the reduction and amination of graphene oxide via two-step liquid phase treatment with hydrobromic acid and ammonia solution in mild conditions [44]. The AGO is biocompatible, has electrical conductivity, and has the tendency to form wrinkled and corrugated graphene layers are observed in the AGO derivative compared to the pristine rGO. AGO can be used for biosensing, photovoltaic, catalysis application, and is used as a starting material for further chemical modifications.

**Figure 3.** Production of graphene oxide (GO) reduced graphene oxide (rGO), and animated graphene oxide (AGO) from graphene [39].

‐ ‐ Table 2 shows the essential properties of graphene, GO, and rGO [3,43]. Graphene has good electron mobility [45], increased surface area [46], good electrical conductivity [15], good thermal conductivity [47], high elastic modulus [48], strength and stiffness [48], and good wear and friction properties [4,7]. Large surface area and the ability to form nanocomposites, graphene-based materials have wide applications in regenerative medicine and drug delivery. High strength, wear-resistant, and low friction are useful in coatings and nanocomposites. Good electrical property is suitable for biosensors, semiconductors, and supercapacitors.

**Table 2.** Physical and mechanical properties of graphene [3,43].


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#### **4. Characterization and Properties of Graphene**

Several methods to study graphene's surface structure are of use, these include transmission electron microscopy, scanning electron microscope, energy dispersive spectroscopy, Raman spectroscopy, X-ray diffraction, and atomic force microscopy, [49–53]. A notable characteristic of GO includes a somewhat rough surface morphology, as observed using a scanning electron microscope [52,53]. Raman spectra of graphene-based materials exhibit a D- and G band at about 1320/cm and 1570/cm, respectively [54]. D bands signify the breathing mode of κ-point phonons with A*1g* symmetry and the G band signifies the tangential stretching mode of the E*2g* phonon of the carbon sp<sup>2</sup> atoms. The ID/I<sup>G</sup> ratio of around 0.84–0.97 has been reported [49].

The crystallinity and spacing of the interplane of graphene can be studied from XRD. The deflection, height, and 3D images are obtained at micron and nanoscale, by the XRD. AFM can reveal the surface structure and allow for the observation of features at the molecular and atomic levels. R<sup>a</sup> (roughness average) can be also calculated from the AFM. XPS makes it possible to study the binding between C−O−C and C−C, and elemental composition [55].

#### **5. Functionalization of Graphene**

The development of nanocomposites has a long history. Although graphene has potential applications in engineering and biomedicine, its properties can be further improved via functionalization and doping due to its sp2 carbon atoms [56–58]. Graphene-based materials can be strengthened by various biopolymers (e.g., epoxy and polyketone) and metals (e.g., Zr, Ag, Cu, Zn, Au, Al, Ni, and Mg) [9,59–62], and nonmetals. As graphene is atomically thin, flat, and conducting material, it is suitable to produce energy storage devices [63]. At, present, the biomedical application of graphene nanocomposites is increasing [9,61,64,65]. The graphene nanocomposites have improved biocompatibility [66–68], surface properties [3,60], and mechanical properties [60] compared to pristine graphene. Graphene oxide, which is more amenable to chemical modification than pristine graphene. These properties permit applications involving protective and anticorrosion coatings [67,68], friction reduction [60], and antibacterial utilizations [69].

In graphene, n- or p-type doping Fermi level production is generally seen by physical or chemical bonds [70,71]. In graphene, chemical functionalization offers an obvious solution to the problems associated with graphene [72]. Electron-donating or -withdrawing groups can be bonded to the graphene network by synthetic chemistry methods, which could contribute to the bandgap widening and good dispersibility in common organic solvents.

The functionalization of graphene can be through covalent or non-covalent. Covalent bonds with graphene can occur using radical species, including nitrene, carbene, and aryl intermediates [72]. Conversely, modification of graphene occurs through noncovalent interactions, such as π–π interactions, van der Waals forces, ionic interactions, and hydrogen bonding, and result in major alteration of its structure and electronic properties [72]. The noncovalent interaction of graphene occurs with aromatic species, organic molecules, other carbon nanostructures, and inorganic species.

An aryl group can be grafted on the sp<sup>2</sup> carbon network of graphene using a diazonium salt and this has been widely applied to form covalently functionalized conducting or semiconducting materials [73,74]. A dinitrogen molecule is eliminated, and then, an electron is transferred from graphene to the diazonium salt to form an aryl radical. Thionine (Th) diazonium cation—covalently attached to the glassy carbon (GC) electrode via graphene nanosheets (GNs) (GC–GNs–Th)—has potential for application in sensors for detecting glucose and nitrite [74]. In addition, perfluorophenyl azides (PFPAs) can be covalently functionalized with graphene [75,76]. The functionalized graphene exhibits new chemical functionalities because the PFPAs groups impart solubility in both water and organic solvents [76].

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Similarly, adsorption of aromatic molecules onto graphene, e.g., borazine (B3N3H6), triazine (C3N3H3), and benzene (C6H6) occurs through non-covalent bonds [77]. Park et al. [78] studied the influence of pyridine adsorption and the applied electric field on the band structure and metallicity of zigzag graphene nanoribbons (ZGNRs) using density functional theory. They found that adsorption of an electron-accepting organic molecule, such as pyridine, on ZGNRs should provide a simple and useful way to widen the band gap and can be used to turn the band structure of nanoscale electronic devices based on graphene applications. ‐ ‐ ‐ ‐

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Zhang et al. [79] developed a biosensor for the detection of microRNAs (miRNAs) based on graphene quantum dots (GQDs) and pyrene-functionalized molecular beacon probes (py-MBs). The pyrene unit served to shorten the distance between py-MBs and GQDs and to generate an increased fluorescence signal from dyes appended on the probes. When hybridized with the target miRNAs, the hairpin structure of py-MBs opened and formed more precise duplex structures.

Another important application of functionalized graphene is antimicrobial applications. Silver nanoparticles (AgNPs) be able to be decorated on the GO to make GO/Ag nanocomposite (Figure 4) [49,66]. This nanocomposite can be applied for coating and antimicrobial applications [49]. The ratio of D and G bands (ID/IG) of the GO/Ag nanocomposite may be elevated as a result of the disorder of the GO/Ag matrix [49,80]. ‐ ‐

**Figure 4.** Characterization of graphene oxide (GO) nanocomposite formed from GO sheets decorated with Ag (GO/Ag). (**a**) Transmission electron microscopy (TEM) image, (**b**) Raman spectra, and (**c**) X-ray diffraction (XRD) [49].

Furthermore, Jeyaseelan et al. [81] developed the AGO for fluoride removal application, which was studied in terms of adsorption isotherms, kinetics (particle/intraparticle diffusion and pseudo-first/second-order models), and thermodynamic studies of AGO. The fluoride removal mechanism of AGO was found to be an electrostatic attraction.

Tissue engineering has emerged as an important approach to bone regeneration/ substitution [82]. Functionalized graphene and its derivates have been also used in bone regeneration and tissue engineering. Graphene can be combined with natural and synthetic biomaterials to enhance the osteogenic potential and mechanical properties of tissue

engineering scaffolds [83–85]. Scaffolds play a central role in tissue engineering as structural support for specific cells and provide the templates to guide new tissue growth and construction [84]. Nishida et al. [86] coated collagen scaffolds with various concentrations of GO and evaluated the bioactivity, cell proliferation, and differentiation both in vivo and in vitro. The results showed that GO affected both cell proliferation and differentiation and improves the properties of collagen scaffolds. Subcutaneous implant tests showed that low concentrations of GO scaffold enhance cell in-growth and are highly biodegradable, whereas high concentrations of GO coating resulted in adverse biological effects. Consequently, scaffolds modified with a suitable concentration of GO are useful as a bioactive material for tissue engineering.

Similarly, Kang et al. [87] studied the covalent conjugation of GO flakes to 3D collagen scaffolds improves the mechanical properties of the scaffolds and promotes the osteogenic differentiation of human MSCs (hMSCs) cultured on the scaffolds. The covalent conjugation of GO flakes to collagen scaffolds increased the scaffold stiffness by 3-fold and did not cause cytotoxicity. hMSCs cultured on the GO/collagen scaffolds showed significantly enhanced osteogenic differentiation compared to cells cultured on non-modified collagen scaffolds. The enhanced osteogenic differentiation observed on the stiffer scaffolds was mediated by MSC mechanosensing because molecules that are involved in cell adhesion to stiff substrates were either up-regulated or activated. The 3D GO/collagen scaffolds could offer a powerful platform for stem cell research and orthopedic regenerative medicine.

Recently, graphene-based bioactive glass is studied as a potential drug/growth factor carrier, which includes the composition–structure–drug delivery relationship and the functional effect on the tissue-stimulation properties [82,88,89]. Wang et al. [88] designed a scaffold composed of mesoporous bioactive glasses (MBG) and GO and studied the composite porous scaffold that promotes local angiogenesis and bone healing. This in vitro study found that the MBG/GO scaffolds have better cytocompatibility and higher osteogenesis differentiation ability with rat bone marrow mesenchymal stem cells (rBMSCs) than the purely MBG scaffold. Moreover, MBG/GO scaffolds promote vascular ingrowth and, importantly, enhance bone repair at the defect site in a rat cranial defect model. The new bone was fully integrated not only with the periphery but also with the center of the scaffold. Hence, the MBG/GO scaffolds possess excellent osteogenic-angiogenic properties which will make them appealing candidates for repairing bone defects.

Finally, biodegradable composites have been used in various regeneration processes applications such as the regeneration of bones, cartilage, and soft tissues. Stepanova et al. [90] synthesized aminated graphene with oligomers of glutamic acid and their use for the preparation of composite materials based on poly(ε-caprolactone) for tissue regeneration applications. The poly(ε-caprolactone) films filled with modified aminated graphene were produced and characterized for their mechanical and biological properties. They found that grafting of glutamic acid oligomers from the surface of aminated graphene improved the distribution of the filler in the polymer matrix that, in turn, improved the mechanical properties of composite materials. In addition, the modification improved the biocompatibility of human MG-63 osteoblast-like cells.

#### **6. Graphene Coating Applications**

The potential application of graphene for various biomedical applications is promising [3,9], such as anticorrosion, antibacterial coatings, and friction reduction [67], as shown in Figure 5. Graphene is chemically inert, atomically smoothness and high durability make it an alternative candidate for implant coatings [91].

‐ **Figure 5.** Biomedical applications of graphene-based coatings.

#### *6.1. Anticorrosion Coating*

There are various applications of metallic materials in medicine and dentistry, such as dental implants, orthopedic fixations, orthodontic, joint replacements, stents, endodontic files, and reamers [92]. However, the disadvantage of such biomaterials is the metal ions release, such as Ni, Ti, Ag, hence, coating of metallic materials plays an important role in such problems [92,93]. Although various coatings are being tried on metallic biomaterials, especially nitinol (NiTi), producing a successful coating has been always a challenge [94–107]. Notable disadvantages of polymer coating include toxicity of the component's roughness, porosity, and detachment of the coatings [108].

ʹ ‐ ‐ ‐ ‐ ‐ Even though graphene is an atom thick, it is inert and it is water-resistant and oxygen [4]. Hence, these properties combined with their durability and atomically stability has proven graphene to be useful as a corrosion barrier film [68,109–112]. Graphene can be directly grown on metallic surfaces (Mg, Zn, Ni, Al, etc.) to produce a protective coating [109,111,113]. Singh et al. [114] successfully developed an anti-corrosion graphene composite coating on Cu. In dentistry, graphene coatings can prevent corrosion of various metallic biomaterials such as archwires, files and reamers, and various metallic prostheses [65,68,109]. Furthermore, Hikku et al. [115] studied the anti-corrosion property of graphene and polyvinyl nanocomposite (GPVA) coating on the aluminum-2219 alloy (Al-2219). The corrosion rate for the coated Al-2219 alloys was better (polyvinyl alcohol coated alloy: 2.57 mm/year and GPVA coated alloy: 3.85 <sup>×</sup> <sup>10</sup>−<sup>4</sup> mm/year), whereas for untreated alloy: 45.25 mm/year in 3.5% NaCl solution (Figure 6). Hence, the GPVA coated Al-2219 alloy showed the best corrosion resistance than the uncoated alloy.

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‐ ‐ ‐ ‐ **Figure 6.** Anti-corrosion graphene blended with polyvinyl alcohol (GPVA) coatings: (**a**) Nyquist plot of bare Al-2219 and PVA coated Al-2219, (**b**) Nyquist plot of GPVA coated Al-2219, (**c**) Bode plot from the impedance analysis, and (**d**) Tafel plot of bare Al, PVA coated Al, and GPVA coated Al [115].

‐ ‐ Graphene coatings can improve implant surface properties and reduce corrosion [91,116,117]. Podila et al. [91] produced graphene on Cu using chemical vapor deposition technique and transported it onto NiTi implant samples and studied the effects of the coatings on cell morphology and adhesion and they noted that the biological responses (cell adhesion and protein adsorption) were increased on the graphene-coated NiTi substrates, in comparison to the uncoated NiTi substrates. Thus, graphene-coated NiTi can be applied to the stent. Additionally, graphene could improve the osseointegration of Ti implants. In addition, Suo et al. [116] produced a homogeneous GO/chitosan/hydroxyapatite (GO/CS/HA) coating using electrophoretic deposition (EPD) on Ti. The GO/CS/HA coating's wettability and bonding strength were greater than the HA, GO/HA, and CS/HA coatings. Moreover, the GO/CS/HA coating significantly enhanced the cell–material interactions in vitro and osseointegration in vivo. Hence, the GO/CS/HA coatings on Ti can be a potential coating in implant dentistry.

‐ ‐ ‐ ‐ ‐ Magnesium (Mg) can be used to make biodegradable implants; however, its major drawbacks of difficult-to-control corrosion. Catt et al. [110] produced a conducting polymer 3,4-ethylene dioxythiophene (PEDOT) and a GO coating for Mg implants to prevent corrosion. It was found that the significant reduction of Mg ions concentrations and pH of the media from the PEDOT/GO coating suggests a significant corrosion resistance. A positive finding was that of decreased hydrogen amounts. Three important factors were due to the passive layer preventing the ingress of a solution, film's negative charges, and development of a corrosion-resistant Mg-phosphate coat. Additionally, promising biocompatibility, in vitro, was observed as the coating did not show signs of toxicity to cultured neurons. Hence, the PEDOT/GO coating is successful in preventing Mg-based implants corrosion.

‐ ‐ GO coating is also useful in tissue engineering and regenerative applications. Root fracture treatment, cementation of prostheses, pulp therapy, filling, repair, and regeneration of bone defects, may all indicate the use of bioactive cement. A bioactive cement typically releases calcium-ions (Ca2+), increases the alkalinity in its surrounding environment, and induces cell differentiation and formation of mineralized tissue. However, the cement tends to possess poor mechanical properties, at risk of fracture due to poor strength and fracture toughness [118]. The mechanical properties are improved by the addition of graphene. A doubling of the strength of 58S bioactive glass was observed by the addition of 0.5 wt.% [119]. The addition of GO [119] and rGO [120] have also shown significant improvements in mechanical parameters, the latter (rGO 1 wt.%) resulted in a 200% increase in the fracture toughness of hydroxyapatite [119]. Additionally, the bone cement's bioactive properties are enhanced due to the addition of graphene. Several cell types, including bone marrow stem cells, periodontal ligament stem cells (PDLSCs), and dental pulp stem cells have shown spontaneous osteogenic differentiation as promoted by chemical vapor deposition-produced pristine graphene scaffolds and substrates [121,122]. Indeed, in vivo bone formation was exhibited by implanting GO-coated collagen scaffolds into tooth extraction sockets of beagle dogs. The GO-coated scaffolds showed increased bone formation and calcium absorption after 14 days, whereas the control scaffold was mostly filled with connective tissue [123]. Similarly, Zhou et al. [112] evaluated the bioactive effects of GO coated Ti substrate on PDLSCs and compared them to sodium titanate substrate. It was seen that the GO coated Ti substrate-induced PDLSCs exhibit suggestively higher alkaline phosphatase (ALP) activity, proliferation rate, and higher gene expression of osteogenesis markers, ALP, runt-related transcription factor 2 (Runx2), bone sialoprotein, and osteocalcin (OCN) compared to the sodium titanate substrate. Protein expressions of Runx2, bone sialoprotein, and OCN were additionally promoted by GO. Together, the findings suggest that GO and PDLSCs represent a favorable combination for regenerative medicine and dentistry.

#### *6.2. Antibacterial Application*

Bacteria and fungiform biofilms on the teeth surface, prostheses, or implant-anchored restorations [124]. If left untreated, the biofilm on dental implants may result in loss of the implant. It is challenging to produce implants with a high degree of osseointegration at the same time as inhibiting bacterial colonization [125–127]. The peri-implant diseases around implant result in implants failure due to supporting bone loss around the implant [128–132].

Various antimicrobial nanomaterials include polymers, nanoparticles such as gold nanoparticles (AuNPs), AgNPs, nanodiamond, and graphene-based materials [133–135]. Even though AgNPs show promising antibacterial properties, clinical applications of AgNPs are frequently impeded by their tendency to aggregate and consequent loss of antibacterial activity [133,136]. Additionally, the cytotoxicity of AgNPs towards human cells has been observed [137]. The amount of AgNPs should be minimal to avoid complications. However, AgNPs can be decorated onto GO to produce GO/Ag nanocomposite for increased antimicrobial activity [49,55]. AuNPs are used more for microbial identification rather than antimicrobial applications [138,139].

The graphene-based materials have powerful antimicrobial properties and inhibit bacterial colonization [69,125,140,141]. Agarwalla et al. [140] studied the graphene coating on Ti and their interaction with a biofilm of *Pseudomonas aeruginosa*, *Enterococcus faecalis*, *Streptococcus mutants*, and *Candida albicans*. They observed that when repeated twice, it reduces the formation of biofilm due to the hydrophobicity of graphene. These all findings show that coating Ti with graphene is useful for biofilm prevention on implants.

Graphene coatings enhance the adhesion of cells and osteogenic differentiation. Gu et al. [142] studied the osteoinductive and antibacterial effects of graphene sheets modified Ti implants. Chemical vapor deposition growth of graphene sheets by thermal treatment at 160 ◦C for 2 h and transferring to Ti discs. It was found that the graphene coatings on Ti enhanced adhesion of cells, osteogenic differentiation, and exhibited antibacterial properties. Similarly, another study also found similar results, i.e., osteogenic differentiation of mesenchymal stem cells using graphene [143]. Hence, graphene is capable to enhance the surface properties of NiTi-based implants.

Similarly, functionalized GO can improve the antimicrobial property, as demonstrated by the GO/Ag nanocomposite (Figure 4) [49,55]. Graphene nanocomposite has excellent antibacterial action against *Escherichia coli* and *Staphylococcus aureus* [55]. Zhao et al. [43] fabricated gelatin-functionalized GO (Gogel) surface coatings on NiTi substrates. The Gogel's biocompatibility and antimicrobial properties were investigated, and it exhibited the highest rate of mouse osteoblastic adhesion, proliferation, as well as differentiation of cells compared to GO coated NiTi. Moreover, they reported that *E. coli* was suppressed on the surfaces of Gogel and GO. Following incubation on Gogel and GO, the integrity of the *E. coli* cell membrane was compromised and showed a low live/dead ratio. Therefore, GO-based coatings have both a high degree of biocompatibility and antimicrobial activity. ‐ ʹ ‐

Chen et al. [144] studied the interaction of GO with four phytopathogens (two bacteria and two fungi). The studied bacteria were *Xanthomonas campestris pv. undulosa* and *pseudomonas* and studied fungus were *Fusarium oxysporum* and *Fusarium graminearum* (Figure 7). It was found that GO killed nearly 90% of the bacteria and repressed 80% macroconidia germination along with partial cell swelling and lysis at 500 µg mL−<sup>1</sup> . They mentioned that GO sheets intertwined the bacterial and fungal spores resulting in the local perturbation of their cell membrane, decreasing the bacterial membrane potential, and resulting in the leakage of electrolytes of fungal spores causing cell lysis (Figure 8). μ <sup>−</sup>

 μ **Figure 7.** <sup>−</sup> Fluorescence microscopy images of cells following exposure to graphene oxide (500 µg mL−<sup>1</sup> ): (**a**) *X. campestris pv. undulosa*, (**c**) *P. syringae*, (**e**) *F. oxysporum*, and (**g**) *F. graminearum* and images following staining of cells with propidium iodide and fluorescence stain (**b**,**d**,**f**,**h**) [144].

**Figure 8.** Antibacterial mechanism of graphene oxide against pathogens and fungal spores [144].

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#### **7. Conclusions**

The available literature shows that graphene-based coatings can improve the bioactivity of biomaterials, provide microbial- and corrosion-protection of implants, both in vitro and in vivo. Peri-implant infections causing peri-implantitis are among the most common reasons for implant loss and may be prevented by the coating of antimicrobial graphene. These additive properties of graphene can be modified by methods of functionalization. Graphene exhibits high biocompatibility, corrosion prevention, and antimicrobial properties to prevent the colonization of bacteria. Graphene coatings enhance adhesion of cells, osteogenic differentiation, and exhibit antibacterial activity to parts of Ti unaffected by the thermal treatment. Graphene-based materials are promising and may hold the key for the next material-based revolution for antimicrobial and coatings applications in dental and medical technology. More research is urged before clinical utilization will be a widespread reality.

**Author Contributions:** Conceptualization, V.S., H.E.S. and D.R.; methodology, V.S., H.E.S., J.S. and D.R.; software, D.R.; validation, V.S. and D.R.; formal analysis, H.E.S. and D.R.; investigation, H.E.S. and D.R.; resources, H.E.S. and D.R.; data curation, H.E.S. and D.R.; writing—original draft preparation, H.E.S. and D.R.; writing—review and editing, V.S., J.S., Z.K., M.S.Z. and D.R.; visualization, V.S., Z.K., M.S.Z., J.S. and D.R.; supervision, V.S. and J.S.; project administration, V.S.; funding acquisition, V.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

