1 Introduction

Since the discovery of graphene in 2003, the number of researches about the two-dimensional based materials/devices have steadily increased in various scientific, technological and industrial fields [1,2,3,4]. Graphene is a two-dimensional carbon material which exhibits remarkable properties such as high electrical conductivity, mechanical strength, and thermal stability [5, 6]. Graphene oxide (GO) and reduced graphene oxide (rGO) are chemically modified graphene that become very popular in laboratories due to the challenges and scalability issues producing pristine graphene [3, 7]. GO is obtained from graphite oxidation, introducing oxygen functional groups such as hydroxyl, carbonyl, carboxyl and epoxy groups on the surface [8, 9]. Removing the oxygen functional group in GO through a reduction process will produce rGO [10, 11]. This reduction process recovers sp2 carbon structure and improves the electronical properties of rGO. However, due to the limited amount of oxygen functional groups in rGO, it becomes less hydrophilic and difficult to disperse in water compared to GO [1, 5].

Theoretically, rGO has larger surface area than GO because of its surface's flatness like a pristine graphene [12]. This large surface area makes rGO very suitable as a scaffold for attaching nanoparticles and creating nanocomposites. Adding nanoparticles such as gold nanoparticles (AuNPs) into the surface of graphene materials could enhance their optical, electrical, and biomedical properties [13,22,23,24]. While depositing AuNPs on rGO surface as rGO-Au nanocomposite, the size of AuNPs influences the activity of that material, such as for antioxidant, antibacterial agent, and catalyst [40, 41]. The highly conductive rGO surface is also probably channeling away the electrons of AuNPs and weakening the plasmon oscillation [42]. Some previous study also reported that depositing AuNPs on graphene sheets could drastically weaken and even eliminate the AuNPs peak [43,44,45]. Therefore, the absence of the AuNPs peak in this UV–Vis spectra does not necessarily mean that AuNPs are not formed on the rGO surface.

3.2 XRD analysis

Different forms of crystallinity were observed among GO, rGO, and AuNPs using XRD as illustrated in Fig. 2. The unmodified GO exhibits a diffraction line at 2θ = 10.26°, attributed to the (001) plane [38]. After the reduction process, GO peak disappears and a new peak of rGO arises at 2θ = 25.04° for the (002) plane. This peak shift in rGO due to the removal of oxygen functional groups in GO, causing the interlayer space to shrink from 8.62 to 3.55 Å. In contrast, in the rGO-Au NM nanocomposite, the GO peak does not entirely vanish due to a partial reduction process; instead, it shifts to 10.97° (Fig. 2b). De Silva et al. [22] also reported a similar diffraction pattern in the reduction process of GO. Extended microwave treatment leads to a more pronounced reduction of GO sheets and increased rGO crystallinity, as indicated by the heightened peak at around 25° 2θ. However, extending the microwave irradiation to 5 min still gives similar peaks and patterns to those observed at 4 min. The reduction process likely reaches its maximum after 4 min s

Fig. 2
figure 2

Diffraction patterns of GO and rGO-Au nanocomposites (a), along with a zoomed-in diffraction pattern specifically focusing on rGO-Au (b)

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Furthermore, the modification of the graphene surface with AuNPs introduces a new diffraction pattern for rGO-Au materials at 2θ = 38.28°, 44.51°, 64.73°, and 77.67°, corresponding to (111), (200), (220), and (311) planes of Au, respectively (Fig. 2a). This pattern match with JCPDS 4–784 data of Au and it confirms the presence of AuNPs on rGO surface.

3.3 Attenuated total reflection–infrared (ATR-IR) analysis

ATR-IR spectroscopy was used to investigate the changes in functional groups within both GO and rGO-Au nanocomposites. Figure 3 shows that GO exhibits several characteristic IR absorption bands which similar to synthesized rGO-Au such as C=C stretching vibration of aromatic group at ~ 1620 cm−1 and C–O stretching vibration at 1422, 1224 and 1055 cm−1 as carboxyl, epoxy and alkoxy group, respectively [S1 also reveals an even distribution of AuNPs on the graphene sheets of the rGO-Au 4 min nanocomposite. Apparently, in this case, ascorbic acid also plays a significant role as a cap** agent, preventing the agglomeration of AuNPs and contributing to the uniformity [23, 53]. Compared to previous research, without ascorbic acid, AuNPs exhibit various shapes, and the reduction of the GO does not occur, although the materials were subjected to microwave irradiation [54]. This suggests the effectiveness of ascorbic acid combining with microwave treatment in enhancing the structural characteristics of the rGO-Au nanocomposite.

Fig. 5
figure 5

SEM images of GO (a), rGO-Au NM (b), rGO-Au 2 min (c) and rGO-Au 4 min (d)

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3.7 TEM analysis and synthesis mechanism

TEM analysis was conducted to investigate the particle size of AuNPs within the rGO-Au nanocomposite as shown in Fig. 6. AuNPs is confirmed in TEM images as a dark spots in the graphene sheet (pointed by the arrow) due to high electron density of gold material [55]. In the absence of microwave irradiation, AuNPs in the rGO-Au NM nanocomposite exhibit agglomeration (Fig. 6b) and the average size is 26.4 nm. In contrast, under microwave radiation, a significant decrease in AuNPs size is observed. Both rGO-Au 2 min and rGO-Au 4 min exhibit very small AuNPs size (< 5 nm) (Fig. 6c and 6d). The size of AuNPs was hardly seen in regular TEM. To validate this finding, TEM analysis was taken three times with different suspension of rGO-Au 4 min and the images were remained same without any agglomeration or larger particle of AuNPs observed. However, this result appears to contradict the larger size of AuNPs observed in previous SEM images (Fig. 5c and 5d). This discrepancy can be attributed to the SEM analysis being performed on solid samples that were not dispersed under sonication, leading to AuNPs agglomeration. Moreover, regular SEM is also unable to observe very small nanoparticles. The SEM–EDX map** image (Fig. S1) also found that many AuNPs were spread on graphene sheets but remain unseen in SEM images.

Fig. 6
figure 6

TEM images of GO (a), rGO-Au NM (b), rGO-Au 2 min (d), rGO-Au 4 min (e) and HRTEM images of rGO-Au 4 min (c, f)

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High-resolution TEM analysis is necessary to accurately measure the size of AuNPs in rGO-Au 4 min nanocomposite. Figure 6e reveals that the average size of AuNPs is 2.2 nm on the rGO surface. The lattice spacing of 2.35 Å corresponds to (111) plane of AuNPs [56] is observed in Fig. 6f. To provide a comprehensive comparison, Table 2 summarizes various methods and findings from previous research on the fabrication of GO/rGO-Au. Previously, to synthesize AuNPs under 5 nm size, separate cap** agents like cetyltrimethylammonium bromide (CTAB) and polyvinylpyrrolidone (PVP) were needed, in addition to the reducing agent [54]. Apparently, microwave heating serves not merely as an alternative heating method but potentially provides a different reaction mechanism in nanoparticle synthesis, resulting in different nanostructured materials. Although microwave synthesis method provides fast synthesis and distinct nanomaterial properties, scale up to industrial level could be very challenging. The depth of microwave radiation penetration is very limited (only few centimeters), thus large vessel in batch production will be ineffective [62].

Fig. 7
figure 7

Schematic representation of microwave-assisted synthesis of rGO-Au nanocomposites

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3.8 Antibacterial properties

For the following discussion, the rGO-Au nanocomposite, subjected to 4 min of microwave treatment, was employed for the antibacterial properties assay and will be referred to as rGO-Au. Antibacterial assay by disk diffusion method did not reveal any observed antibacterial activity using GO and rGO-Au at 100 ppm against human pathogens E. coli and S. aureus, as demonstrated in Fig. S2. Positive control ciprofloxacin is the only substance that shows antibacterial activity. In this case, it seems that the lack diffusion of GO and rGO-Au prevent these materials to spread in agar media. Some researcher also reported that graphene-based materials demonstrated no inhibition zone with the disk diffusion method [63, 64]. However, this result contradicts Shalini’s [65] and Kadiyala’s [33] findings where GO, GO-Au and rGO-Au nanocomposite demonstrate > 10 mm inhibition zone in disk diffusion method against E. coli and S. aureus [65]. This inconsistency may be attributed to differences GO characteristic [35]. In Shalini's experiment [65], a distinct GO synthesis method was used, which may resuliting GO that exhibits better dispersibility in water and agar media. Improved dispersibility of GO can maximize the contact between the material and bacteria [66].

On the other hand, the OD assay demonstrates that both GO and rGO-Au has bacteriostatic effect that suppress E. coli and S. aureus growth at a low concentration of 10 ppm (see Fig. 8a and 8b). By comparing OD of control and treated bacteria suspension, the relative growth inhibition ability of GO and rGO-Au can be determined. However, the synthesized nanocomposites did not kill the bacteria completely, explaining the lack of observed antibacterial activity in the disk diffusion method. These results suggest that the disk diffusion method is inadequate for accurately evaluating the antibacterial properties of graphene-based materials, which may demonstrate bacteriostatic activity rather than bactericidal effects.

Fig. 8
figure 8

Bacterial growth of E. coli (a) and S. aureus b under rGO-Au exposure. Comparison of bacterial growth inhibition by GO and rGO-Au, determined by comparing OD values with the negative control (c). Antibacterial activity of rGO-Au in various concentration (d)

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As illustrated in Fig. 8a and 8b, the bacterial number increased with prolonged incubation time, as observed in the negative control's OD increase. From the beginning rGO-Au showed higher bacterial growth inhibition than GO for both E. coli and S. aureus. Interestingly, GO enhanced E. coli growth in the beginning, and finally inhibit the bacterial growth 14 h incubation. The bacterial growth enhancement by GO due to the GO itself could act as framework for bacterial to attach and proliferate [63]. After 18 h incubation rGO-Au nanocomposite exhibited superior bacteriostatic activity compared to unmodified GO, inhibiting 29.0% of E. coli and 37.6% of S. aureus growth, whereas GO only inhibited 14.3% of E. coli and 25.8% of S. aureus growth (Fig. 8c).

The presence of AuNPs on the rGO-Au nanocomposite clearly enhances bacteriostatic ability, suggesting the crucial role of AuNPs in the observed effects. rGO-Au was found to be more effective than GO in inhibiting bacterial growth against E. coli and S. aureus. After monitoring the bacterial growth at intervals, rGO-Au was retested at various concentrations to determine its effectiveness. Figure 8d illustrates that rGO-Au displayed 53.1% inhibition on E. coli at just 2.5 ppm concentration, while S. aureus showed 28.7% inhibition. The optimal concentrations of rGO-Au were determined to be 2.5 ppm for E. coli and 5 ppm for S. aureus. At these concentrations, bacterial growth was suppressed by 53.1% and 50.0%, respectively. These number indicate that the synthesized rGO-Au was not an antibacterial agent, but has good potential as a bacteriostat since it could inhibit half of bacterial growth. The synthesized rGO-Au nanocomposite demonstrated greater efficacy against E. coli, a Gram-negative strain, than the Gram-positive strain S. aureus. Gram-negative bacteria like E. coli have a thinner peptidoglycan layer in their cell wall, making them more prone to damage. On the other hand, a higher concentration of rGO-Au is required to kill Gram-positive bacteria such as S. aureus because the cell wall structure is thicker [26, 64].

In general, increasing rGO-Au concentration above 5 ppm did not result in a significant difference and could even slightly decrease the antibacterial activity at 50 ppm concentration (Fig. 9d). The growth inhibition stagnancy of E. coli and S. aureus was likely due to the limited amount of gold on rGO sheets. Compared to other studies (as depicted in Table 3), the GO-AuNPs and rGO-AuNPs showed high antibacterial activity at around 40–60 ppm, but AuNPs content was around 2%, more than three times AuNPs amount in our rGO-Au nanocomposite. Although AuNPs are crucial for enhancing graphene-based material antibacterial properties, their amount should be considered and optimized. The AuNPs content of 0.62% in the rGO-Au material proved insufficient to completely kill all E. coli and S. aureus bacteria even at 50 ppm concentration. Furthermore, the ineffectiveness of high concentrations of rGO-Au in our study can be attributed to agglomeration and precipitation occurring as the concentration of rGO-Au increases. Despite the minimal amount of AuNPs content, our rGO-Au exhibits remarkable effectiveness even at low concentrations. Table 3 shows that the synthesized rGO-Au with very low Au content at a concentration of 2.5 ppm exhibits comparable bacteriostatic performance to bare AuNPs with a particle size of 20–40 nm against E. coli. The tiny size of the AuNPs attached to the rGO sheets, could be the reason behind its efficacy by providing a larger surface area for direct contact and interaction with bacteria [67]. In summary, AuNPs’ size and quantity on graphene based-materials have a vital role in bacterial growth inhibition. Nevertheless, silver nanoparticles, as well-known antibacterial nanomaterials, completely inhibit bacterial growth of E. coli and S. aureus at a concentration of 1 ppm [73] and other motile cells [74], oxidative stress [28], interruption in the glycolysis process of the cells [75], DNA damaging [76], metal ion release [77] and contribution in generation and explosion of nanobubbles [78]. Antibacterial properties of synthesized rGO-Au Au mainly through the direct contact and wrap** mechanism. The direct contact and bacteria wrap** mechanism was observed in Fig. 9. However, the carbon tape substrate makes rGO sheets challenging to observe. Therefore, the substrate was changed to a glass substrate, and the SEM images reveal that rGO sheets wrapped the bacteria (Fig. 9c and 9d). Due to the high conductivity of rGO sheets and AuNPs, they appear very bright under the SEM microscope compared to the bacterial cell. SEM images display that AuNPs in rGO-Au attached to E. coli cell walls (Fig. 9b). Apparently, the morphology of E. coli was also damaged after incubation with the rGO-Au nanocomposite. Direct contact of bacteria with the sharp edges of rGO surfaces rupture the bacteria membrane, eventually resulting in bacterial death [28]. The rGO sheets also wrap the bacterial cells, preventing nutrients intake to the cells and leading to growth inhibition [73, 79]. Furthermore, the presence of AuNPs on the rGO-Au nanocomposite enhances antibacterial properties by interacting with bacterial cell walls and cytoplasm. Bacteria cell walls contain phospholipid bilayers, creating negatively charged cell surfaces surface [80]. This characteristic makes AuNPs easily bind with the cell wall through electrostatic interaction and potentially penetrating the inner cell. It also possible that rGO-Au damaging the DNA due to AuNPs exhibit an affinity for binding with thiol groups in DNA molecules and enzymes in the cytoplasm, causing disruptions in bacterial metabolic processes that eventually lead to bacterial death [26]. Hence, this study demonstrated the synergistic effect between rGO and AuNPs could effectively inhibit bacterial growth at remarkably low concentrations.

4 Conclusion

In conclusion, the microwave-assisted synthesis of rGO-Au using ascorbic acid proves to be a promising method for rapid and efficient production of nanocomposites with ultrasmall size of AuNPs. Microwave heating might offer a unique reaction mechanism, preventing agglomeration during the nucleation of AuNPs through molecular vibration and collisions induced by electromagnetic waves. The optimal microwave irradiation time was found to be 4 min, ensuring a high reduction degree of rGO-Au. Characterization of the synthesized nanocomposite clarifies the successful reduction of GO to rGO, showcasing a uniform distribution of ultrasmall AuNPs with the average size 2.2 nm on the rGO surface. This synthesized nanocomposite displays remarkable bacteriostatic activity at very low concentration despite the limited quantity of AuNPs. Compared to disk diffusion method, the OD method more suitable to observed antibacterial properties in graphene-based materials. The inhibition of bacterial growth is highly influenced by both the size and quantity of AuNPs present in graphene-based materials. These findings pave the way for further exploration in nanomaterials for biomedical applications such as develo** new antibiotics and antimicrobial coatings. Although the microwave heating provides advantages in nanomaterial synthesis, the scalability of this method presents challenges. Further research is necessary to optimize AuNPs quantities in graphene-based materials while maintaining their ultrasmall size. It could make rGO-Au nanocomposite as a remarkable antibacterial agent in very low concentration.