AuNps were chemically synthesized by the reduction of HAuCl4 with sodium citrate. About 1 mL of 1 mM HAuCl4 was mixed with 10 mL of deionized water and 1 mL sodium citrate 38.8 mM with vigorous stirring (600 rpm) for 10 min. The mixture was boiled under stirring and then the hot plate was turned off. A color change was observed from colorless to wine red within 10 min and stirring was continued for 20 min (De Souza et al., 2019).

To purify the synthesized gold nanoparticles from the mixture containing ginger extract, the solution was centrifuged using an ultracentrifuge in four steps. In the first step, centrifugation was carried out at 10,000 rpm for 20 min at 4°C. The supernatant was separated from the settled suspension. Deionized water was added to the pellet, and centrifugation was repeated at 20,000, 30,000, and 40,000 rpm for 20 min each at 4°C. During the purification process, the pH of the solutions was measured using a calibrated pH meter. The pH levels of the ginger extract, curcumin suspension in water, and 1 mM HAuCl₄ solution were recorded. Additionally, the final pH values of both biologically and chemically synthesized AuNPs were also evaluated.

The characterization of the synthesized gold nanoparticles (AuNPs) was performed using several analytical techniques. UV-Vis spectroscopy was conducted with a Thermo Scientific Genesys 30 spectrophotometer to identify the characteristic surface plasmon resonance peak, confirming the formation of AuNPs (Burda et al., 2005). Fluorescence measurements were taken using a Horiba FluoroMax4 fluorometer to assess the photoluminescent properties of the nanoparticles (Tiwari et al., 2011). The ginger extract components were analyzed using an Agilent Technologies 5973/6890 GC-MS system, equipped with an HP-5 30 × 0.25 capillary column, to identify the phenolic compounds involved in the reduction process, with the analysis performed at the national library (Yadi et al., 2022) Dynamic Light Scattering (DLS) and zeta potential measurements were carried out using a Malvern instrument to determine the size distribution and stability of the AuNPs (Malvern Instruments). Fourier-Transform Infrared Spectroscopy (FT-IR) was performed with a Nicolet 4700 FT-IR, utilizing 32 scans and a resolution of 4 cm⁻¹, to identify the functional groups on the AuNPs’ surface (Colthup, 2012). Energy Dispersive X-ray Spectroscopy (EDX) analysis was conducted using a Bruker system to confirm the elemental composition and purity of the AuNPs (Goldstein et al., 2017). Transmission Electron Microscopy (TEM) images were obtained with a Tecnai F20 transmission electron microscope, operating at 200 kV, to visualize the morphology and size of the nanoparticles (Sharma, 2005).

To assess the antimicrobial activity, AuNPs were passed through a syringe filter with a pore size of 0.45 µm to remove any bacteria/fungi that may have come through the preparation. This process was carried out after measuring the sizes of the gold nanoparticles using dynamic light scattering (DLS).

Bacteria indicator strains of the Gram-positive Staphylococcus aureus ATCC 29213 and Gram-negative Pseudomonas aeruginosa ATCC 27833 and Escherichia coli ATCC 25922 were used to determine antimicrobial activity of gold nanoparticles synthesized with ginger extract, curcumin, and those produced chemically. The minimum inhibition concentration (MIC) values were determined through the microdilution method providing insights into the effectiveness of the nanoparticles in inhibiting bacterial growth (Gouyau et al., 2021).

Additionally, biomolecules from ginger and curcumin were tested to evaluate their impact on antimicrobial activity. This approach helped to understand the combined effect of the biocompounds and gold nanoparticles in suppressing the growth of pathogenic microorganisms.

The synthesis of gold nanoparticles was successfully achieved using three distinct methods: ginger extract, curcumin, and sodium citrate. The Zingiber officinale extract facilitated a notable color transition from pale yellow to purple, which was confirmed by UV-Vis spectroscopy with a strong absorbance peak at approximately 530 nm, indicating the effective formation of gold nanoparticles primarily within the size range of 10–50 nm. This result suggests that ginger extract not only serves as a reducing agent but also stabilizes the nanoparticles, potentially due to the presence of bioactive compounds such as phenolics and flavonoids. In contrast, the use of curcumin resulted in a change from yellow to pink, with an absorbance peak around 540 nm and a particle size distribution of 20–60 nm. This method underscores curcumin’s dual role as both a reducing and stabilizing agent, further highlighting its potential for eco-friendly nanoparticle synthesis. The chemical synthesis with sodium citrate exhibited a color change from colorless to wine red, confirming nanoparticle formation with a peak around 520 nm and a size distribution of 15–45 nm. These findings indicate that both natural extracts and chemical agents can effectively synthesize gold nanoparticles with varying characteristics, suggesting potential applications in biomedical fields, drug delivery, and catalytic processes.

UV-Vis absorption spectroscopy is a crucial technique for evaluating the synthesis of metal nanoparticles in aqueous solutions (Figure 8). The stability is indicated by the maximum absorbance value remaining constant when samples are analyzed over different time periods (Chanana and Liz-Marzan, 2012). According to the charts, the UV-Vis spectra measurements of biosynthesized gold nanoparticles with ginger extract and curcumin, as well as chemically produced AuNPs, show a peak around 550 nm. This peak is characteristic of the surface plasmon resonance (SPR) of gold nanoparticles, which is typically observed in the range of 500–550 nm, depending on the size and shape of the nanoparticles (Haiss et al., 2007). The presence of a consistent peak over time in the UV-Vis spectra indicates that the nanoparticles are stable in solution without significant aggregation or shape change, which is critical for their potential biomedical applications (Dreaden et al., 2012).

The spectrum of Ginger-AuNPs shows a broad peak, indicating successful synthesis and characteristic optical properties of AuNPs (Figure 9). This broad peak is typically associated with the localized surface plasmon resonance (LSPR) of gold nanoparticles, which is consistent with observations in other studies of gold nanoparticle synthesis using plant extracts such as ginger (Yadi et al., 2022). The higher intensity observed in the fluorometer results for Curcumin-conjugated AuNPs suggests a strong interaction and successful reduction of gold ions by curcumin. This is supported by our data here indicating that curcumin acts effectively as both a reducing and capping agent, leading to stable gold nanoparticles with enhanced optical properties. The successful formation of these nanoparticles are evident in their intense fluorescence, which aligns with previous studies on curcumin gold nanoparticles (Sindhu et al., 2014). In comparison, the results for chemically synthesized AuNPs show lower intensity than the green-synthesized AuNPs. However, they still exhibit typical optical properties of gold nanoparticles, such as the LSPR peak, which confirms the formation of gold nanoparticles albeit with less pronounced optical characteristics compared to their biologically synthesized counterparts. This difference in intensity and optical properties between green-synthesized and chemically synthesized AuNPs can be attributed to the presence of organic molecules in the green synthesis process, which can enhance the stability and functional properties of the nanoparticles (Yadi et al., 2022; Link and El-Sayed, 2003).

Monitoring fluorescence offers valuable insights into the stability of nanoparticles under various environmental conditions. As demonstrated by Pallem et al. (2009), the fluorescence properties of gold nanoparticles are significantly influenced by their size, shape, and surface functionalization, which are critical for determining their stability in solution. Moreover, excitation-emission fluorescence data can act as a sensitive indicator of nanoparticle morphology, revealing structural changes over time or in response to external factors. The results from fluorescence measurements of synthesized gold nanoparticles using biological compounds such as ginger and curcumin, compared to chemically produced counterparts, indicate that the biologically synthesized nanoparticles exhibit superior size and morphology, enhancing their stability. Although some noise in the fluorescence data was observed—attributed to the water solvent used during synthesis—this also highlights the environmentally friendly conditions under which these nanoparticles were produced. The use of water emphasizes the intrinsic properties of the synthesized gold nanoparticles and their potential efficacy in combating bacteria. These findings underscore the advantages of biological synthesis methods for developing stable materials suitable for various applications.

To analyze the gold nanoparticles using FTIR spectra, the samples were freeze-dried. The spectra results provided information about the cap compositions of the gold nanoparticles. Figure 10 (A) shows the FTIR spectra results of biosynthesized gold nanoparticles using ginger root extract, with a peak at 3,395 cm⁻¹ attributed to the O-H stretching of phenolic compounds. The band at 1,654 cm⁻¹ corresponds to the C=C stretching of alkenes. Figure (B) illustrates the functional group of the ginger extract, where the signal at 3,381 cm⁻¹ is associated with O-H bonds of phenolic compounds, and the signal at 1,635 cm⁻¹ is related to N-H stretching of amides. Figure (C) presents the FTIR spectra of the biosynthesized AuNPs with Curcumin, showing a signal at 3,432 cm⁻¹ due to the O-H bonds of phenolic compounds. Additionally, the sharp peak at 1,591 cm⁻¹ corresponds to the C-C bonds of aromatic compounds. In Figure(D), pure Curcumin displays a signal at 3,510 cm⁻¹ due to its O-H bonds, with two other signals at 1,627 and 1,602 cm⁻¹ corresponding to C=O bonds. The sharp peak at 1,509 cm⁻¹ indicates the C=C bonds of Curcumin. Figure (E) reveals the components associated with chemically produced AuNPs, with signals at 3,418 cm⁻¹ due to R-C-OH groups. In Figure (F), the signals at 3,453 and 3,271 cm⁻¹ are attributed to the OH group of sodium citrate. It is interesting that the biomolecules when associated with the AuNPs provide a more simplified and peak broadened spectrum, which might be the result of the influence of surface plasmons.

Ginger extract was analyzed using gas chromatography-mass spectrometry (GC-MS). Figure 11 shows a graph with a strong signal at 48.974. This signal was compared with mass spectra patterns from the standard library data of the National Institute of Standards and Technology (NIST). The library search analysis indicates that the signal can be associated with Gingerol. Since Gingerol is a phenolic phytochemical compound and its strongest signals are due to its high concentration in the extract and considering the FTIR spectra analysis of the biosynthesized AuNPs, which shows that phenolic groups are associated with the AuNPs, it suggests that Gingerol is highly likely to be present in the caps of the gold nanoparticles.

GC-MS was chosen for the analysis because ginger root contains many volatile substances, making it more suitable than LC-MS, which is typically used for high molecular weight compounds. Since water was used as the solvent and the extract was filtered before GC-MS analysis, the baseline was understandably weaker. However, the strongest signal was attributed to the presence of gingerol. It is important to note that using a different solvent might have resulted in the detection of hydrophobic compounds, but in this case, hydrophilic compounds were predominantly observed.

The biologically and chemically synthesized gold nanoparticles (AuNPs) were evaluated for pH changes resulting from the synthesis and purification process. Table 1 shows the initial pH levels of the synthesized AuNPs without purification, which could influence the outcomes of antimicrobial activity assessments. The pH assessment revealed that the ginger extract and curcumin suspension had a neutral pH of around 6.5. The pH of 1 mM HAuCl₄ was strongly acidic, which lowered the overall pH of the solution. Although the pH of 3.86 mM sodium citrate was basic at approximately 8.0, its minimal addition did not significantly alter the acidic nature of the HAuCl₄ solution. The purification process, including centrifugation and washing with deionized water, led to an increased pH of the purified AuNPs. The assessment of pH before and after purification revealed significant changes, which are crucial as they can influence the antimicrobial activity of the gold nanoparticles. Understanding these pH variations is essential for accurately interpreting the efficacy of the nanoparticles.

The efficacy of biosynthesized gold nanoparticles (AuNPs) with ginger extract and curcumin, compared to chemically produced AuNPs, was tested against planktonic cells of clinical isolates of P. aeruginosa, E. coli, and S. aureus. To evaluate the effect of biological materials and chemical reagents on antimicrobial activity, the materials, gold salt, and the mixture of HAuCl₄ with the biological reagents before the formation of gold nanoparticles were assessed (Table 2). The results demonstrated that curcumin AuNPs exhibited strong antimicrobial activity compared to ginger-AuNPs and chemically produced gold nanoparticles. However, green synthesized AuNPs with ginger showed better activity than chemically produced AuNPs. The concentrations of ginger extract (0.8 mg/mL) and curcumin (0.36 mg/mL) used for synthesizing gold nanoparticles did not exhibit any antimicrobial activity when tested on their own at the same concentrations used in the nanoparticle synthesis.

Our AuNPs synthesized with ginger root extract and curcumin exhibited well-defined peaks in the FTIR spectra, indicating successful capping by organic molecules. The purification process effectively removed biological residues and salts, and the nanoparticles were freeze-dried to prevent water-related artifacts. This led to clear and interpretable FTIR spectra, with minimal interference.

Here we compare to other biogenic synthesis of AuNPs compiled in Table 3. Yang et al. (2014) synthesized AuNPs using waste mango peel extract, resulting in particles ranging from 6 to 18 nm. However, their FTIR analysis was less precise, likely due to the lack of a purification process and freeze-drying. As a result, water molecules surrounding the nanoparticles introduced unwanted signals in their spectra. Additionally, the absence of zeta potential measurements limited the evaluation of nanoparticle stability (Yang et al., 2014).

Chandan Tamuly (2013) biosynthesized AuNPs using Gymnocladus assamicus leaf extract, but the synthesis took 4 h under stirring, making it less time-efficient. The UV-Vis spectra revealed broad peaks, likely due to residual biological compounds. TEM images showed aggregation, suggesting poor stability, and the study lacked DLS and zeta potential measurements, leaving size distribution and stability uncertain (Tamuly et al., 2013).

In contrast, our study demonstrated better results in UV-Vis spectra due to proper purification, which removed residual biological compounds. Our TEM images showed no aggregation, and the zeta potential measurements indicated a stable colloidal solution with values greater than −30 mV, confirming better size distribution and stability (Fourt, 1968; Heimenz, 1978). Also Singh et al. (2013) showed that curcumin capped gold nanoparticles have better size distribution and stability for several months that also had a superior antioxidant activity.

In other study, Wu et al. (2013) biosynthesized AuNPs using Cacumen platycladi leaf extract. However, the age and type of leaves (seasonality) were not specified, which raises concerns about reproducibility. The nanoparticles were of mixed shapes, including nanospheres and nanoplates, and their sizes were inconsistent, indicating poor control over shape and size distribution (Wu et al., 2013). In our study, using ginger extract and curcumin, we achieved well-controlled and uniform nanoparticle sizes, with excellent shape distribution, making the nanoparticles suitable for various applications. Our method is highly repeatable due to its reliance on globally available plant extracts.

In 2015, Paul et al. (2015) synthesized AuNPs from Pogestemon benghalensis leaf extract, resulting in nanoparticles of various shapes (spherical, triangular, hexagonal) and sizes ranging from 10–50 nm. The lengthy 12-hour synthesis time and variable shape distribution indicated limitations in their method, compounded by a lack of zeta potential data, which is crucial for assessing stability. In contrast, our method produced nanoparticles with uniform shape distribution, measuring 10–20 nm (Paul et al., 2015).

Moreover, in the same year, Sujata Patra et al. focused on AuNPs synthesized from Butea monosperma leaf extract. The limited geographic availability of this plant limits the reproducibility of their findings, especially outside South and Southeast Asia. Their method required 24 h of stirring, yielding nanoparticles with varying shapes and sizes (10–100 nm) and low zeta potential values (−12.8 to −16.4 mV), suggesting weak stability. Our approach, using widely available ginger and curcumin, resulted in nanoparticles with high surface charge and zeta potential values of −30 to −40 mV, indicating superior stability and no signs of aggregation (Patra et al., 2015).

Adewale et al. (2020) synthesized AuNPs using Crassocephalum rubens leaf extract, which presents challenges for replication in areas like Canada due to its specific geographic distribution. Their study did not include zeta potential or DLS measurements, hindering the assessment of stability and size distribution. Our work, however, incorporated both measurements, confirming high stability and controlled size distribution of our nanoparticles. Furthermore, we highlighted the role of biological compounds in stabilizing AuNPs, which enhanced antimicrobial activity compared to chemically synthesized counterparts (Adewale et al., 2020).

Nadagouda et al. (2014) utilized extracts from various fruits to synthesize AuNPs, with the synthesis process extending overnight and lacking specified stirring conditions that shows the unrepeatability of their methods. Their TEM images revealed particles exceeding 100 nm in size, with evident aggregation, indicating poor size distribution and stability (Nadagouda et al., 2014). In contrast, our method is efficient and specifies clear reaction parameters, resulting in nanoparticles with superior size distribution and stability, confirmed by zeta potential analysis.

Aljabali et al. (2018) synthesized AuNPs from Ziziphus zizyphus leaf extract, which resulted in heterogeneous nanoparticles that did not exhibit antimicrobial activity even at higher concentrations (5 mg/mL). Conversely, our study showed that AuNPs synthesized with ginger extract and curcumin demonstrated significant antimicrobial activity at much lower concentrations of 1.4 mg/mL, emphasizing the effectiveness of our method (Aljabali et al., 2018).

Yadi et al. (2022) prepared AuNPs from ginger root extract using ethanol, but they did not assess the impact of ethanol on antimicrobial efficacy. Their reported particle size was 314 nm, with a zeta potential of −7.11 mV, indicating poor stability (Yadi et al., 2022). Our study, which utilized water for the extraction process and included rigorous purification steps, produced AuNPs ranging from 10–20 nm with a zeta potential between −30 to −40 mV. This approach allowed us to clearly demonstrate the antimicrobial properties attributed to the AuNPs alone, without interference from residual compounds.