Green Synthesis of l‑Cysteine-Functionalized rGO-Silver Nanoparticles for Enhanced Photocatalytic and Antibacterial Applications

Abstract

In this study, we developed an eco-friendly two-step synthesis of an l-cysteine/reduced graphene oxide-silver nanoparticle (l-Cys-rGO-AgNPs) hybrid material using l-cysteine and l-ascorbic acid as reducing agents. The successful formation and uniform distribution of AgNPs (20–40 nm) on rGO sheets were confirmed by FTIR, XRD, FESEM, and EDX analyses. The nanocomposite achieved 71% photocatalytic degradation of methylene blue and demonstrated potent antibacterial properties against Escherichia coli, with over 99.9% reduction, and weaker performance against Staphylococcus aureus, producing 30–70% reduction. FESEM imaging demonstrated substantial damage to bacterial membranes, with E. coli showing the most disruption, which proves stronger antibacterial effects. These results show that l-Cys-rGO-AgNPs can act as a multifunctional material supporting sustainable environmental remediation and antimicrobial uses.

1. Introduction

Silver nanoparticles (AgNPs) have attracted widespread interest in nanomedicine and biology due to their potent antimicrobial, antifungal, and antioxidant properties. − These properties are largely corresponded to the release of ionized silver (Ag+) from the nanoparticles, which interacts directly with bacterial cellular components and thus renders AgNPs especially effective against bacteria. As described by Vasilev et al., Ag+ ions employ multiple modes of antibacterial action: (i) binding to bacterial DNA and disrupting replication and transcription; (ii) interacting with protein sulfhydryl (−SH) groups, thereby inactivating essential enzymes; and (iii) damaging bacterial cell walls and membranes, leading to the leakage of intracellular contents and ultimately to cell death.

Recent experimental studies indicate that colloidal silver nanoparticles demonstrate antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa strains. Colloidal silver nanoparticles display potential as substitute treatments for conventional antimicrobial agents. According to Richter et al., quasi-spherical silver nanoparticles reduced biofilm formation and bacterial survival across both laboratory and animal experiments. The characteristics of these nanoparticles suggest that they have great potential for medical applications. Traditional production methods for AgNPs depend on harmful chemicals, which produce dangerous waste and high energy consumption. This raises environmental concerns. Researchers have created green synthesis methods to solve environmental challenges related to traditional nanoparticle production. For example, they have developed green synthesis methods that employ eco-friendly reducing agents to produce AgNPs with minimal environmental damage. ,

Graphene oxide’s (GO) unique two-dimensional framework and superior physicochemical characteristics make it an exceptional platform for nanoparticle decoration. − GO sheets interact with bacterial cells by causing oxidative damage and disrupting membrane integrity, which enhances their antimicrobial activity. A combination of GO and AgNPs enhances photocatalytic activity as well as antibacterial effectiveness by utilizing their synergistic properties. , The greatest difficulty lies in developing environmentally sustainable synthesis techniques that enable an even distribution of AgNPs on GO while maintaining their stability and functionality. ,,

l-cysteine plays a dual role in nanoparticle synthesis through its thiol group (−SH), acting as both a reducing agent to form AgNPs and a stabilizing agent to prevent their aggregation. , l-cysteine is known for being a naturally occurring sulfur containing amino acid and has been used as the reducing agent in many versatile chemical and biological applications due to its dominant thiol group (−SH) (Scheme , left). While l-cysteine alone can reduce silver ions, we found that incorporating l-ascorbic acid (l-AA) as a coreducing agent improves the reduction process. We chose L-AA (vitamin C) for its strong reducing properties, which come from the enediol group in its structure (Scheme , right). The two hydroxyl groups (−OH) on neighboring carbon atoms make the molecule highly reactive in redox reactions. l-AA was also used as a coreducing agent to improve the reduction process. Its enediol structure gives it strong reducing power while following green chemistry principles. It is worth noting that combining these two reducing agents with GO has not been previously explored for making multifunctional hybrid materials.

1. Chemical Structures of l-Cysteine (Left) and l-Ascorbic Acid (Right) .

^a Adapted with permission from ref published under a creative commons attribution (CC BY) license.

Our research group demonstrated that using visible light to irradiate urea-doped TiO₂ nanoparticles sensitized with porphyrin led to powerful antibacterial photodynamic inactivation, which shows the capacity of photocatalytic nanomaterials for antimicrobial applications. Photodynamic therapy (PDT) depends on photosensitizers to generate singlet oxygen, while photocatalysis-based antimicrobial strategies explored in this work produce reactive oxygen species (ROS) through semiconductor activation, which leads to bacterial suppression.

We present here a new green two-step synthesis method for l-cysteine/reduced graphene oxide-silver nanoparticles (l-cys/rGO-AgNPs). The process has two main steps. l-cysteine interacts with GO during hydrothermal reduction to produce l-cys/rGO. AgNPs are deposited in a controlled manner when l-AA serves as a coreducing agent. The structure and morphology of the hybrid material were analyzed through FTIR, XRD, FESEM, and EDX techniques. The hybrid material demonstrated photocatalytic activity by degrading the methylene blue. The antibacterial properties of the material were tested against Gram-positive and Gram-negative bacteria in both light and dark environments. This work responds to the growing interest in eco-friendly synthesis methods by employing a water-based two-step process using l-cysteine and l-ascorbic acid as natural, nontoxic reducing and stabilizing agents. Unlike conventional methods using hazardous chemicals, our approach is safer and sustainable and avoids environmental risks.

2. Materials and Methods

Materials and Characterization Methods

Silver nitrate (AgNO₃), potassium permanganate (KMnO₄), sodium nitrate (NaNO₃), sulfuric acid (H₂SO₄), l-cysteine, and methylene blue (MB) (C₁₆H₁₈ClN₃S·3H₂O) were purchased from Sigma-Aldrich Co. Graphene oxide (GO) was supplied by the Kaneh Azar Co. (Tabriz, Iran). All chemicals were analytical grade and used without further purification. The shape and surface features of the samples were examined by using a TESCAN MIRA FESEM. Elemental composition was analyzed with energy-dispersive X-ray spectroscopy (EDS). Functional groups were identified using Fourier transform infrared spectroscopy (Shimadzu 8400S FT-IR) over a range of 400–4000 cm^–1. Crystallinity and phase purity were assessed using a PHILIPS X’Pert Pro D6792 powder X-ray diffraction (XRD) system with monochromatic Cu Kα radiation (λ = 1.5418 Å), and data were processed with X’Pert software. The photocatalytic performance of the samples in methylene blue degradation was evaluated by using a Shimadzu 1700 PharmaSpec UV–vis spectrophotometer.

Synthesis of l-Cys-GO

Graphene oxide (0.25 g) was dispersed in 20 mL of deionized water by ultrasonication (40 kHz, 250 W, 30 min). Separately, l-cysteine (0.125 g) was dissolved in 10 mL of deionized water and sonicated under the same conditions. The combined solutions were further sonicated for 1 h before being transferred to a 50 mL Teflon-lined autoclave. The mixture was heated at 160 °C for 7 h and then naturally cooled to room temperature. The product was vacuum filtered through a 0.45 μm PTFE membrane, washed with deionized water (3 × 50 mL) and ethanol (3 × 30 mL), and then dried under vacuum at 60 °C for 12 h.

Synthesis of l-Cys-rGO-AgNPs

l-Cys-GO powder (0.5 mg/mL) was dispersed in deionized water by ultrasonication (30 min). The suspension pH was adjusted to 10 using an ammonium hydroxide solution. AgNO₃ solution (5 mM) was added dropwise to the suspension under continuous stirring in dark conditions, followed by heating at 60 °C in an oil bath. l-ascorbic acid solution was then added, maintaining an l-AA:AgNO3 weight ratio of 2.0. The reaction continued at 60 °C for 1 h, after which the mixture was cooled to room temperature. The product was collected by centrifugation and washed several times with deionized water and ethanol. The final product was obtained by freeze-drying. Scheme illustrates the detailed synthesis pathway.

2. Schematic Illustration of the Two-Step Synthesis of l-Cys-rGO-AgNPs: Functionalization of GO with l-Cysteine Followed by In Situ Reduction and Decoration with Silver Nanoparticles.

Photocatalytic Degradation of MB

The photocatalytic activity of l-Cys/rGO-AgNPs was tested for MB degradation. A 15 W LED lamp was placed 10 cm from the reaction vessel with continuous air bubbling at 1 atm. The photocatalyst (0.003 g) was dispersed in 12 mL of MB solution (20, 30, or 80 mg/L) and sealed with parafilm. The solution was first kept in the dark for 30 min to reach adsorption–desorption equilibrium, followed by 90 min of LED irradiation at room temperature. A methylene-blue-only sample (without photocatalyst) was included as a control under the same light conditions to assess baseline degradation. MB degradation was monitored using a UV–vis spectrophotometer (Shimadzu 1700Pharrma Spec).

The removal efficiency was calculated using the equation below.

$$\mathrm{removal}\mathrm{efficiency}(\%)=\left[\frac{(C_0-C}{C_0}\right]\times 100$$

where C ₀ is the initial MB concentration and C is the final concentration. Both values were measured using UV–vis absorption at 665 nm.

Reusability Studies

The photocatalytic stability of l-Cys-rGO-AgNPs was tested through four sequential degradation cycles of MB solution (30 ppm) under LED irradiation (15 W). The photocatalyst underwent centrifugation for collection after each run and was then washed with deionized water and dried before the beginning of the next cycle. Throughout all experimental cycles, identical conditions were maintained while using a fresh MB solution for each run. Postcycle physical or compositional analysis was not performed.

Antibacterial Activity Assessment (Time-Kill Assay)

A time-kill assay , was used to evaluate antibacterial activity. Bacterial cultures were prepared and adjusted to a 0.5 McFarland standard (1.5 × 10⁸ CFU/mL) using physiological serum or phosphate buffer. After dilution, a specific volume (0.4–1 mL) of the bacterial suspension was mixed with the samples. Samples were tested at a final concentration of 50 μg/mL. At 1, 3, and 6 h, aliquots were collected, plated on culture media, and incubated at 30 °C for 24–48 h. At the final step, colony-forming units (CFU) were counted to determine the bacterial viability. Antibacterial activity was evaluated for both l-Cys-GO and l-Cys-rGO-AgNPs samples under LED and dark conditions. The reduction percentage (RP%) and logarithmic reduction (LR) were calculated relative to those of the control samples.

$$\mathrm{CFU}/\mathrm{mL}=\frac{(\mathrm{number}\mathrm{of}\mathrm{clonie}\times \mathrm{dilution}\mathrm{factor})}{\mathrm{volume}\mathrm{of}\mathrm{culture}\mathrm{plate}}$$

Statistical Analysis

All photocatalytic and antibacterial experiments were conducted in triplicate unless otherwise stated. Results are presented as average values. No additional statistical analysis was performed.

3. Results and Discussion

Characterization

Scheme demonstrates the two-step synthesis process of l-Cys-rGO-AgNPs through GO functionalization with l-cysteine, followed by in situ reduction and subsequent AgNP decoration. FTIR spectroscopy demonstrates the successful production of l-Cys-rGO-AgNPs through identified vibrational shifts, which show the attachment of l-cysteine and the formation of AgNPs. Peak positions, bond types, and vibrational modes are detailed in the spectral data within Table .

1. FTIR Spectral Data of GO-l-Cys and GO-l-Cys-AgNPs Showing Peak Positions, Bond Types, Vibrational Modes, and Corresponding Observations for Each Functional Group.

peak (cm–1)	l-Cys-GO	l-Cys-rGO-AgNPs	bond type	vibrational mode	observation
OH	3234	3437	O–H (hydroxyl)	stretch	broader peak due to heterogeneous environment
CO (stretch)	1740	1630	CO (carbonyl, ketone/acid)	stretch	shift indicates carbonyl-silver coordination
N–H	1567	1571	N–H (amine/amide)	bending	minor shift due to AgNP interaction
CC	1140	1104	CC (sp carbon)	stretch	shift due to GO structural changes
C–S	633	611	C–S (thiol/sulfur bond)	stretch	shift confirms Ag–S interaction
Ag–X	-	472	Ag–X (Ag–S, Ag–O, or Ag–N)	stretch/bending	may indicate AgNP interactions, as Ag–Ag bonds are IR-inactive

l-cysteine bonded to GO through N–H (1567 cm^–1) and C–S (633 cm^–1) vibrations demonstrate strong interactions between the amine and thiol groups of l-cysteine and the functional groups of GO. The second step, AgNPs deposition, is evidenced by shifts in key peaks, e.g., C–S (to 611 cm^–1), N–H (to 1571 cm^–1), and CO (to 1630 cm^–1). − A new peak appears at 472 cm^–1, which corresponds to the expected range for Ag–X interactions (e.g., Ag–S, Ag–O, or Ag–N). This agrees with previous reports of metal–ligand vibrations (between 400 and 700 cm^–1). − Multiple binding interactions are evidenced by the broadening of the OH band at 3437 cm^–1 and in the C–S and C–C regions.

l-cysteine plays a dual role in the synthesis by modifying the surface of GO and stabilizing AgNPs. During hydrothermal treatment, its −NH₂ and −SH groups likely bind covalently or coordinately to oxygen-containing functional groups on GO, as supported by FTIR spectral shifts. While l-cysteine may exhibit mild reducing properties, the primary reduction of Ag⁺ is driven by l-AA, added at a 2:1 (w/w) ratio relative to that of AgNO₃ to balance nucleation and growth. The combination of l-cysteine and l-AA improves redox efficiency; l-cysteine facilitates Ag⁺ anchoring, while l-AA promotes rapid electron transfer and controlled nanoparticle formation.

The UV–Vis spectrum of l-Cys-rGO-AgNPs exhibited a broad absorption band centered at around 405 nm, characteristic of AgNPs, and a shoulder near 280 nm attributed to π → π* transitions in partially reduced GO (Figure ). In contrast, AgNPs alone showed a sharp surface plasmon resonance (SPR) peak at ∼ 420 nm, confirming their nanoscale formation.

1.

UV–Vis spectra of AgNPs and l-Cys-rGO-AgNPs.

The FESEM micrograph shows silver nanoparticles deposited on the surface of l-cysteine-functionalized reduced graphene oxide sheets. Silver nanoparticles between ∼ 20 and ∼ 40 nm appear evenly spread over the surface of rGO. The FESEM images (Figure ) show silver nanoparticles distributed on l-Cys-rGO sheets, suggesting the formation of the l-Cys-rGO-AgNPs nanocomposite. EDS analysis was used to investigate the elemental composition of the l-Cys-rGO-AgNPs. The EDS spectrum presented in Figure (top), together with the elemental composition in Figure (middle), shows that rGO sheets contribute C (41.62 wt %, 71.66 atom %) and O (12.34 wt %, 15.95 atom %), l-cysteine contributes S (7.86 wt %, 5.07 atom %), while silver nanoparticles contribute Ag (38.18 wt %, 7.32 atom %). Elemental mapping depicted in Figure (bottom) shows all elements spread evenly throughout the nanocomposite structure.

2.

FESEM images of (A) l-Cys-rGO before silver decoration, showing the layered morphology of functionalized graphene oxide, and (B) l-Cys-rGO-AgNPs, illustrating the uniform distribution of AgNPs (∼20–40 nm) on the rGO sheets.

3.

EDS analysis of l-Cys-rGO-AgNPs. (Top) EDS spectrum confirms the presence of C, O, S, and Ag. (Middle) Elemental composition table showing weight % (W%), atomic % (A%), ZAF factors, and peak-to-background ratios. (Bottom) SEM image with elemental mapping demonstrating the uniform distribution of AgNPs on rGO.

Finally, the XRD analysis further confirms the successful synthesis of l-Cys-rGO-AgNPs (Figure ). The intense, broad peak located at 2θ = 25° (002) in the XRD pattern of l-Cys-GO confirms that GO sheets exhibit interlayer stacking. After formation of the nanocomposite, l-Cys-rGO-AgNPs shows significantly reduced intensity of the peak at 2θ = 25°, indicating successful reduction of GO to rGO. The formation of crystalline AgNPs in the nanocomposite is evidenced by sharp diffraction peaks at 2θ = 38°, 44°, 65°, and 78°, indexed to (111), (200), (220), and (222) planes of the face-centered cubic silver structure. ,

4.

XRD patterns of (a) l-Cys-GO showing a characteristic peak at 2θ = 25° and (b) l-Cys-rGO-AgNPs showing a reduced intensity of 2θ = 25° and characteristic fcc silver peaks at 2θ = 38° (111), 44° (200), 65° (220), and 78° (222).

Photocatalytic Degradation of MB

The photocatalytic performance of the l-Cys-rGO-AgNPs nanocomposite was tested for MB degradation by using LED light (15 W). The absorption peaks of MB at 665 and 614 nm diminished across different irradiation periods and demonstrated removal rates of 71%, 60%, and 38.5% after 120 min for starting MB concentrations of 20, 30, and 80 ppm. It demonstrates that photocatalytic activity depends on MB concentration, e.g., the availability of active sites and light penetration limitations (Figure ). Compared to our previous TiO₂-based system (PNT), which achieved ∼ 65% degradation of MB under similar light conditions, the current l-Cys-rGO-AgNPs show improved degradation (71%) and added antibacterial properties. This enhancement is likely due to synergistic effects from AgNPs and the rGO matrix functionalized with l-cysteine. Table gives a quick comparison with other reported photocatalysts and shows how our system stands out in both performance and added antibacterial function.

5.

UV–vis absorption spectra showing the photocatalytic degradation of methylene blue (MB) at different initial concentrations (20, 30, and 80 ppm) using l-Cys-rGO-AgNPs under 15W LED irradiation for 120 min. A control experiment with MB alone (without a catalyst) was also included to confirm that no significant degradation occurred under light exposure alone.

2. Comparison of MB Degradation Efficiency and Key Features of Various Photocatalysts under Different Light and pH Conditions.

photocatalyst	light source	pH	degradation (%)	time (min)	additional features	references
PNT (TiO2-porphyrin)	LED (45 W)	∼7	∼65%	30	antibacterial, porphyrin-sensitized, visible-light active
TiO2 (unmodified)	sunlight	10	80%	60	simple, low-cost TiO2
ZnO (green, Padina extract)	sunlight	10	98%	60	antibacterial, green method
5%NT/TiO2	visible (300 W Xe)	∼7	56.5%	150	interstitial N-doping enhancement
l-Cys-rGO-AgNPs (this work)	LED (15 W)	∼7	71%	30	antibacterial, green synthesis	

A stepwise mechanism describes the degradation process of MB when l-Cys-rGO-AgNPs are used, as shown in Figure . Silver nanoparticles (AgNPs) activate surface plasmon resonance (SPR) when exposed to light-emitting diode (LED) illumination at 15 W, which produces electron–hole pairs (e^–/h⁺). Electrons (e^–) move to reduced graphene oxide (rGO), while holes (h⁺) stay within the AgNPs. The generated charge carriers interact with molecular oxygen (O₂) and water (H₂O) or hydroxide ions (OH^–) to produce superoxide radicals (^•O₂ ^–) and hydroxyl radicals (^•OH), which together form reactive oxygen species (ROS). These species play a critical role in the photocatalytic degradation of MB, as demonstrated in our previous study and further supported by literature. , The generated radicals degrade MB into small intermediates that continue to oxidize until they are converted into carbon dioxide (CO₂) and water (H₂O).

6.

Photocatalytic degradation mechanism of MB using l-Cys-rGO-AgNPs under LED light (15W, 420–430 nm), involving photoexcitation, SPR-induced charge separation, ROS generation, and MB oxidation to CO₂ and H₂O. Ag⁺ release may act as a secondary contributing pathway.

We did not adjust the pH of the methylene blue solution during the photocatalytic experiments. We chose to use a neutral pH to maintain consistency with the antibacterial tests, which were also conducted under neutral conditions. While it is well-known that alkaline conditions can enhance hydroxyl radical (^•OH) formation and improve dye degradation, , our nanocomposite still demonstrated strong photocatalytic activity at natural pH. Moreover, the l-cysteine functionalization of the rGO-AgNPs may alter the surface charge of the nanocomposite. l-cysteine adds carboxyl and thiol groups to the nanocomposite surface, giving it a negative charge. This may increase the electrostatic attraction toward the cationic MB dye, thus enabling more effective adsorption and degradation.

l-Cys-rGO-AgNPs photocatalyst reusability was assessed over four consecutive photocatalytic cycles. The photocatalyst showed good stability and recyclability, as the MB removal efficiency remained high, with only a moderate decrease from 65% to 57% after four cycles. Figure shows the recycling performance data. Although postcycle structural or leaching analyses were not performed, the consistent photocatalytic performance over four cycles indicates that the catalyst maintained reasonable operational stability.

7.

Recycling performance of l-Cys-rGO-AgNPs for MB (30 ppm) photodegradation under LED irradiation (15W), showing degradation efficiency values of 67%, 63%, 59%, and 57% for runs 1–4, respectively.

Antibacterial Activity

The antibacterial effectiveness of l-Cys-GO and l-Cys-rGO-AgNPs against S. aureus ATCC 25923 was tested under LED light exposure and dark conditions. The antibacterial performance of l-Cys-GO under LED light was negligible (<5%) for the initial 3 h and increased to 50% by 6 h, while l-Cys-rGO-AgNPs demonstrated a steady 30% reduction during the entire test period. In darkness, l-Cys-rGO-AgNPs demonstrated enhanced antibacterial effectiveness, increasing their reduction rates from 30% to 70% throughout 6 h, while l-Cys-GO displayed improvement from less than 5% to 50%. Table contains the full antibacterial performance details

3. Antibacterial Activity of l-Cys-GO and l-Cys-rGO-AgNPs against S. aureus ATCC 25923 under LED Lamp Exposure and Dark Conditions .

		gram-positive bacteria (S. aureus ATCC 25923)
		exposure to LED lamp			no light (dark)
time	antimicrobial efficacy parameters	l-Cys-GO	l-Cys-rGO-AgNPs	control	l-Cys-GO	l-Cys-rGO-AgNPs	control
1 h	VC1(CFU/mL)	>9.5 × 104	7 × 104	1 × 105	>9.5 × 104	7 × 104	1 × 105
	RP2 (%)	<5%	30%	-	<5%	30%	-
	LR3 (log10)	<0.022	0.155	-	<0.022	0.155	-
3 h	VC (CFU/mL)	>9.5 × 105	7 × 105	1 × 106	8 × 105	5 × 105	1 × 106
	RP (%)	<5%	30%	-	20%	50%	-
	LR (log10)	<0.022	0.155	-	0.097	0.301	-
6 h	VC (CFU/mL)	5 × 106	7 × 106	1 × 107	5 × 106	3 × 106	1 × 107
	RP (%)	50%	30%	-	50%	70%	-
	LR (log10)	0.301	0.155	-	0.301	0.523	-

^aFootnotes: VC¹ (Viable Cell Count), RP² (Reduction Percentage), LR³ (Log Reduction), CFU (Colony-Forming Unit), and LED (Light-Emitting Diode, 15 W). Tests were performed at 37 °C with an initial bacterial concentration of ∼ 10⁵ CFU/mL. (−) indicates control values used as a reference for calculations.

The antibacterial activity of l-Cys-GO and l-Cys-rGO-AgNPs demonstrated notably different efficacy against Escherichia coli compared to S. aureus. l-Cys-rGO-AgNPs achieved an exceptional antibacterial effect against E. coli with over a 99.9% reduction (LR > 4–6) under both LED light exposure and dark environments throughout the entire testing period. In contrast, its effect on S. aureus was considerably lower, showing only a 30–70% reduction. l-Cys-GO reached a 70% reduction in E. coli after 3 h under both conditions, with an LR value of 0.523, but required 6 h to reach a 50% reduction in S. aureus. Table displays a summary of the antibacterial effects against E. coli. The varied antibacterial activity between E. coli and S. aureus originates from their unique cell wall structures as the thick peptidoglycan layer in Gram-positive S. aureus delivers enhanced resistance to antimicrobial agents. , In contrast, the outer membrane of E. coli contains negatively charged lipopolysaccharides (LPS), which may interact more readily with the l-cysteine-functionalized nanocomposite, enhancing nanoparticle binding and uptake.

4. Antibacterial Activity of l-Cys-GO and l-Cys-rGO-AgNPs against E. coli ATCC 25922 under LED Lamp Exposure and Dark Conditions.

		gram-negative bacteria (E. coli ATCC 25922)
		exposure to LED lamp			no light (dark)
time	antimicrobial efficacy parameters	l-Cys-GO	l-Cys-rGO-AgNPs	control	l-Cys-GO	l-Cys-rGO-AgNPs	control
1 h	VC1(CFU/mL)	5 × 104	<1 × 101	1 × 105	7 × 104	<5 × 102	1 × 105
	RP2 (%)	50%	>99.9%	-	30%	>99.5%	-
	LR3 (log10)	0.301	>4	-	0.155	>2.301	-
3 h	VC (CFU/mL)	3 × 105	<11 × 101	1 × 106	4 × 105	<1 × 101	1 × 106
	RP (%)	70%	>99.9%	-	60%	>99.9%	-
	LR (log10)	0.523	>5	-	0.398	>5	-
6 h	VC (CFU/mL)	3 × 106	<1 × 101	1 × 107	3 × 106	<1 × 101	1 × 107
	RP (%)	70%	>99.9%	-	70%	>99.9%	-
	LR (log10)	0.523	>6	-	0.523	>6	-

A study has demonstrated that amino acid (l-cysteine)-functionalized graphene oxide exhibits superior antibacterial action against E. coli compared to S. aureus, with enhanced membrane disruption attributed to interactions between Ag⁺ and bacterial proteins, as well as thiol-mediated surface binding. This is consistent with our findings, where we observed over a 99.9% reduction in E. coli cells, compared to only a 30–70% reduction in S. aureus populations.

In contrast, prismatic AgNPs combined with GO and GO-Ag nanocomposites demonstrate enhanced antibacterial effects on S. aureus, which lead to complete annihilation at elevated concentrations. , The pronounced antibacterial effects of prismatic AgNPs stem from their reactive (111) crystal facets, together with enhanced ROS production. In our system, however, antibacterial activity was observed under both light and dark conditions. This suggests that silver ion release and membrane disruption are the primary contributors, while light exposure may enhance activity through ROS generation, particularly in E. coli, but plays a secondary role.

FESEM imaging demonstrated the morphological differences between S. aureus and E. coli when they were exposed to l-Cys-rGO-AgNPs (Figure ). FESEM images A and B display S. aureus cells that maintained their spherical form but exhibited distorted membranes with rough surfaces. Images C and D demonstrate substantial structural damage to E. coli cells, characterized by reduced size and membrane breakdown, which indicates that the thin peptidoglycan layer of Gram-negative bacteria increases their vulnerability to nanoparticle damage. The AgNPs were uniformly distributed on the rGO surface with sizes ranging from ∼ 20 to 40 nm, as seen in FESEM images, with only minor clustering. This nanoscale dispersion likely enhances bacterial contact and facilitates effective Ag⁺ ion release. The results confirm the quantitative antibacterial studies, as l-Cys-rGO-AgNPs show greater effectiveness against E. coli.

8.

FESEM images of S. aureus (A, B) and E. coli (C, D) after treatment with l-Cys-rGO-AgNPs. E. coli cells exhibit pronounced morphological damage, including membrane rupture and surface collapse, while S. aureus shows more localized surface alterations. These structural differences support the observed higher antibacterial efficacy against E. coli.

4. Conclusions

The present study successfully demonstrated a new green synthesis approach for an l-cysteine/reduced graphene oxide-silver nanoparticle (l-Cys-rGO-AgNPs) hybrid material. FTIR, XRD, FESEM, and EDX analyses confirmed the successful functionalization of GO with l-cysteine and uniform decoration of AgNPs (56.72 and 97.09 nm) on the rGO surface. The nanocomposite showed efficient photocatalytic activity in MB degradation, achieving 71% removal efficiency for 20 ppm of MB concentration, with good reusability over four following cycles. Furthermore, the composite displayed remarkable antibacterial behaviors, particularly against E. coli (>99.9% reduction) compared to S. aureus (30–70% reduction), demonstrating the influence of bacterial cell wall structure on antimicrobial efficacy. As shown in the FESEM studies, E. coli exhibited significant membrane damage, confirming its higher susceptibility to l-Cys-rGO-AgNPs. The better performance in both photocatalytic and antibacterial applications, combined with a green, water-based synthesis using biocompatible reagents under mild conditions, makes l-Cys-rGO-AgNPs a promising and scalable multifunctional material for environmental remediation and antimicrobial applications. Future work should address challenges such as scalability, toxicity, and cost. Machine learning may also help optimize synthesis conditions to further improve performance. This strategy is also applicable to other metals such as Cu, as shown in our ongoing work, highlighting its versatility for multifunctional applications.

Glossary

Abbreviations

AgNPs

silver nanoparticles

CFU

colony-forming unit

EDX

energy-dispersive X-ray spectroscopy

FESEM

field emission scanning electron microscopy

FTIR

Fourier transform infrared spectroscopy

GO

graphene oxide

l-AA

l-ascorbic acid

l-Cys

l-cysteine

l-Cys-GO

l-cysteine functionalized graphene oxide

l-Cys-rGO-AgNPs

l-cysteine functionalized reduced graphene oxide-silver nanoparticles

MB

methylene blue

ppm

parts per million

RP

reduction percentage

rGO

reduced graphene oxide

UV–vis

ultraviolet–visible spectroscopy

XRD

X-ray diffraction

LED

light-emitting diode

SPR

surface plasmon resonance

ROS

reactive oxygen species

LR

log reduction

VC

viable cell count

The authors declare no competing financial interest.