Enhanced Microbial Sensing via Resazurin Reduction Catalyzed by Graphene Oxide, A Versatile Approach for Diagnostics and Electrochemical Applications

TL;DR

Graphene oxide speeds up resazurin reduction by bacteria, improving microbial detection for environmental monitoring and diagnostics.

Contribution

GO enhances resazurin reduction by Staphylococcus aureus, enabling rapid and sensitive microbial sensing.

Findings

GO accelerates resazurin reduction by S. aureus at non-cytotoxic concentrations.

The GO–resazurin system enables rapid detection of viable bacteria in environmental samples.

Resazurin/GO/bacteria interactions show enhanced resorufin color loss, useful for pollution monitoring and microbial fuel cells.

Abstract

Resazurin is a cell viability phenoxazine dye widely employed for bacterial monitoring, as its colorimetric and fluorometric conversion reflects microbial metabolic activity. In this work, we demonstrate that graphene oxide (GO), a two‐dimensional nanomaterial with high surface reactivity, markedly accelerates the reduction of resazurin in the presence of
Staphylococcus aureus
, enabling rapid microbial detection at non‐cytotoxic concentrations. Importantly, this GO‐mediated enhancement directly supports applications in environmental toxicology. Rapid identification of bacterial contaminants in water and environmental samples is essential for assessing toxic exposures, such as those caused by pathogenic contamination of drinking water. By lowering the time required to detect viable bacteria, the GO–resazurin system provides a sensitive and practical tool for evaluating environmental quality and potential health risks associated with microbial contamination. While the GO and resazurin lack interaction in the absence of cells, the
S. aureus
metabolism specifically increases electron shuttling of the probe towards the carbon material compared to other tested bacteria. Furthermore, with a comprehensive spectrofluorimetric characterization of resazurin/GO/bacteria interaction, we demonstrate an enhanced resorufin color loss that unveils potential applications in revolutionizing environmental monitoring, pollutant degradation, and microbial fuel cells design. These findings highlight the role of GO as a catalytic enhancer, enabling more sensitive early warning systems in exposure assessment, improving accuracy in pollution monitoring, and facilitating efficient and sustainable remediation strategies for toxic substances removal.

Figures

Resazurin and GO interaction effects on fluorescence and absorption spectra. (A) Resazurin and resorufin fluorescence emission spectra using excitation at 540 nm. (B) Absorbance spectra of resazurin and resorufin normalized to background. Resorufin has been obtained after letting the probe interact with metabolically active S. aureus cells. (C) Fluorescence peak (590 nm) intensity of resazurin and GO, soon after mixing or after 24 h of interaction. (D) Fluorescence peak (640 nm) intensity of resazurin and GO, soon after mixing or after 24 h of interaction. In the inset, the linear correlation between GO concentration and peak signal is reported, the signal of the sample with resazurin and GO at 500 μg/mL was not detectable. (E) Picture of GO‐resazurin mix in 96 wells with a maximum GO concentration of 500 μg/mL and dilutions 1:2. Ration between 590/640 nm fluorescence peaks at different time points. (F) Normalized absorbance spectra of GO and resazurin mix at different concentrations were obtained by subtracting GO signal. The resazurin spectrum is reported in pink.

Resorufin peak intensity in the presence of different concentrations of S. aureus: 590 nm primary peak in (A), 640 nm secondary peak in (B), and ratio 590/640 nm in (C).

Viability of S. aureus in the presence of GO without agitation in water. Optical density (OD) values 4 h after incubation in fresh LB broth from samples incubated with GO.

GO accelerates S. aureus‐mediated resazurin conversion. Peak intensity (590 nm) of bacteria incubated with resazurin alone (A), GO at 16 μg/mL and resazurin (B), or GO at 125 μg/mL and resazurin (C). Pictures of 96 wells containing GO‐S. aureus and resazurin (first line in each picture) at different GO concentrations, GO without the probe (second line), or GO and resazurin without bacteria (third line), taken at different time points (D). Pictures of supernatants after centrifugation; samples were incubated with resazurin and GO at different concentrations with or without bacteria after 1 h of interaction (E). Scheme of GO effects on resazurin in the presence of S. aureus (F).

Effect of GO on resazurin absorption properties. OD peak (560 nm) intensity in a time of 24 h (A). OD peak (610 nm) intensity in 24 h and 560/610 nm ratio (dashed lines) is reported in (B).

Resazurin absorption in presence of GO can be exploited for fast diagnostics (A) GO‐resazurin absorption spectra after incubation with different concentrations of S. aureus (107 CFU/mL, 0.5 × 107 CFU/mL or 0.25 × 107 CFU/mL, symbolized with +++; ++; +, respectively). Resazurin spectra without GO are reported in pink. (B) Percentage of the efficacy of 560/610 nm ration in the presence of different GO concentrations (16GO or 31GO refers to 16 μg/mL or 31 μg/mL, respectively) normalized to resazurin alone. (C) Pictures of 96 wells after incubation of GO and/or resazurin with or without E. coli, S. pasteurii or S. aureus in solution ( indicates the complete color loss after 24 h of incubation for S. aureus). The ratio measured after 20 min or 72 h of incubation for each species is also reported.

Full text

Introduction

1

Environmental toxicology increasingly relies on rapid, sensitive, and cost‐effective microbial monitoring tools to assess risks associated with contaminated water, food, and soil [1, 2]. Bacterial pathogens in drinking water and recreational waters are among the most relevant toxic exposures, as they can trigger acute or chronic health effects when not promptly detected. Conventional culture‐based approaches, while reliable, are time‐consuming and often unsuitable for timely exposure assessment. Resazurin or Alamar blue (7‐hydroxy‐10‐oxidophenoxazin‐10‐ium‐3‐one) is a widely employed phenoxazine dye in cell viability assays [3]. This blue indicator has long been applied in biological laboratory testing due to its ability to change color in response to metabolic activity, making it an essential tool in detecting microbial growth and evaluating eukaryotic cell viability [4]. The resazurin reduction mechanism involves the enzymatic action of living cells and causes the blue, oxidized form of resazurin to change into a pink, fluorescent resorufin, signaling the presence of viable organisms. Bleaching out of the pink color of the reduced state of the dye usually occurs after prolonged incubation times with the formation of a colorless product called dihydroresorufin [4]. Compared to other cell fluorescent assays, resazurin has the advantage of being non‐toxic and therefore suitable to keep cells viable during measurements, while tetrazolium‐based dyes such as 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and (sodium 3′‐[1‐(phenylaminocarbonyl)‐3,4‐tetrazolium]‐bis (4‐methoxy6‐nitro) benzene sulfonic acid hydrate) (XTT) require an end‐point assessment after cell lysis [4]. Resazurin is also water‐soluble and acts as an intermediate electron acceptor without inhibiting the normal electron chain. Indeed resazurin can be reduced by NADPH, FADH_2_, FMNH, NADH as well as cytochromes, mitochondrial reductases, and other enzymes such as the diaphorases, NAD(P)H:quinone oxidoreductase, and flavin reductase [4]. Besides laboratory testing, the resazurin color conversion has been exploited in microcapillaries for the evaluation of antibiotic susceptibility of uropathogenic bacteria in urine samples [5, 6, 7, 8]. In bone research, Mg‐implant biodegradation has been detected noninvasively in near‐real‐time through the skin by measuring hydrogen release using resazurin‐based patches [9]. Other resazurin applications include oral bacteria testing [10] or quantification of microbial contamination in pharmaceutical samples [11], with the advantages of direct application to the patient/or to the sample without toxicity and with high sensitivity [12]. Resazurin, however, is also known for its electron shuttling characteristics used to enhance bio‐electricity generation in microbial fuel cells, bio‐electrochemical devices that convert chemical energy stored in organic compounds into electrical energy through the metabolic activity of microorganisms [13].

Building on these foundations, we aimed to investigate the effects of the carbon‐based nanomaterial graphene oxide (GO) in the interactions between resazurin dye and bacteria, specifically to reduce the incubation required to detect pathogenic bacteria, that is, Staphylococcus aureus , and to improve the electron shuttling mechanism described for the pure probe.

When evaluating the interaction of resazurin and nanomaterials, additional layers of complexity emerge, particularly in the context of photocatalytic interactions. In particular, it was demonstrated that the probe can be photo‐catalytically reduced by the GO under UV light [14]. GO is well known for biosensing and drug delivery due to its high surface area and biocompatibility [15, 16, 17]. In microbiological applications, GO exhibits antimicrobial properties, making it effective in inhibiting bacterial growth and biofilm formation at specific concentrations and incubation conditions [18, 19]. Additionally, GO is employed in photocatalysis for environmental applications, such as pollutant degradation, owing to its ability to enhance the efficiency of light‐induced reactions [20, 21]. Indeed, graphene‐based materials can be used as catalysts to drive chemical reactions thanks to their high surface area, excellent electrical conductivity, and ability to separate electron–hole pairs efficiently. These advantages are exploited in environmental cleanup, water splitting for hydrogen production, and degradation of organic pollutants. Catalysis without light can also occur thanks to specific defects and groups on the GO surface [22, 23, 24].

In this work, we demonstrate via spectrofluorimetric study and microbial growth quantification that the interaction between resazurin and low concentrations of GO in the presence of microbial cultures enables a rapid reduction of the probe, leading to the formation of resorufin and subsequently dihydroresorufin. This interaction highlights the potential for GO‐resazurin systems to enhance the sensitivity and response speed in pathogen environmental monitoring, particularly for wastewater treatment applications. We also verified that another Gram‐positive bacterium, Sporosarcina pasteurii , or Gram‐negative Escherichia coli, did not exhibit an analogous effect on resazurin in the presence of GO, suggesting a specific metabolic mechanism or interaction unique to S. aureus . This specificity could be harnessed for targeted microbial detection in environmental samples, offering a valuable tool for assessing water quality and contamination. Additionally, the integration of resazurin and GO in environmental sensing platforms, such as wearable devices for real‐time monitoring, holds significant promise. The light‐independent catalytic properties of GO further open avenues for its use in microbiological‐based treatments of wastewater, accelerating the breakdown of organic contaminants in resource‐limited settings. This study emphasizes the expanding applications of resazurin beyond diagnostics, providing insights into the convergence of microbiology, materials science, and environmental technology for pollution control, resource management, and ecosystem restoration.

Materials and Methods

2

Graphene Oxide Preparation

2.1

GO (GrapheneA) was used at the final concentration reported in each graph from a batch at 4 mg/mL using ultrapure water for serial dilution. Samples were periodically vortexed to avoid precipitation. GO size and chemical properties are reported in our previous work [25].

Bacteria Cell Growth

2.2

S. aureus (ATCC strain 29213) or E. coli (ATCC strain 25922) were grown in Luria Bertani (LB) medium at 37°C overnight, and cells were harvested via centrifugation (4000 rpm for 10 min) and washed three times with deionized water to remove residual macromolecules and other growth medium constituents. The pellets were then suspended in ultrapure water and kept at 37°C for resazurin assay. S. pasteurii (ATCC‐11859) was grown in Urea Broth at 30°C overnight and washed as reported for S. aureus. Experiments with resazurin have been performed at 30°C. Bacterial cell suspensions were diluted to obtain cell samples having OD = 0.3 that corresponded to 10^7^ CFU/mL. 50 μL of cells were mixed with GO (GrapheneA) to obtain a final GO concentration comprised between 8 and 500 μL. Cell viability in controls or after resazurin and GO incubation was evaluated by the colony counting method, spreading 100 μL of colonies onto LB plates and letting colonies grow overnight at 37°C. Full characterization of GO is provided in our previous work [26].

Resazurin Assay and UV‐Spectroscopy

2.3

Resazurin assay has been performed using the manufacturer's protocol (Cell Signaling) with a dilution of the original probe batch of 1:10 and incubating the samples for up to 24 h. Absorbance and fluorescence spectra have been recorded in a UV transparent 96‐well plate using Cytation 3 Cell Imaging Multi‐Mode Reader. Image analysis was performed using ImageJ software while spectra were analyzed and normalized using Microsoft Excel.

Results and Discussion

3

Resazurin Conversion to Resorufin Does Not Occur in the Presence of GO, However, Resazurin Fluorescence Is Proportional to GO Concentration

3.1

The resazurin‐based viability assay can be carried out either with colorimetric, fluorometric, or absorption detection [27]. To detect the fluorescence conversion of resazurin to resorufin, the excitation range employed is 530–540 nm, using a main emission peak at 590 nm and a secondary peak at 640 nm, present both in resazurin and resorufin (Figure 1A). For absorbance, either an increase in the intensity of the peak at the wavelengths corresponding to resorufin maximum (540–570 nm) or a decrease in absorbance at the wavelengths of resazurin maximum (585–620 nm) can be monitored during the probe conversion (Figure 1B). Alternatively, a ratiometric index can be used for the partial elimination of technical or instrumental biases, bearing in mind that the remaining non‐consumed resazurin might interfere with resorufin formed during the reaction [27].

Resazurin and GO interaction effects on fluorescence and absorption spectra. (A) Resazurin and resorufin fluorescence emission spectra using excitation at 540 nm. (B) Absorbance spectra of resazurin and resorufin normalized to background. Resorufin has been obtained after letting the probe interact with metabolically active S. aureus cells. (C) Fluorescence peak (590 nm) intensity of resazurin and GO, soon after mixing or after 24 h of interaction. (D) Fluorescence peak (640 nm) intensity of resazurin and GO, soon after mixing or after 24 h of interaction. In the inset, the linear correlation between GO concentration and peak signal is reported, the signal of the sample with resazurin and GO at 500 μg/mL was not detectable. (E) Picture of GO‐resazurin mix in 96 wells with a maximum GO concentration of 500 μg/mL and dilutions 1:2. Ration between 590/640 nm fluorescence peaks at different time points. (F) Normalized absorbance spectra of GO and resazurin mix at different concentrations were obtained by subtracting GO signal. The resazurin spectrum is reported in pink.

In the presence of GO, resazurin fluorescence peaks at 590 and 610 nm (EXC = 540 nm) are quenched by increasing GO concentration (Figure 1C,D, respectively). The quenching is stable and more pronounced at high GO concentrations. Interestingly, the 640 nm signal is dependent on GO concentration, highlighting the possibility of using resazurin dye to quantify traces of GO in solution (inset in Figure 1D). This approach might be useful in many procedures that foresee GO centrifugation and manipulation to distinguish, as an example, the amount of small unprecipitated flakes of GO.

When incubated with GO alone, resazurin is not converted to resorufin as demonstrated by ratiometric analysis (590/640 nm) and colorimetrically visible by the picture of samples after 24 h of incubation (Figure 1E and inset, respectively). The slight increase in the ratio after 24 h is not significant compared to values obtained with living bacteria reported below and is due to the increase of the 590 nm signal after the initial quenching. This might indicate a non‐reversible interaction between GO and the probe that allows for the restoration of the signal. Besides the evident color bleaching at concentrations > 125 μg/mL, absorbance is not affected by GO to the same extent as fluorescence, as shown in Figure 1F. In this case, the reduced signal at 590 nm is not proportional to GO concentration.

GO Increases

S. aureus Conversion of Resazurin Into Resorufin and Subsequent Quenching Into Dihydroresorufin, This Mechanism Can Improve the Speed of Microbial Detection

3.2

When living S. aureus cells are incubated with resazurin, the probe is converted over time by bacteria according to cell concentration, causing an increase in resorufin peaks. In Figure 2A,B, the intensity of 590 nm and 640 nm peaks is reported when the probe is incubated with 3 different bacteria concentrations (0.25, 0.5, or 1 × 10^7^ CFU/mL). The higher the number of cells, the faster the change in the speed of resazurin conversion to resorufin. The recommended manufacturer test time is between 1 and 4 h; interestingly, after 24 h, the overall fluorescence signal (590 and 640 nm) diminishes at high S. aureus concentrations due to the probe transformation in dihydroresorufin. However, the ratiometric index 590/640 nm is still proportional to the initial cell inoculum (Figure 2C).

Resorufin peak intensity in the presence of different concentrations of S. aureus: 590 nm primary peak in (A), 640 nm secondary peak in (B), and ratio 590/640 nm in (C).

When GO is added to the mixture in a concentration range comprised between 0 and 500 μg/mL, the nanomaterial effects on bacteria should be considered prior to investigating effects on the probe.

The antibacterial effects of GO, through blade cutting, trapping, or reactive oxygen species generation, involve GO's sharp edges destroying bacterial membranes or its sheets enveloping bacteria, disrupting cellular functions. These effects depend on GO concentration—higher concentrations enhance interactions—and are influenced by experimental conditions like pH, temperature, bacterial species, and medium composition, which can modify the extent and nature of GO's antibacterial activity [19, 28, 29]. We optimized the incubation temperature (37°C) to minimize cell death in the absence of nutrients (i.e., in water) and minimized the effect of blades by avoiding sample agitation and using bacteria in a stationary phase rather than an exponential phase. MIC was used to calculate the viability of bacteria after exposure to the nanomaterial during the 4 h resazurin assay (Figure 3). As is visible, the lowest concentration of bacteria is more affected by GO in solution with ~47% and ~35% of cells killed after 4 h of exposure to GO. With a higher number of CFUs, the GO effect was lower or absent (10^7^ CFUs/mL). Therefore, to analyze probe conversion to resorufin, GO concentrations below 125 μg/mL can be used since the bacteria viability is not significantly affected (effect < 20%).

Viability of S. aureus in the presence of GO without agitation in water. Optical density (OD) values 4 h after incubation in fresh LB broth from samples incubated with GO.

In Figure 4, we compared the probe conversion by using different GO concentrations using the 590 nm peak. As expected, the process occurs after the initial resorufin conversion; therefore, it happens earlier when the number of bacteria is higher, even at a very low GO concentration (16 μg/mL) (Figure 4A,B and pictures in Figure 4D). The high GO concentration (125 μg/mL) speeds up the process, and the dye color is rapidly bleached (Figure 4C and pictures in Figure 4D), as demonstrated also by centrifugation of samples and analysis of supernatant after 1 h of interaction (Figure 4E).

GO accelerates S. aureus‐mediated resazurin conversion. Peak intensity (590 nm) of bacteria incubated with resazurin alone (A), GO at 16 μg/mL and resazurin (B), or GO at 125 μg/mL and resazurin (C). Pictures of 96 wells containing GO‐S. aureus and resazurin (first line in each picture) at different GO concentrations, GO without the probe (second line), or GO and resazurin without bacteria (third line), taken at different time points (D). Pictures of supernatants after centrifugation; samples were incubated with resazurin and GO at different concentrations with or without bacteria after 1 h of interaction (E). Scheme of GO effects on resazurin in the presence of S. aureus (F).

The centrifugation confirms that 500 μg/mL and 250 μg/mL cause the total disappearance of the resazurin color from the supernatant after 1 h of incubation, even without bacteria. We hypothesize that the high availability of GO surface limits the probe interaction with bacterial enzymes and that the free probe (available only at concentrations ≤ 125 μg/mL) is quickly converted by S. aureus into the non‐fluorescent form at these high concentrations, as shown in the scheme in Figure 4F.

GO is enriched with oxygen‐containing functional groups and exhibits a combination of chemical and physical properties that make it an efficient catalyst in various transformations. Its ability to accelerate the reduction of resazurin to resorufin can be attributed to its acidic nature, surface functionalities, and interaction with aromatic compounds. The oxygen‐containing groups, such as epoxides, hydroxyls, and carboxyls, play a critical role in facilitating redox reactions by mediating electron and proton transfer [30, 31]. Additionally, the inherent acidity of GO promotes protonation processes that stabilize reaction intermediates, enhancing the reaction rate. The aromatic scaffold of GO allows for π–π interactions with resazurin and resorufin, concentrating these molecules on their surface and improving the likelihood of reaction events. GO's catalytic activity has been demonstrated in processes such as sulfide oxidation, olefin hydration, and condensation reactions, suggesting parallels to its ability to enhance enzymatic or microbial reduction of resazurin [30, 31]. These combined properties likely enable GO to function as both a chemical and physical catalyst, increasing the efficiency and sensitivity of resazurin conversion, as observed in microbial systems, while maintaining biocompatibility at low concentrations.

Accelerated Resorufin Production: Implications for Point‐Of‐Care Diagnostic Applications and Environmental Microbial Detection

3.3

Compared to the fast fluorescence quenching, the absorbance spectra, which are less quickly affected, can be used to analyze the mechanism of probe conversion into resorufin when GO and bacteria are incubated together. As reported above in Figure 1F, the absorbance peak of resazurin (610 nm) can be distinguished from resorufin (560 nm) only below 125 μg/mL.

Absorbance peak values are consistent after 24 h (Figure 5A) at concentrations < 125 μg/mL. Conversely, the high GO concentrations, which are however rarely used in biological experiments and impair microbial growth, impair the signal of the probe at least in the first hours of incubation. In Figure 5A, it is visible how the probe signal is recovered after 24 h of incubation with GO at high concentrations; this confirms the reversible interaction between the GO surface and the probe as observed for fluorescence. The ratio between 560 nm and 610 nm peaks is affected by high GO concentrations, probably due also to partial GO sheet stacking or aggregation (dashed lines in Figure 5B).

Effect of GO on resazurin absorption properties. OD peak (560 nm) intensity in a time of 24 h (A). OD peak (610 nm) intensity in 24 h and 560/610 nm ratio (dashed lines) is reported in (B).

Rapid detection of microorganisms is crucial for environmental monitoring and timely diagnosis to minimize the risk of disease transmission. When GO, S. aureus, and resazurin are incubated, absorbance peak analysis confirms the rapid conversion of the probe to resorufin and then dihydroresorufin (Figure 6A). The timing of conversion strictly depends on the concentration of bacteria, as shown by the disappearance of the 610 nm peak in favor of the 560 nm peak. As explained previously, the high concentrations of GO not only cause quenching of the signal but also might impair bacteria viability, which is not convenient for the design of a diagnostic sensor. Therefore, in Figure 6B, we focus on the low GO concentrations (16 and 31 μg/mL) to observe the increase in the development of signal by analyzing probe dynamics alone 20 min after incubation. The efficiency of probe conversion, quantified with the 560/610 nm ratio, is here compared with the probe alone and reaches almost 200% with the highest concentration of cells and 31 μg/mL. The effects observed, that is, the fast reduction of resazurin GO without UV light, can be facilitated by the defects in the GO structure, such as vacancies and functional groups acting as active sites for electron transfer combined with the S. aureus metabolism producing redox‐active intermediates, such as reactive oxygen species (ROS) and other metabolic by‐products, which can enhance the electron transfer between GO and resazurin [32].

Resazurin absorption in presence of GO can be exploited for fast diagnostics (A) GO‐resazurin absorption spectra after incubation with different concentrations of S. aureus (107 CFU/mL, 0.5 × 107 CFU/mL or 0.25 × 107 CFU/mL, symbolized with +++; ++; +, respectively). Resazurin spectra without GO are reported in pink. (B) Percentage of the efficacy of 560/610 nm ration in the presence of different GO concentrations (16GO or 31GO refers to 16 μg/mL or 31 μg/mL, respectively) normalized to resazurin alone. (C) Pictures of 96 wells after incubation of GO and/or resazurin with or without E. coli, S. pasteurii or S. aureus in solution (* indicates the complete color loss after 24 h of incubation for S. aureus). The ratio measured after 20 min or 72 h of incubation for each species is also reported.*

We also tested whether a different Gram‐positive bacterium, S. pasteurii , or the gram‐negative E. coli had a similar effect on resazurin in the presence of GO. The quick loss of resazurin color occurs with S. aureus alone rather than with S. pasteurii or E. coli , pointing out the existence of a specific metabolic mechanism or interaction (Figure 6C). In Figure 6C, the ratio obtained with 0.5 × 10^7^ CFU/mL cells for S. aureus , E. coli , or S. pasteurii is reported after 20 min of incubation or 72 h. Only S. aureus improves probe efficiency in the first minutes of incubation in the presence of GO. In all the other experimental conditions, the probe is poorly affected by bacteria and/or by GO. The metabolic behavior and oxygen consumption of S. aureus and S. pasteurii differ significantly due to their distinct physiological characteristics; this might explain the specific behavior with GO and the probe since the former is a facultative aerobe while S. pasteurii is an obligate aerobe. Also, S. aureus exhibits higher metabolic rates, which could boost the reduction of resazurin to resorufin. In contrast, S. pasteurii , with its different metabolic pathways and lower interaction efficiency with graphene oxide, shows reduced conversion. Similarly, Gram‐negative E. coli did not rapidly accelerate resazurin conversion. It seems likely that the bacterium probe consumption process occurs at a much slower rate, and that GO only contributes to a slight color quenching at these low concentrations, which again reflects a specific metabolic pathway existent in Gram‐positive S. aureus . Indeed, after 72 h, the color loss, that is, the conversion into dihydroresorufin, is much reduced (Figure 6C). This difference could be further investigated in future studies since specific GO interactions with different bacteria might occur at the membrane, changing the electronic behavior and consequently catalytic properties. These data pave the way for future studies in microbial fuel cell design, as S. aureus is not a conventionally electrogenic bacterium, yet GO seems to modify its behavior in the presence of resazurin.

Beyond its implications for diagnostics, the GO–resazurin system holds significant potential for environmental toxicology. Waterborne pathogens, including Staphylococcus aureus , are well‐recognized contributors to toxic exposures through drinking water and recreational activities. Our findings demonstrate that GO accelerates resazurin reduction specifically in the presence of metabolically active S. aureus , allowing bacterial detection within minutes rather than hours. Such sensitivity could be directly applied to the monitoring of environmental matrices, where rapid quantification of microbial contamination is essential for preventing toxic exposures. Moreover, the catalytic properties of GO suggest additional relevance for bioremediation strategies, as GO can both signal microbial growth and participate in pollutant degradation pathways. Indeed, microbial fuel cells (MFCs) exhibit tremendous potential for the sustainable management of dye‐contaminated wastewater by degrading azo dyes while simultaneously generating electricity. These systems integrate physicochemical and biological principles to convert chemical waste into electrical energy and to detoxify pollutants [33]. Over the past decade, significant advances have been achieved in MFC configurations and electrode materials, with resazurin being employed both to enhance electron‐shuttling mechanisms and to support bacterial survival [13, 34]. Within MFCs, strategies such as shortening the electron diffusion path, decreasing internal cell resistance, and improving biofilm–electrode interactions are key to enhancing bioelectricity production. Our findings suggest that the combined use of unconventional bacterial strains, GO, and resazurin may inspire the development of new MFC designs for biomass refinery and environmental remediation.

Conclusions

4

In this article, we analyze the unique interaction of the resazurin (Alamar Blue) probe in the presence of graphene oxide and bacterial species. We demonstrate rapid quantification of viable S. aureus within 20 min of incubation due to GO acceleration of probe metabolization. Under the same experimental conditions, GO can act as either a catalyst or a quencher by increasing the concentration of the nanomaterial. The optimal GO concentration to improve the resazurin detection performance is very low, allowing the cells to remain viable and unaffected by the presence of the nanomaterial. We also demonstrate that S. aureus can accelerate the conversion of resorufin to colorless dihydroresorufin more efficiently than another gram‐positive ( S. pasteurii ) or gram‐negative E. coli, probably due to its higher production of reactive oxygen species (ROS). Indeed, S. aureus possesses metabolic pathways that are particularly effective in electron transfer, facilitating a quicker reduction process. This process is further accelerated by GO in the solution, presenting new possibilities for microbial detection and microbial fuel cell designs based on resazurin. However, future work will extend our investigation to additional Gram‐positive and Gram‐negative bacteria, allowing a comparative analysis of GO‐mediated effects across different microbial metabolisms. Indeed, while this study primarily focuses on demonstrating the feasibility and rapidity of this microbial sensing approach, the inclusion of bacteria with diverse metabolic profiles will provide further insights into the specificity of this mechanism and its potential applicability in environmental and diagnostic settings. Furthermore, in future work, complementary characterization techniques, such as electrochemical impedance spectroscopy and cyclic voltammetry, could be particularly relevant in complex environmental matrices, where organic and inorganic contaminants may influence the reaction dynamics.

In conclusion, this study highlights the unique interaction of resazurin with GO in the presence of bacterial species, demonstrating its potential for rapid, efficient environmental monitoring. The catalytic enhancements provided by GO enable applications such as bioremediation, real‐time water quality assessment, and integration into microbial fuel cell systems. These advancements not only improve detection and treatment methods for environmental contaminants but also provide sustainable approaches for energy generation through bio‐based systems based on nanomaterials. The results presented here provide a strong proof‐of‐concept for the acceleration of resazurin reduction by GO, forming a solid basis for further research aimed at optimizing this method for practical applications in rapid microbial diagnostics and environmental monitoring.

Author Contributions

Valentina Palmieri: conceptualization, methodology, data curation, formal analysis, writing – original draft. Marco de Spirito: validation, visualization, writing – review and editing. Massimiliano Papi: resources, visualization, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.