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Photoactive nanocatalysts as DTT-assisted BSA-AuNCs with enhanced oxidase-mimicking ability for sensitive fluorometric detection of antioxidants

Journal of Nanobiotechnology volume 22, Article number: 585 (2024) Cite this article

Abstract

Redox imbalance and oxidative stress are increasingly recognized as significant factors in health disorders such as neurodegenerative disorders, premature aging and cancer. However, detecting antioxidant levels that is crucial for managing oxidative stress, can be challenging due to existing assays’ limitations, such as insensitivity to thiol-containing antioxidants. This study presents a simple fluorescence-based assay for antioxidant detection employing the enhanced photocatalytic oxidase-like activity of dithiothreitol (DTT)-assisted bovine serum albumin (BSA)-stabilized gold nanoclusters (DTT@BSA-AuNCs). The reported nanozyme exhibits remarkable stability, versatility, and catalytic activity. Under LED irradiation, DTT@BSA-AuNCs generate singlet oxygen, which converts non-fluorescent thiamine to fluorescent thiochrome, utilizing dissolved oxygen for catalysis. Antioxidants inhibit thiochrome formation, leading to fluorescence quenching. This method enables sensitive detection of antioxidants such as ascorbic acid and glutathione with limits of detection of 0.08 µM and 0.32 µM, respectively, under neutral pH, outperforming previous studies. The assay successfully detects antioxidants in human saliva and cancer cell models. The DTT@BSA-AuNCs-based assay offers a cost-effective, sensitive, and straightforward approach for detecting antioxidants in biological samples, facilitating improved monitoring of oxidative stress in various diseases.

Introduction

Reactive oxygen species (ROS) are free radicals derived from molecular oxygen such as hydroxyl radicals (•OH), peroxyl radicals (ROO•), singlet oxygen (1O2), and superoxide anions (O2•−). Regular exposure to exogenous sources such as pollution, UV radiation, unhealthy diet, etc., leads to accumulation of ROS in the body causing oxidative stress and cellular damage which leads to several inflammatory and degenerative diseases. Antioxidants play an important role in neutralizing ROS and protecting cells. By stabilizing these reactive molecules, antioxidants prevent oxidative damage to cellular components such as DNA, proteins, and lipids, thereby protecting against various oxidative stress-related health issues [1]. Low-molecular-weight antioxidants constitute a crucial cellular defense system against oxidants by directly terminating free radical chain reactions. Examples include- ascorbic acid, flavonoids, glutathione and carotenoids [2]. When insufficient intake of dietary antioxidants and ROS overproduction lead to oxidative stress, the overuse of antioxidant supplements may lead to antioxidant-induced stress termed “antioxidative stress” [3]. Maintaining a delicate balance between oxidants and antioxidants, or achieving tight redox regulation, is crucial. In this context, regular monitoring of antioxidants in the body can help to maintain the balance between antioxidants and free radicals and prevent the aforementioned complications. However, some conventional assays for detecting antioxidant compounds involve time-intensive protocols, and expensive, unstable reagents and requiring skilled personnel. A significant drawback of certain existing assays such as the ferric reducing antioxidant power (FRAP) assay and oxygen radical absorbance capacity (ORAC) assay, is their insensitivity to thiol-containing molecules [4, 5], such as glutathione (GSH), the primary intracellular thiol-containing antioxidant that directly scavenges free radicals [6]. Together, these limitations hinder their frequent and accurate application.

The field of nanobiocatalysis, which utilizes enzyme-mimicking nanomaterials (nanozymes), has garnered significant research interest in recent years due to its potential applications in medical diagnostics, therapeutics, pathogen identification, drug delivery and bioanalysis [7,8,9,10,11,12]. Due to their intrinsic enzyme-like activity, comparable catalytic efficiency to that of natural enzymes, enhanced durability, multifunctionality, extended shelf life, and cost-effectiveness, these nanomaterials have emerged as attractive alternatives for broader applications in various fields [13]. After the pioneering discovery of Fe3O4 nanoparticles as peroxidase-mimicking nanozymes in 2007 [14], different nanomaterials, such as metal-based nanostructures, metal oxides, and carbon-based nanostructures have been used as peroxidase-mimics for colorimetric antioxidant detection [15,16,17]. In addition to offering advantages such as robustness and low cost compared to natural enzymes, these peroxidase-mimicking nanozymes require harsh reaction conditions, such as toxic and unstable H2O2, which limits their practical application. In addressing this issue, oxidase-like nanozymes have emerged as safer alternatives since they do not require H2O2. However, it is worth noting that oxidase nanozymes, while promising, are currently less prevalent than their peroxidase counterparts [18]. While traditional colorimetric methods have sometimes been criticized for lower sensitivity, recent advancements, such as those demonstrated by Pompa et al. with platinum nanozymes [19], have significantly improved the sensitivity of these assays, enabling effective detection at lower concentrations.

To further increase the catalytic activities of nanozymes, scientists have utilized several approaches by controlling their size [20], composition [21], and providing external stimuli [22, 23]. Among these approaches, the use of light stimulation to enhance the efficiency of nanozymes is highly promising. Light-responsive nanozymes offer superior control over their catalytic activity due to light-induced high spatiotemporal control, minimizing background activity and enabling “on-demand” initiation and modulation. This makes them ideal for challenging reactions. Additionally, the tunability of light source parameters (duration, intensity, and wavelength) allows fine-tuning of activity. These nanozymes also function under mild conditions and utilize light as a clean trigger, eliminating the need for chemical activators. This combination makes them attractive and sustainable alternatives for various applications [24, 25].

Atomically precise gold nanoclusters (AuNCs), approximately 2 nm in size, hold promise for biosensing due to their unique properties such as high surface area, excellent stability, biocompatibility, efficient light absorption, and long-lived excited states [26, 27]. The large surface-to-volume ratio of AuNCs results in improved catalytic activity compared to larger gold nanoparticles (AuNPs). Their ultra-small size provides unique electronic and optical properties, such as discrete energy levels and strong luminescence, enhancing sensitivity for fluorescence-based detection. Additionally, the quantum confinement effect in AuNCs enhances ROS generation efficiency, making AuNCs effective light-responsive oxidase mimics. In contrast, AuNPs lack this photo-oxidase mimicking activity, which is independent of irradiation wavelength and surface stabilizing ligands [28]. Singh et al. demonstrated the responsiveness of bovine serum albumin-stabilized-AuNCs (BSA-AuNCs) in a sensitive fluorescence-based detection method for H₂O₂. They found that the bright red fluorescence of BSA-AuNCs decreased significantly upon interaction with H2O2, showcasing their potential for detection [29]. Other researchers have exploited their peroxidase-mimicking activity (requiring H2O2) to detect various molecules such as antibiotics [30], heavy metals [31] etc. However, reports on oxidase-mimicking AuNCs, which utilize readily available dissolved oxygen for catalysis, are relatively scarce. Moreover, the catalytic activity of AuNCs under neutral conditions, which is crucial for biological applications, has been largely overlooked. For instance, Liu et al. demonstrated that glutathione-stabilized AuNCs catalyze the photoinduced reduction of nitrobenzene to aniline under highly acidic conditions (pH 1), showcasing their function as ‘photonanozymes’ [32]. Similarly, Jiang et al. reported the application of AuNCs for glucose detection in an acidic environment at pH 4 [33]. This highlights their potential but indicates their limited efficacy at neutral pH, hindering their use in biological settings such as antioxidant detection.

Given the growing need for sensitive detection of antioxidants (including thiol-containing ones) and the vast potential of AuNCs for oxidase-mimicking applications at neutral pH, new approaches must be explored. In this study, we introduce dithiothreitol (DTT)-assisted BSA- stabilized AuNCs (DTT@BSA-AuNCs), a unique photoresponsive oxidase-mimicking nanozyme for detecting antioxidants. DTT@BSA-AuNCs were utilized to achieve effective oxidation of non- fluorescent thiamine (TH) to fluorescent thiochrome (TC) by generating singlet oxygen in a neutral environment in the presence of visible light via an LED. DTT@BSA-AuNCs efficiently utilize dissolved oxygen present in the environment for catalysis, eliminating the need for supplemental oxygen (a common limitation of oxidase-mimic nanozymes). The inherent ability of antioxidants to scavenge reactive species resulted in quenching of the fluorescence signal, enabling sensitive detection of antioxidants namely ascorbic acid (AA) and glutathione (GSH) with limits of detection of 0.08 µM and 0.32 µM, respectively.

While previous studies have successfully utilized the intrinsic fluorescence of AuNCs to detect cancer cells based on overexpression of surface markers such as Glut-1 [34], the present study takes a different approach by employing the oxidase-mimicking ability of AuNCs to detect cellular antioxidants. Unlike some surface markers, which may exhibit variability and complex regulatory mechanisms [35], intracellular antioxidants may provide a more comprehensive reflection of the cellular redox environment. As cancer cells often elevate intracellular antioxidants especially GSH levels to counteract oxidative stress [36,37,38,39], a comprehensive assessment of cellular antioxidant capacity is essential. By focusing on antioxidants, this study complements existing biomarker-based approaches and offers potential for deeper insights into the complex cancer cell biology.

The detection of AA and GSH in human saliva and cellular antioxidants in cancer cells demonstrates the practical application of this method. The principle of the study is displayed in Fig. 1. Importantly, this study explored the potential of DTT@BSA-AuNCs as light-activated oxidase mimics for the sensitive detection of antioxidants, especially the challenging GSH molecule. DTT@BSA-AuNCs address the limitations of existing methods by overcoming low AuNCs activity at neutral pH, eliminating external oxygen requirements, and offering superior sensitivity through a fluorescence-based assay. Furthermore, this method utilizes basic instrumentation for rapid (within 10 min) and cost-effective analysis. Thus, this study contributes to the development of highly efficient photocatalytic nanozyme systems for sensitive fluorometric detection of antioxidants in biological samples.

Fig. 1
figure 1

Schematic illustration of the principle behind the antioxidant detection method. Under LED irradiation, DTT@BSA-AuNCs generate ROS, primarily singlet oxygen, which oxidizes non-fluorescent thiamine to fluorescent thiochrome. This reaction leads to an increase in the fluorescence signal. Conversely, antioxidants scavenge ROS, preventing thiamine oxidation and resulting in a decreased fluorescence signal or fluorescence quenching. Therefore, the assay enables the detection of antioxidants by monitoring changes in fluorescence intensity

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Methods

Synthesis of DTT@BSA-AuNCs and BSA-AuNCs

DTT@BSA-AuNCs

The experimental procedure involved precise temperature control. Initially, an aqueous solution of HAuCl4·3H2O was preheated to 37 °C, while DTT and BSA were maintained at 25 °C. First, 6 mL of 10 mM HAuCl4·3H2O was combined with 2 mL of 2.5 mg/mL DTT, and the mixture was gently shaken until a dark color developed. This mixture was then added to 6 mL of a 50 mg/mL BSA solution with continuous slow stirring for proper mixing. After 2 min, 350–400 µL of 1 M NaOH was added to adjust the pH to approximately 12. The mixture was then stirred at 50 °C and 500 RPM for 1 h. During this period, the color of the solution changed to orange, indicating the successful formation of fluorescent AuNCs. The synthesized DTT@BSA-AuNCs were then freeze-dried and stored at 4 °C for future use.

BSA-AuNCs

BSA-AuNCs without the use of DTT were synthesized following a previous procedure without any modification [40].

Photo-responsive oxidase mimicking behavior of DTT@BSA-AuNCs

In a typical experiment, 245 µL of phosphate buffer (pH = 7.4), 25 µL of DTT@BSA-AuNCs (final concentration = 0) and 50 µL of TH (final concentration = 1 mM) with 180 mL of double distilled water (DDW) were added to an Eppendorf vial. Then, the reaction mixture was subjected to irradiation under 420 nm LED for 10 min followed by fluorescence measurement (Synergy H1 Microplate reader, Biotek) at 440 nm.

Effect of different ROS scavengers on the catalytic activity of DTT@BSA-AuNCs

245 µL of phosphate buffer (pH = 7.4), 50 µL of various scavengers, 25 µL of DTT@BSA-AuNCs (final concentration = 0.5 mM), 50 µL of TH (final concentration = 1 mM) and DDW were added up to 500 µL. The final concentrations of the scavengers were as follows: sodium azide (100 mM), catalase (100 U/mL), mannitol (5 mM), p-benzoquinone (1 mM) and disodium ethylenediaminetetraacetate (EDTA-2Na, 1 mM). The mixture was thoroughly mixed and the reaction mixture was subjected to irradiation under a 420 nm LED for different times followed by fluorescence measurement.

Detection of singlet oxygen by DTT@BSA-AuNCs under light

A stock solution of 50 mM 3,3’-diaminobenzidine (DAB) was prepared in N, N-dimethylformamide (DMF). 5 µL of DAB solution and 25 µL of DTT@BSA-AuNCs were added to heavy water to achieve a final concentration of 0.5 mM each. The mixture was then subjected to irradiation for different periods of time followed by absorbance measurements (Synergy H1 microplate reader, Biotek).

Detection of antioxidants by DTT@BSA-AuNCs

Typically, 245 µL of phosphate buffer (pH = 7.4), 25 µL of DTT@BSA-AuNCs (final concentration = 0.5 mM), 50 µL of TH (final concentration = 1 mM) and various concentrations of AA or GSH were introduced into the eppendorf vial. Then, the required amount of DDW was added to achieve a final volume as 500 mL, followed by irradiation with a 420 nm LED for 10 min and subsequent fluorescence measurements. For AA and GSH detection from saliva, saliva was centrifuged for 45 min at 13,000 RPM and superdiluted with DDW (v/v-1:100). Known concentrations of AA and GSH were spiked into the samples, and recovery percentages were calculated.

Selectivity of antioxidants detection by DTT@BSA-AuNCs

245 µL of phosphate buffer (pH = 7.4), 25 µL of DTT@BSA-AuNCs (final concentration = 0.5 mM), 50 µL of TH (final concentration = 1 mM), 5 µL of various interferents (100 µM) and 5 µL of AA and GSH (100 µM) with DDW were added separately to make the reaction mixture up to 500 µL. The mixture was thoroughly mixed and the reaction mixture was subjected to irradiation under a 420 nm LED for 10 min, followed by fluorescence measurement.

Preparation of cell lysates and detection of cellular antioxidants by DTT@BSA-AuNCs

MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F-12) with 10% FBS and 1% penicillin-streptomycin (10,000 U/mL). HS 578Bst cells were cultured in Hybri-Care Medium with 1.5 g/L sodium bicarbonate, 30 ng/mL mouse EGF, 10% FBS, and 1% penicillin-streptomycin (10,000 U/mL). Both cell lines were maintained at 37 °C in a 5% CO₂ atmosphere. Upon reaching 80% confluence, 5 × 106 cells were harvested, washed with chilled PBS (three times), and centrifuged at 1000 RPM for 5 min each wash. Then, the cells were disrupted using low-power ultrasonication at low temperature for 2 min. Then, the cell lysate was centrifuged at 13,000 RPM for 20 min at 4 °C. The supernatant, containing the cell lysate (5 × 106 cell equivalents), was transferred to sterile tubes for subsequent assays. For each assay, 10 µL of DTT@BSA-AuNCs (final concentration = 0.2 mM), 50 µL of 1 mM TH, and various amounts of cell lysates were mixed with phosphate buffer (pH 7.4) and the final volume was made up to 0.5 mL. The mixture was thoroughly mixed and the reaction mixture was subjected to irradiation under a 420 nm LED for 10 min, followed by fluorescence measurement.

Results and discussion

Characterization of DTT@BSA-AuNCs

A schematic illustration of the synthesis process for DTT@BSA-AuNCs is presented in Fig. 2a. A detailed explanation of each step in the DTT@BSA-AuNCs synthesis, including the optimization of conditions such as pH and temperature, is provided in the supplementary section (Figure S1-S4 in ESM). The high-resolution transmission electron microscopy (HRTEM) image of the prepared DTT@BSA-AuNCs indicates monodispersed nanoclusters with an average diameter of 2.267 nm (Fig. 2b and c), which falls within the expected size range of Au25NCs, consistent with previous reports [41, 42]. Moreover, the HRTEM images confirmed the presence of a standard face-centered unit cell (FCC) structure of Au. As displayed in Fig. 2d, the S 2p XPS peak at 162.8 eV corresponds to the sulfur atom bound to the gold surface as thiolate species [43] and the peak at 164.4 eV corresponds to the unbound or free thiol group. Additional characterizations of DTT@BSA-AuNCs, as revealed by X-ray diffraction (XRD) analysis, Au 4f spectrum and survey scan of X-ray photoelectron spectroscopy (XPS), Fourier Transform Infrared (FTIR) spectrum analysis are detailed in the supplementary section, including Figures S5–S8 in ESM.

Fig. 2
figure 2

Synthesis and Characterization of DTT@BSA-AuNCs. (a) Schematic illustration of the synthesis of DTT@BSA-AuNCs. Au(III) and DTT were introduced into BSA, followed by NaOH addition until the pH reached 12. The exposed thiol groups in BSA led to increased gold-thiol interaction. The temperature of the solution was raised to 50 °C with continuous stirring for 1 h, leading to the successful formation of fluorescent DTT@BSA-AuNCs. (b) HRTEM image of monodispersed DTT@BSA-AuNCs. The inset in the upper right corner shows an enlarged picture of a single AuNC. The scale bar is 5 nm. (c) The corresponding size distribution of the DTT@BSA-AuNCs shows an average diameter of 2.267 nm. (d) XPS analysis of the sulfur S 2p peak. The S 2p peak at 162.8 eV corresponds to the Au-S bond, and the peak at 164.4 eV corresponds to the free thiol group (S-H). (e) Fluorescence emission spectra of DTT@BSA-AuNCs. The DTT@BSA-AuNCs (red curve) exhibit maximum fluorescence intensity at 690 nm, whereas BSA alone (black curve) has negligible fluorescence at that wavelength. Inset: photographs of the DTT@BSA-AuNCs under sunlight (left) showing an orange-brown color, and under 365 nm UV light (right) showing bright red fluorescence

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As shown in Fig. 2e, the DTT@BSA-AuNCs exhibited maximum red fluorescence emission at approximately 690 nm, consistent with previous studies involving BSA-AuNCs [44, 45] when excited at 510 nm. In contrast, negligible fluorescence emission was observed from BSA alone. Thus, the strong red fluorescence exhibited by DTT@BSA-AuNCs was attributed to the AuNCs rather than the fluorescence from the BSA protein. The obtained fluorescence emission peak near 700 nm indicates the presence of Au25 clusters based on the spherical Jellium model. The UV–vis absorption spectrum displays an absorbance band between 280 and 290 nm for DTT@BSA-AuNCs and a prominent absorbance peak at 280 nm for only BSA (Figure S3 in the ESM). This phenomenon attributed to the π–π* transitions in aromatic amino acid residues such as tryptophan, tyrosine, and phenylalanine in the native BSA structure [46]. The slight redshift in the absorbance of DTT@BSA-AuNCs is attributed to the interaction between the AuNCs and the aromatic amino acids in BSA [47]. The absence of a surface plasmon resonance peak at 520 nm, characteristic of AuNPs larger than 3 nm, confirms that no larger nanoparticles were formed during synthesis, consistent with previous reports [48].

Photo-responsive oxidase mimicking activity of DTT@BSA-AuNCs

In any detection process employing AuNCs as nanozymes, a substrate undergoes a catalytic reaction, leading to the production of an electrochemical, fluorescent, or colorimetric response [49]. Notably, the prevalent choice for readout in conjunction with AuNCs has been colorimetric. However, due to the superior sensitivity and selectivity of fluorescence over colorimetric detection, this study opted for the fluorescence method. TH is a popular substrate for fluorescence analysis due to its cost-effectiveness, high water solubility, and wide availability [50]. Originally, being non-fluorescent, TH can be oxidized into a fluorescent substance called thiochrome in the presence of appropriate oxidants in an alkaline environment. The need for such alkaline conditions limits its utility in biomedical applications. However, in our study, we observed the oxidation of TH to TC under neutral conditions in the presence of light.

An LED light with a wavelength of 420 nm was utilized to irradiate the DTT@BSA-AuNCs. Figure 3a, clearly shows that when subjected to light irradiation, DTT@BSA-AuNCs catalyzed the conversion of non-fluorescent TH to fluorescent TC, as indicated by the strong fluorescence peak at approximately 440 nm. Intriguingly, in the absence of light exposure, there was a negligible fluorescence signal when TH was treated with DTT@BSA-AuNCs. Furthermore, even under light irradiation, TH alone fails to generate fluorescent TC in the absence of DTT@BSA-AuNCs. These findings indicate the indispensable role of visible light in the catalytic activity of DTT@BSA-AuNCs nanozyme. Additionally, this result overcomes the common limitation of requiring alkaline conditions for TH oxidation, demonstrating that TH can be efficiently oxidized to TC under neutral conditions. As shown in Fig. 3b, by successively turning off and on the source of light, the oxidase-mimicking activity of the DTT@BSA-AuNCs revealed a staircase-like behavior, indicating a photoregulated oxidase-like activity of the DTT@BSA-AuNCs. Hence, through external light irradiation, the oxidase-like activity of the DTT@BSA-AuNCs could be precisely controlled, favoring high spatial-temporal resolution of its catalytic activity. The gradual increase in the oxidase-mimicking activity of DTT@BSA-AuNCs with repeated light irradiation cycles can be attributed to a cumulative effect. Each “light on” period leads to TH oxidation and the generation of TC leading to TC accumulation over time; hence the fluorescence intensity increases gradually.

Fig. 3
figure 3

Light-activated oxidase-mimicking activity of DTT@BSA-AuNCs. (a) Fluorescence spectra of the three samples containing DTT@BSA-AuNCs + TH without light irradiation, TH only, and DTT@BSA-AuNCs + TH under light irradiation. DTT@BSA-AuNCs + TH under light irradiation displayed strong fluorescence emission peak at 440 nm. However, DTT@BSA-AuNCs + TH without light irradiation and TH only with light irradiation, did not show any fluorescence (b) Photocontrollable oxidase-mimicking activity of DTT@BSA-AuNCs demonstrated by the staircase-like behavior when the light source is turned on and off, as indicated by the blue and black arrows, respectively. (c) Oxidase-like photocatalytic activities of BSA-AuNCs synthesized by conventional (BSA-AuNCs, dotted curves) and DTT-assisted (DTT@BSA-AuNCs, bold curves) methods under different irradiation times. Color codes indicate irradiation times: pink for 15 min, orange for 30 min, and violet for 60 min. (d) Determination of increasing fluorescence spectra with rising TH concentration (up to 1 mM) represented by lighter to darker shades of red under a fixed BSA-AuNCs concentration during 10 min of light irradiation

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To confirm whether the inclusion of DTT could enhance the oxidase-mimicking photocatalytic performance of BSA-AuNCs, we synthesized BSA-AuNCs in the presence of DTT (with a one-hour reaction at 50 °C) and in the absence of DTT (with a 12-hour reaction at 37 °C). Then, we incubated the prepared DTT@BSA-AuNCs and BSA-AuNCs in TH and subjected them to extended light irradiation for 15, 30, and 60 min. As displayed in Fig. 3c, we initially observed similar oxidase mimicking photocatalytic abilities in both variants when the irradiation time was limited to 15 min. However, interestingly, as the irradiation time increased to 30 and 60 min, a remarkable enhancement in photocatalytic performance was evident in the DTT@BSA-AuNCs. In contrast, conventionally synthesized BSA-AuNCs exhibited minimal improvement in photocatalytic behavior, even with extended irradiation. This finding confirmed the superior oxidase-mimicking photocatalytic efficiency of the DTT@BSA-AuNCs synthesized via the DTT-assisted method, which was likely attributed to increased nanocluster formation. Therefore, DTT@BSA-AuNCs were selected for the present study. Existing studies on DTT-modified AuNCs for biosensing have demonstrated how DTT enhances the binding affinity for specific target molecules such as mercury ions [51] and 6-mercaptopurine [52] via surface modification. This binding alters the fluorescence of the AuNCs, enabling detection. In contrast, our research focused on the photocatalytic activity of DTT-BSA-AuNCs. We explored how DTT influences the efficiency of light-driven reactions. While previous reports emphasize surface modification for target binding, in our study, DTT acted as a reducing agent, leading to the synthesis of more AuNCs, resulting in more active sites for photocatalysis. Next, the irradiation wavelength dependency of the reported photoresponsive oxidase-like activity of DTT@BSA- AuNCs was investigated using several LED wavelengths ranging from ultraviolet to visible, to near-infrared light (NIR), including 365, 420, 470, 530, 570, 617, 735, and 940 nm. Figure S9 in the ESM shows that the UV-visible spectrum, especially at wavelengths ranging from 365 nm to 530 nm, was the most suitable region for triggering the function of the nanozyme, which decreased as the light wavelength approached the NIR range. Based on these observations and considering the range from 365 to 530 nm, the LED with a wavelength of 420 nm was selected as the initial light source for the current study because it falls within this range and is close to the midpoint. Photo-responsive nanozymes have traditionally been activated by powerful light sources such as xenon lamps [53, 54]. This study, however, demonstrated that a standard LED emitting light within the UV-vis spectrum can effectively activate the reported nanozyme. While Xenon lamps produce light with a higher photon flux and greater intensity, our findings highlight that LEDs, despite their lower intensity, remain a viable and more sustainable alternative. The use of LEDs offers advantages in terms of reduced energy consumption and maintenance requirements, enhancing the cost-effectiveness and environmental friendliness of the applications.

To comprehensively understand the combined effects of varying concentrations of DTT@BSA-AuNCs, TH, and irradiation time, we conducted systematic experiments in which each parameter was adjusted. Initially, DTT@BSA-AuNCs were used at 0.1 mM, TH concentrations ranging from 0.1 to 1 mM, and irradiation times of 5, 10, 15, 20, and 25 min (Figure S10 in the ESM). Subsequently, DTT@BSA-AuNCs were maintained at 1 mM, with similar variations in TH concentrations and irradiation times (Figure S11 in the ESM). In the next set, the TH concentration was fixed at 0.1 mM, the DTT@BSA-AuNCs concentration was varied from 0.1 to 1 mM, and the irradiation time was adjusted over multiple durations (Figure S12 in the ESM). Finally, with TH fixed at 1 mM, the DTT@BSA-AuNCs concentrations were varied similarly (Figure S13 in the ESM). The results showed that the fluorescence intensity of TC increased proportionally with increasing concentrations of DTT@BSA-AuNCs and TH, especially with increasing irradiation time. The summary plots in Figures S10f, S11f, S12f, and S13f in the ESM highlight these trends, demonstrating the tunability of the assay based on these parameters. Moreover, the observed maximum fluorescence intensity peak of TC, spanning from 425 nm to 445 nm, aligns with the expected characteristic peak of TC, as reported by many other studies [55,56,57]. Hence, this extensive exploration confirmed the significant ability of DTT@BSA-AuNCs to enhance photoresponsiveness by increasing the concentration of DTT@BSA-AuNCs, TH, or irradiation time, thereby demonstrating the efficiency of the nanozyme. For further studies, the concentrations of DTT@BSA-AuNCs and TH were maintained at 0.5 mM and 1 mM, respectively (unless otherwise specified), with an irradiation time of 10 min. This choice was made to reduce the consumption of the nanozyme and enhance the cost-effectiveness of the applications.

As displayed in Fig. 3d, upon increasing the concentration of TH while keeping the concentration of DTT@BSA-AuNCs fixed, the fluorescence intensity of TC gradually increased until TH reached a concentration of 1 mM during 10 min of irradiation. The increase in the fluorescence intensity of TC demonstrated a linear relationship with an R2 value of 0.99 (Figure S14a in the ESM), validates the effectiveness of our assay. This significant linear correlation confirms that our fluorescence-based measurement accurately reflects the concentration of TC derived from TH, thereby establishing the assay’s reliability and robustness for precise quantitative analysis. However, when the concentration of TH was further increased beyond 1 mM, a gradual decrease in fluorescence intensity was observed (Figure S14b in the ESM). This intriguing phenomenon was attributed to ‘substrate inhibition,’ wherein at high substrate concentrations, the excessive substrate may nonspecifically bind to an alternative site of the nanozyme, producing antagonistic effects [58, 59].

To explore the versatility of the photoregulated oxidase mimetic activity of DTT@BSA-AuNCs, two widely used chromogenic enzymatic substrates, 3,3,5,5-tetramethylbenzidine (TMB) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), were tested. Figure S15a and b in the ESM shows that light irradiation for 1 h could oxidize these enzymatic substrates into their respective oxidized forms as observed from their characteristic absorbance peaks at 650 nm (TMBox) and 420 nm (ABTSox) in the UV-vis spectra. These results demonstrated the versatile photoresponsive oxidase-like properties of DTT@BSA-AuNCs, with TH being the most sensitive substrate because it requires a shorter duration of light irradiation to drive its photocatalytic activity than TMB and ABTS.

Catalytic mechanism of oxidase-mimicking DTT@BSA-AuNCs photocatalysis

To quantify the oxidase-mimicking activity of DTT@BSA-AuNCs, steady-state kinetic analysis of the oxidation of TH was performed. The typical kinetic parameters, such as the Michaelis-Menten constant (Km) and the maximum initial velocity (νmax), were obtained by fitting the data to the Michaelis-Menten equation, and typical double-reciprocal Lineweaver-Burk plots were constructed (Figure S16a and b in the ESM). Our study revealed a lower Km value of 0.159 mM for the TH substrate than for the DTT@BSA-AuNCs, indicating the high affinity of the nanozyme for the substrate. Additionally, we observed a substantially high νmax value of 3.79 × 10− 3 M/s, indicating the high catalytic efficiency of DTT@BSA-AuNCs. Comparisons of the Km and νmax values obtained in our study with those reported in prior studies are provided in the Table S1 in the ESM. The lower Km and higher νmax values obtained in our study indicate the superior performance of our developed nanozyme system. To understand the importance of dissolved oxygen in the photocatalytic activity of DTT@BSA-AuNCs, we purged the reaction mixture with nitrogen gas to minimize dissolved oxygen levels, followed by irradiation. As illustrated in Fig. 4a, a substantial decrease (37.1%) in photocatalytic activity was observed upon oxygen depletion, suggesting that dissolved oxygen plays a vital role in the photocatalytic activity of DTT@BSA-AuNCs. However, the possibility of residual dissolved oxygen in the purged mixture cannot be entirely ruled out.

Fig. 4
figure 4

Catalytic mechanism of DTT@BSA-AuNCs-TH system. (a) Fluorescence spectra showing photocatalytic activity of DTT@BSA-AuNCs in the presence of dissolved O2 (from air) and N2 (from N2 purging). Inset picture shows the fluorescence intensity at 440 nm expressed as % of control where the control condition represents the fluorescence intensity measured in dissolved O2 (from air), and the experimental group represents the fluorescence intensity measured in presence of N2 (from N2 purging). (b) Effect of different ROS scavengers (catalase for H2O2, EDTA for h+, mannite for •OH, p-benzoquinone for O2•−, sodium azide for 1O2) on the photocatalytic oxidation on TH by the DTT@BSA-AuNCs under light irradiation. (c). Absorbance spectra of DAB (1O2 probe) and DTT@BSA-AuNCs, with increasing irradiation times from 10 min to 120 min represented by darker to lighter shades of blue. DAB with DTT@BSA-AuNCs shows increased absorbance around 460 nm with prolonged irradiation. (d) Change in absorbance at 460 nm for DTT@BSA-AuNCs + DAB with and without light irradiation, and for DAB in presence of light. Only DTT@BSA-AuNCs + DAB under light irradiation showed increased absorbance at 460 nm. No change in absorbance at 460 nm was observed for DTT@BSA-AuNCs + DAB without light and only DAB in presence of light. Error bars represent standard deviation of three independent measurements

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For the oxidase-like activity of DTT@BSA-AuNCs, various reactive species could be involved as reactive intermediates, such as hydroxyl radicals (•OH), singlet oxygen (1O2), hydrogen peroxide (H2O2), superoxide anions (O2•−), and photogenerated holes (h+). To identify the key reactive species responsible for TH oxidation, we employed selective quenchers to eliminate the ROS individually. Mannitol and catalase were used to scavenge •OH and H2O2, respectively. Sodium azide was used for 1O2, p-benzoquinone for O2•−, and EDTA for h+. Figure 4b shows that sodium azide effectively inhibited TH oxidation completely, indicating the dominant role of 1O2 in the process. P-benzoquinone and mannitol also inhibited TH oxidation but to a lesser extent than sodium azide. Interestingly, EDTA enhanced the catalytic activity because it acts as a sacrificial hole acceptor, reducing the recombination of photogenerated electrons and holes and thereby increasing the overall oxidase-like activity [60]. Additionally, the presence of catalase increased TH oxidation, likely due to its peroxidase-like activity at low H2O2 concentrations, which oxidizes the substrate [61, 62]. Hence, these results indicate that 1O2 plays the dominant role in the photocatalytic behavior of DTT@BSA-AuNCs followed by •OH and O2•−. To further verify the generation of 1O2 from DTT@BSA-AuNCs upon light irradiation, DAB was used as a 1O2 probe. As shown in Fig. 4c and d, DAB exhibited an increase in absorbance at approximately 460 nm upon reaction with 1O2, which increased with prolonged irradiation time. Conversely, in the absence of light, the absorbance of DAB did not increase, indicating that light is required for the DTT@BSA-AuNCs to release 1O2. Therefore, these findings collectively indicate that the primary driver of the photocatalytic activity of DTT@BSA-AuNCs is singlet oxygen.

Stability of the photoresponsive oxidase-mimicking activity of DTT@BSA-AuNCs

Natural enzymes typically lose stability and catalytic activity over time, whereas nanozymes are expected to offer better stability and long-term storage. To assess this, DTT@BSA-AuNCs were stored at room temperature (25 °C), 4 °C, and − 20 °C for one month in both dark and lighted environments. The photocatalytic activity was monitored weekly (Fig. 5a-c). The results indicate that DTT@BSA-AuNCs remained stable and interestingly, their catalytic activity increased over time. This increase is likely due to the temperature-dependent synthesis of DTT@BSA-AuNCs, which gradually enhanced the reduction of AuNCs. A higher population of DTT@BSA-AuNCs led to a greater oxidase-like catalytic activity. This observation is further supported by the fact that at a slightly higher temperature (25 °C), the catalytic activity was greater compared to 4 °C (Fig. 5a and b). At -20 °C, no change in catalytic activity was observed, indicating a complete arrest of the reducing property of BSA at this low temperature (see Fig. 5c).

Fig. 5
figure 5

Storage stability of DTT@BSA-AuNCs at different temperature conditions. Stability of the photoresponsive oxidase-mimicking ability of DTT@BSA-AuNCs stored for one month at (a) 25 °C (room temperature), (b) 4 °C, and (c) -20 °C, in both dark (represented by grey bars) and lighted (represented by faint yellow bars) environments. Fluorescence intensity was recorded weekly. Error bars represent standard deviation of three independent measurements

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Detection of antioxidants

As noted in earlier mechanistic studies discussed in this article, the cause of TH oxidation by DTT@BSA-AuNCs in the presence of light is strongly linked to the generation of ROS by DTT@BSA-AuNCs. Antioxidants, recognized for their capacity to neutralize free radicals or ROS, are expected to decrease the oxidase mimicking capability of DTT@BSA-AuNCs. To test the above hypothesis, we selected ascorbic acid (AA), a common and essential antioxidant in the human body, and glutathione (GSH), an important biological thiol antioxidant found in the human body, as model antioxidants. As displayed in Fig. 6a and c, the fluorescence intensity of oxidized TC decreased with increasing concentrations of AA and GSH, which supports the rationality of earlier discussion and demonstrates the detection feasibility. The change in fluorescence intensity (∆F) of TC, measured as the difference between its fluorescence in the absence (F0) and presence (F) of the antioxidants, AA and GSH, consistently increased and remained stable when the AA and GSH concentrations were above 10 µM (Fig. 6b and d). The ∆F exhibits a linear relationship with AA concentration in the range of 0.25-2 µM, with a strong correlation (R2 = 0.99) and a calibration curve described by the equation \(\:y=1030.99x+964.26\) (refer to the inset of Fig. 6b). Similarly, for GSH in the range of 0.25-4 µM, ∆F shows a strong correlation (R2 = 0.996) with a calibration curve given by \(\:y=276.56x+735.89\:\) (refer to the inset of Fig. 6d). The reproducibility of the assay is demonstrated by an average coefficient of variation (CVav %) of 3.5% for AA and 6.4% for GSH, indicating consistent performance across multiple measurements.

Fig. 6
figure 6

Detection of AA and GSH. Fluorescence spectra of irradiated solutions of DTT@BSA-AuNCs and TH in PBS (pH 7.4) at different concentrations (0 − 100 µM) of (a) AA or (c) GSH show a decrease in fluorescence intensity with increasing concentrations of the respective antioxidants. The relationship between the change in fluorescence intensity; ΔF and (b) AA concentration or (d) GSH concentration consistently increased up to 10 µM. The insets of (b) and (d) represent a linear relationship with AA concentration within the range of 0.25-2 µM, with a strong correlation (R2 = 0.99), and with GSH concentration within the range of 0.25-4 µM, with a strong correlation (R2 = 0.996), respectively. Error bars demonstrate the standard deviation of more than three independent measurements

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The limits of detection (LOD) and limits of quantification (LOQ) for AA were 0.08 µM and 0.26 µM, respectively whereas the LOD and LOQ for GSH were 0.32 µM, and 0.98 µM, respectively. As the lowest AA concentration that can be quantitatively detected is 0.26 µM, and the concentration of AuNCs in the assay is 500 µM, the Antioxidant-to-AuNC ratio is 0.00052:1. This means that 1 µM of AuNCs is capable to detect 0.00052 µM of AA. Similarly, the lowest GSH concentration that can be quantitatively detected is 0.98 µM, giving an Antioxidant-to-AuNC ratio of 0.00196:1 which indicates 1 µM of AuNCs can detect 0.00196 µM of GSH. The Antioxidant-to-AuNC ratio for AA is significantly lower than that of GSH, indicating higher sensitivity towards AA. This is also consistent with the obtained steeper slope of the calibration curve and lower LOD for AA as compared to GSH. This difference is attributed to the different antioxidant mechanisms of AA and GSH. AA, a primary antioxidant and radical chain-breaker, rapidly scavenges ROS, resulting in greater fluorescence quenching. Whereas, GSH, being a secondary antioxidant, operates at a slower rate, leading to a less pronounced reduction in fluorescence. This variation in reaction dynamics explains the observed differences in sensing levels between AA and GSH [63, 64].

Comparing the performance of AA and GSH detection with previously reported results, concerning LOD, detection time, and the medium of detection, as summarized in Table S2 and Table S3 in the ESM respectively, our findings reveal notable achievements. Notably, our ultralow LOD for AA outperforms all previously reported works, except for one with a LOD of 0.01 µM (cited in Table S2 in the ESM). Furthermore, our LOD for GSH is superior to several reported values. Unlike some conventional assays, such as the FRAP assay which cannot detect thiol-containing antioxidants such as GSH due to its detection mechanism, our assay effectively detects GSH [4]. This is because assays such as FRAP rely on a single electron transfer (SET) reaction, whereas most physiologically relevant antioxidants, including GSH, neutralize free radicals in the body through hydrogen atom transfer (HAT). Our assay overcomes this limitation, enabling GSH detection. Moreover, ascorbic acid can utilize both HAT and SET mechanisms [65]. This suggests that our assay has the potential to detect a wider range of antioxidants with diverse functional mechanisms.

It is noteworthy that while all other studies predominantly employed an acidic pH range (pH 3 ̴ 5) as the medium of detection, our approach utilized a neutral pH (pH 7.4), enhancing the practical utility of the detection method. The use of a mild detection system at neutral pH and a short detection time of 10 min contributed to the distinct appeal of our study demonstrating its promise for antioxidant detection.

Detection of antioxidants in human saliva

To further extend the practical utility of the detection strategy, human saliva was chosen as a representative biological sample matrix. As depicted in Table S4 in the ESM, the detection method proved effective in detecting the model antioxidants, AA and GSH in saliva. The table indicates that the spiked AA and GSH recovery rates in saliva fall within the ranges of 100.56-102.03% and 99.56-100.63% respectively. Given the presence of inherent antioxidants, such as AA and GSH, in human saliva [19], the observed detection values slightly exceeded the initial spiked amount. The nearly accurate recovery results obtained indicate that the detection strategy remains unaffected in the biological sample matrix suggesting the robustness of the detection strategy.

Selectivity of antioxidant detection

To be qualified as a suitable sensor material, a nanozyme must not only exhibit lower sensitivity but also demonstrate high selectivity. To evaluate the selectivity of the detection system for antioxidants, various relevant electrolytes commonly found in human saliva such as sodium, potassium, calcium, iron, magnesium and common biomolecules including urea, glucose, sucrose and some amino acids, such as lysine, arginine, glycine and glutamic acid were selected. The concentrations of the tested interferences were kept the same as those of the antioxidants studied, AA and GSH i.e., 100 µM creating a standardized testing environment that allowed for a fair comparison. As shown in Fig. 7, the fluorescence responses of the DTT@BSA-AuNCs-TH system to all the interferents were almost unchanged compared to those of the control (blank solution). However, at the same concentration of antioxidants, AA and GSH exhibited a significant decrease in fluorescence. Therefore, this detection strategy exhibits high selectivity for antioxidant detection.

Fig. 7
figure 7

Selectivity of the detection system towards the presence of antioxidants over various relevant interferences. Fluorescence spectra of irradiated solutions of DTT@BSA-AuNCs and TH in PBS (pH 7.4) with 100 µM of AA, GSH, or interferents reveal a decrease in fluorescence intensity only in the presence of AA or GSH, but not in the presence of interferents. Each error bar shows the standard deviation of three independent measurements. *p < 0.01 compared with the control group; n.s. represents ‘not significantly different from the control group’

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Detection of cellular antioxidants within cancer cells

GSH is the most essential endogenous antioxidant in cells. Elevated levels of GSH are closely associated with cancer development, as it serves as a survival strategy to counteract increased oxidative stress in cancer cells [38, 39, 66]. The increase in GSH levels is also linked to tumor promoters such as Nuclear factor erythroid 2-related factor 2 (NRF2) that activate the GSH synthesis [67]. This study employed DTT@BSA-AuNCs to assess the presence of antioxidants within cancer cells. MCF-7 cells, a model breast cancer cell line, were compared with HS 578Bst cells, normal breast cell line. As shown in Fig. 8a, gradual decrease in the characteristic fluorescence intensity of TC was observed with increasing numbers of both MCF-7 and HS 578Bst cells (1.5 × 103 to 4.5 × 105 cells/mL). This decrease was due to the inhibition of the oxidase-like activity of DTT@BSA-AuNCs by cellular antioxidants. While most intracellular AA is known to be depleted during cell culture, the observed decrease aligns with the known ability of other cellular antioxidants, including GSH, to quench the fluorescence of TC. Notably, MCF-7 cells exhibited approximately twice the inhibition effect compared to HS 578Bst cells (Fig. 8b). This is likely due to the presence of two-fold higher levels of GSH in breast cancer cells as compared to normal cells, reported in previous studies [36, 68]. A schematic representation of detecting antioxidants in cancer cells and normal cells is elucidated in Fig. 8c, displaying more cellular antioxidants in cancer cells effectively scavenges ROS, reducing thiamine to thiochrome conversion and resulting in lower fluorescence. Conversely, normal cells with less cellular antioxidants leads to less ROS scavenging (much more ROS buildup), increasing thiamine to thiochrome conversion resulting in higher fluorescence.

Fig. 8
figure 8

Detection of cellular antioxidants by DTT@BSA-AuNCs. (a) Decrease in fluorescence intensity of irradiated solutions of DTT@BSA-AuNCs and TH with increasing concentrations of MCF-7 breast cancer cells (upper panel) and HS 578Bst normal breast cells (lower panel), with cell numbers ranging from 1.5 × 103 to 4.5 × 105 cells/mL. (b) Plot of normalized (F0 − F)/F0 versus the cell number ranging from 1.5 × 103 to 4.5 × 105 cells/mL; where F0 represents fluorescence intensity at 440 nm in the absence of cells and F represents fluorescence intensity at 440 nm in the presence of cells. (c) Schematic illustration of the detection of antioxidants in cancer cells (left side) versus normal cells (right side). In cancer cells, higher levels of cellular antioxidants effectively scavenge ROS, which reduces thiamine to thiochrome conversion and results in lower fluorescence. In contrast, normal cells, with fewer cellular antioxidants, exhibit reduced ROS scavenging, leading to increased ROS buildup, higher thiamine to thiochrome conversion, and thus, higher fluorescence. Each error bar shows the standard deviation of three independent measurements

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Therefore, this result demonstrated the potential of DTT@BSA-AuNCs for detecting cellular antioxidant levels, enabling diverse biological applications.

Conclusions

This study successfully developed an advanced method for sensitive antioxidant detection using DTT@BSA-AuNCs as light-activated oxidase-mimicking nanozymes. DTT@BSA-AuNCs effectively catalyze the oxidation of non-fluorescent TH to fluorescent TC under neutral pH conditions using a simple LED. Singlet oxygen is identified as the key oxidant in this light-activated process. DTT@BSA-AuNCs show excellent stability and versatility with diverse substrates, enabling highly sensitive detection of representative antioxidants such as AA and GSH, with detection limits as low as 0.08 µM and 0.32 µM, respectively. The practicality of the assay was validated through the detection of AA and GSH in human saliva and cellular antioxidants in a cancer cell model. This method offers several advantages over existing approaches. First, DTT@BSA-AuNCs function efficiently under neutral conditions, overcoming the limitations of previous AuNCs applications in acidic environments, making them more suitable for biological settings. Secondly, DTT@BSA-AuNCs utilize readily available dissolved oxygen, eliminating the need for H2O2 or O2, as commonly required by all peroxidase-mimicking and most oxidase-mimicking nanozymes respectively. Thirdly, this assay employs a fluorescence-based readout, resulting in superior sensitivity compared to previously used colorimetric methods. Fourthly, this assay facilitates streamlined antioxidants detection using a basic fluorescence spectrophotometer, enabling swift analysis within 10 min, with economic feasibility. Finally, this method offers particular promise for the detection of thiol-containing antioxidants such as GSH, which are often challenging to detect using conventional methods. Overall, this study paves the way for the development of highly efficient photocatalytic nanozyme systems for sensitive and practical detection of antioxidants in biological samples. This advancement holds significant promise for applications in diagnostics, monitoring oxidative stress, and potentially guiding interventions related to antioxidant levels. Future research could explore optimizing these nanozymes for a broader range of antioxidants and enhancing their biomedical applications.

Data availability

Data available with the paper or supplementary information.

Abbreviations

AuNCs:

Gold Nanoclusters

BSA:

Bovine Serum Albumin

DTT:

Dithiothreitol

DTT@BSA-AuNCs:

Dithiothreitol-assisted Bovine Serum Albumin-stabilized gold Nanoclusters

DDW:

Double Distilled Water

TH:

Thiamine

TC:

Thiochrome

HRTEM:

High-Resolution Transmission Electron Microscopy

XPS:

X-ray Photoelectron Spectroscopy

XRD:

X-Ray Diffraction

FTIR:

Fourier Transform Infrared

EDTA:

Disodium Ethylenediaminetetraacetate

DAB:

3,3’-Diaminobenzidine

DMF:

N, N-Dimethylformamide

AuNPs:

Gold Nanoparticles

NIR:

Near-Infrared Radiation

TMB:

Tetramethylbenzidine

ABTS:

2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)

LOD:

Limit of Detection

AA:

Ascorbic Acid

GSH:

Glutathione

FRAP:

Ferric Reducing Antioxidant Power

SET:

Single Electron Transfer

HAT:

Hydrogen Atom Transfer

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Acknowledgements

We thank the National Central University Precious Instrument Usage Center for providing access to the XPS instrument (Thermo Fisher ESCALAB Xi+) (with UPS function), instrument code ESCA002000, for detailed XPS analysis of the studied nanomaterial.

Funding

This study was financially supported by the National Science and Technology Council (111-2112-M-008 -021 -MY3).

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Authors and Affiliations

  1. Department of Biomedical Sciences and Engineering, National Central University, Taoyuan, Taiwan

    Sanskruti Swain, Ting-Yi Lin, I-Hsuan Chou, Shu-Chen Liu & Chen-Han Huang

  2. Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan

    I-Hsuan Chou & Hsing-Ying Lin

  3. Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli, Taiwan

    Bikash C. Mallick

Authors
  1. Sanskruti Swain
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  2. Ting-Yi Lin
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  3. I-Hsuan Chou
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  4. Shu-Chen Liu
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  5. Bikash C. Mallick
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  6. Hsing-Ying Lin
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  7. Chen-Han Huang
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Contributions

S.S. and C.-H.H. designed the study, performed the research, analyzed data, prepared the figures, and wrote the manuscript. T.-Y. L., I.-H. C. and B.C. M. assisted in material synthesis and analysis. S.-C. L. and H.-Y. L. assisted in designing the cell study. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Hsing-Ying Lin or Chen-Han Huang.

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This study used anonymized human data (saliva sample) with participant consent.

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The authors declare no competing interests.

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Swain, S., Lin, TY., Chou, IH. et al. Photoactive nanocatalysts as DTT-assisted BSA-AuNCs with enhanced oxidase-mimicking ability for sensitive fluorometric detection of antioxidants. J Nanobiotechnol 22, 585 (2024). https://doi.org/10.1186/s12951-024-02850-5

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Journal of Nanobiotechnology

ISSN: 1477-3155

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