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DOI: 10.1039/D4AN01013B (Paper) Analyst, 2024, Advance Article

A noble metal-enhanced Au@CuO heterostructure with multienzyme-mimicking activities for colorimetric detection of tannic acid†

Xin Kangab, Yiping Rena, Jin Wanga, Xu Zhua, Ning Xina, Fenglei Gao*a and Dehong Yu*ac
aJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Jiangsu 221004, Xuzhou, China. E-mail: 15380117222@163.com; jsxzgfl@sina.com
bThe First Clinical Medical College, Xuzhou Medical University, Xuzhou, Jiangsu, China
cThe Affiliated Pizhou Hospital of Xuzhou Medical University, Jiangsu 221399, China

Received 24th July 2024 , Accepted 30th July 2024

First published on 31st July 2024

Abstract

Nanozymes, serving as synthetic alternatives to natural enzymes, offer several benefits including cost-effectiveness, enzyme-like catalytic abilities, enhanced stability, adjustable catalytic activity, easy recyclability, mild reaction conditions, and environmental friendliness. Nonetheless, the ongoing quest to develop nanozymes with enhanced activity and to delve into the catalytic mechanism remains a challenge. In our research, we effectively developed Au@CuO nanocomposites (Au@CuO Nc), replicating the functions of four enzymes found in nature: peroxidase (POD), catalase (CAT), glutathione peroxidase (GPx), and oxidase (OXD). The catalytic efficiency of Au@CuO Nc for TMB oxidation (oxTMB) was approximately 4.8 times greater than that of plain Cu2O cubes, attributed to the synergistic catalytic impact between the Au element and Cu2O within Au@CuO Nc. Mechanistic studies revealed that the novel Au@CuO Nc nanozyme greatly enhances the decomposition of H2O2 to reactive oxygen species (ROS) intermediates (˙OH, ˙O2− and 1O2), resulting in increased POD-like activity of the single-component Cu2O cubes. When an antioxidant like TA was added to the chromogenic system, it converted oxTMB into a colorless form of TMB, enabling further evaluation of TA. Hence, a colorimetric sensor was developed for the rapid and precise quantitative measurement of TA, demonstrating strong linearity between 0.3 and 2.4 μM and featuring a low detection threshold of 0.25 μM. Moreover, this sensor was effectively utilized for the assessment of TA in actual tea samples. This work innovatively proposes a simplified and reliable strategy for the advanced design of highly effective Cu-based nanozymes, enhancing enzyme-like reactions for simultaneous, on-site colorimetric probing of antioxidants.

1. Introduction

Tannic acid (TA), a polyphenolic compound featuring a central glucose and 10 galloyl groups, possesses distinctive chemical and physiological attributes.1–3 TA displays a broad spectrum of significant biological activities such as antioxidant, anti-inflammatory, antibacterial, and anticancer properties4–6 Its ability to capture free radicals and metal ions, regulate cell signaling pathways, and inhibit the release of inflammatory factors collectively contributes to its positive impact on human health. Additionally, TA also has a certain preventive and therapeutic effect on cardiovascular diseases, diabetes, obesity, and other ailments. Nevertheless, excessive intake of TA can induce indigestion, decrease iron absorption, and inflict oxidative stress damage on cells and tissues, posing hazards to the human body. Hence, notwithstanding its myriad benefits, prudent deliberation is essential in light of the potential health hazards linked to its diverse applications. It is essential to devise quick and uncomplicated approaches for determining the level of TA with exceptional selectivity and sensitivity in the realms of medicine and pharmaceuticals. Traditional approaches, such as electrochemistry, fluorimetry, chemiluminescence, and chromatography,7–9 suffer from drawbacks such as expensive equipment, demanding skill requirements, and complex sample processing procedures, whereas colorimetric techniques can surmount these constraints.10,11

Nanozymes play a critical role in colorimetric sensor platforms for detecting TA as a type of nanomaterial. Nanozymes offer a multitude of advantages over natural enzymes, encompassing cost-effectiveness, remarkable resilience, streamlined manufacturing, customizable architecture and composition, as well as adaptable catalytic functionality.12–15 The development of extremely efficient nanozymes has been one of the central concerns in the area of catalytic treatment. Ever since the discovery of Fe3O4 NPs’ peroxidase-like activity in 2007, a wide range of nanomaterials have been engineered to function as nanozymes, such as noble metals like Ag, Ru, and Ir, metal oxides like V2O5, Fe2O3, RuO2, Cu2O, and CeO2, carbon-based nanozymes including carbon nanotubes, graphene oxide, and carbon dots, as well as metal–organic frameworks (MOFs).16–21 Metal oxides, known for their straightforward synthesis and modifiability, adjustable efficacy, outstanding biocompatibility, and biodegradability, have garnered significant interest as nanozymes in catalytic therapy.22,23 Yang's group designed an Au-modified layered CoAl double oxide (Au/LDO) colorimetric platform for the detection of three liver-related biomarkers: aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), demonstrating great potential.24 Cu2O, a p-type semiconductor oxide, has shown significant promise as a nanozyme due to its adjustable electronic properties (Cu2+/Cu+), oxygen vacancies, low biotoxicity, and capability to mimic multiple enzyme activities.25 Nevertheless, the catalytic performance of previously documented Cu2O nanomaterials is considerably inadequate, significantly constraining their potential use as nanozymes in catalytic therapy. In recent years, gold nanoparticles (AuNPs) have demonstrated catalytic activity when combined with other metal oxides such as Co3O4, Fe2O3, or TiO2, which is attributed to their straightforward synthesis, outstanding biocompatibility, easy surface modification, and tunable optical properties.26–28

In this study, we have effectively developed Au@CuO Nc for the colorimetric detection of TA. Benefiting from the evenly distributed Au element and the combined action of Au element with Cu2O, the engineered nanozyme demonstrates improved peroxidase-like activity, positioning it as a promising substitute for peroxidase mimics. Utilizing Cu2O as a rigid template enabled the in situ formation of Au. Additionally, the cooperative effects stemming from the interaction between Cu2O and Au within the Au@CuO Nc contributed to its superior mimicry of multiple enzyme activities. The performance of multiple nanozymes, along with their steady-state kinetics and the mechanisms underlying various enzymatic activities, was meticulously explored. The potent mimicry of enzymatic catalysis by Au@CuO Nc is likely a key factor in its effective oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), facilitating the transition of TMB from a colorless state to a blue oxidized form (oxTMB). What's more, a highly sensitive colorimetric detection platform for TA was developed by utilizing the reduction of blue oxTMB to colorless TMB upon its introduction into the sensing system. This study presents the Au@CuO Nc nanozyme, which demonstrates four types of enzyme-like activities, and provides novel methods for exploring nanomaterials with various enzyme-mimicking properties (Scheme 1a). It also demonstrates significant potential for creating nano-based platforms aimed at the precise quantification of biomolecules in the medical and healthcare sectors.


image file: d4an01013b-s1.tif
Scheme 1 (a) Multiple enzyme-like activities for the prepared Au@CuO. (b) Schematic illustration of the synthetic route.

2. Experimental section

2.1 POD-like activity assay

The experiments to evaluate POD-like activities involved using the substrate TMB, which underwent catalytic oxidation in the presence of H2O2. In a typical setup, the mixture for the assay included 0.59 mM TMB, 35 mM H2O2, and 30 μg mL−1 concentration of the catalyst (either Au@CuO Nc or Cu2O cubes) in a 1.5 mL solution of reaction buffer (0.2 M NaAc, with a pH of 4.0). The effectiveness of the catalyst as a peroxidase was assessed by monitoring the change in absorbance at 652 nm over time. In a standard experiment, TMB was maintained at a constant concentration of 0.59 mM, while the concentration of H2O2 varied between 5 and 40 mM. Similarly, the experiments were repeated with TMB concentrations ranging from 0.065 to 0.6 mM, keeping the H2O2 concentration fixed at 35 mM. The study investigated the kinetic parameters following the Michaelis–Menten model:
image file: d4an01013b-t1.tif
where V represents the initial reaction rate (M s−1), Vmax denotes the maximum reaction rate (M s−1), [S] is the concentration of the substrate (M), and Km stands for the Michaelis–Menten constant (Mm). The Km value is commonly used to assess the binding affinity between nanozymes and their substrates, with a smaller Km indicating a higher affinity.

2.2 CAT-like activity assay

Tests were conducted with 30 μg mL−1 Au@CuO Nc in a 1.5 mL NaAc buffer solution (pH 4.0), combined with 35 mM H2O2 at a temperature of 50 °C. Following a 5 minute incubation period, the amount of H2O2 used was determined by measuring the reduction in absorbance at 240 nm.

2.3 OXD-like activity assay

The oxidation activity of Au@CuO Nc on TMB was investigated through observing the absorbance at 652 nm with a UV-vis spectrophotometer. In a standard experiment, 30 μg mL−1 of Au@CuO Nc was introduced into a NaAc buffer solution (pH 4.0), followed by the addition of TMB at concentrations varying from 0.9 mM to 7 mM.

2.4 GPx-like activity assay

To examine the GPx-mimicking behavior of Au@CuO Nc, several tests were conducted. The mixture included 0.15 mM NADH, 0.6 mM GSH, glutathione reductase (GR, 1.0 U), 30 μg mL−1 of Au@CuO Nc, and H2O2 (35 mM) in equal proportions. The dynamic behavior of Au@CuO Nc over time was analyzed by altering its concentration (from 10 to 30 μg mL−1) in a solution containing all the mentioned substances. This solution was then subjected to detection using a UV-vis spectrophotometer at a wavelength of 340 nm.

2.5 Optimization of conditions

The study analyzed the impact of multiple variables on the behavior of Au@CuO Nc by monitoring changes in absorbance after the reaction within a pH range of 2–8 and temperatures between 30 and 70 °C over a period of 5 min. Additionally, the research investigated how the interaction with TMB varied in different buffer solutions with TMB concentrations from 0.2 to 0.79 mM, while also assessing the influence of different H2O2 levels ranging from 5 to 70 mM.

2.6 Mechanism detection assay

We exposed Au@CuO Nc to a NaAc-HAc solution (0.2 M, pH 4.0) for 5 min at 50 °C, along with H2O2 (35 mM), TA (1.5 mM), and varying amounts of Au@CuO Nc (ranging from 0 to 30 μg mL−1). To further confirm the action mechanism of these nanozymes, MB and RhB were employed as reactants that interact with ˙OH. The procedure for testing was akin to that used for TA.

2.7 Colorimetric detection of TA

TA detection by the Au@CuO Nc was performed as follows: 30 μg mL−1 Au@CuO Nc, 0.59 mM TMB, and 35 mM H2O2 in NaAc buffer solution were incubated at 50 °C for 5 min before adding different concentrations of TA solution (0.3–75 μM). After thoroughly mixing, the absorbance at 652 nm was measured using a UV–vis spectrophotometer.

2.8 Detection of TA in real samples

To assess the practical value of the colorimetric detection method, a 0.1 g green tea sample was steeped in 20 mL of boiling water. After ten minutes, the tea was filtered through a 0.22 μm filter membrane and then diluted with ultrapure water for further experiments.

3. Results and discussion

3.1 The characterization of Au@CuO Nc

Scheme 1b demonstrates the creation of Au@CuO Nc through a two-phase synthesis process. Initially, wet chemical methods were used to generate Cu2O nanocubes, each roughly 500 nm in diameter. Subsequently, these nanocubes were transformed into Au@CuO Nc upon reacting with a HAuCl4 solution, facilitated by the surfactant PVP. The in situ development of the Au@CuO Nc atop the Cu2O base was enabled by an appropriate PVP additive.29 With the assistance of PVP, uniform fine particulate matter forms on the surface of Au@CuO. According to previous reports, in the absence of PVP, large uneven agglomerates would form. The SEM images reveal that the Au@CuO Nc exhibit a nanocube morphology with fine particles scattering on the surfaces (Fig. 1a and b), indicating enhanced specific surface area and catalytic performance (Fig. S1a and b†), which is consistent with the characterization results of TEM (Fig. 1c–e and Fig. S2†). The elemental composition was as follows: Cu (93.7%), O (6.0%) and Au (0.3%) (Fig. 2a). The EDX elemental mapping images indicate that the distribution of Cu elements within Au@CuO Nc is uniform, while the Au element is predominantly concentrated in the external region (Fig. 1f). A comparison of the XRD patterns of Au@CuO Nc and Cu2O nanocubes was conducted to determine the crystal structure of the samples (Fig. 2b). The XRD pattern of the Cu2O nanocubes reveals diffraction peaks corresponding to cubic Cu2O (JCPDS 034-1354). The identification of four diffraction peaks at 2θ values of 36.4°, 42.5°, 61.9°, and 74.1° corresponds to the Au@CuO Nc's (111), (200), (220), and (311) facets, respectively. The composition and electronic structure of the Au@CuO Nc were further explored through XPS, with the comprehensive XPS spectrum of Au@CuO Nc depicted in Fig. 2c. The Au 4f spectrum of the Au@CuO Nc reveals a pair of doublet peaks (see Fig. 2d), with the 87.6 eV and 83.7 eV peaks corresponding to the spin–orbit components Au 4f5/2 and Au 4f7/2, respectively. In the spectrum of Cu 2p, the 932.6 eV and 952.4 eV peaks are associated with the Cu 2p3/2 and Cu 2p1/2 states of metallic copper, as shown in Fig. 2e and Fig. S4.† Furthermore, the peaks at 933.5 eV and 953.2 eV are linked to monovalent copper (Cu+), and those at 934.7 eV and 954.6 eV are associated with divalent copper (Cu2+). The detection of oxidized copper forms suggests that the Au@CuO Nc increase the oxygen vacancies after the reaction. What's more, O 1s XPS spectra of Au@CuO and Cu2O are shown in Fig. 2f and Fig. S3.†
image file: d4an01013b-f1.tif
Fig. 1 SEM images (a and b) and TEM images (c and d) of Au@CuO. HAADF-STEM image (e) and corresponding elemental mapping (f) of Au@CuO.

image file: d4an01013b-f2.tif
Fig. 2 (a) EDS survey spectrum of Au@CuO. (b) XRD pattern of as-synthesized Au@CuO and Cu2O. (c) Survey XPS spectra of Au@CuO and Cu2O. (d) Au 4f XPS spectra of Au@CuO. (e) Cu 2p XPS spectra of Au@CuO. (f) O 1s XPS spectra of Au@CuO.

3.2 Multiple enzyme activities of Au@CuO Nc

3.2.1 POD-like activities. POD-like nanozymes transform H2O2 into free radicals through a process similar to the Fenton reaction. In this context, colorless substances such as 3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) are used as color-developing agents. These agents undergo oxidation in the presence of H2O2 under acidic conditions, leading to a change in color. Specifically, TMB changes to a blue-colored oxTMB with an absorption peak at 652 nm, while OPD and ABTS transition to oxOPD and oxABTS, respectively, showing an absorption peak at 420 nm and 417 nm when in acidic NaAc solution (Fig. 3 and Fig. 4a). The color changes in the ABTS and OPD colorimetric reactions are shown in Fig. S5a and b.† A significant color shift was observed upon introducing Au@CuO Nc nanozymes and H2O2 into the acidic NaAc solution, in contrast to other combinations like TMB alone, H2O2 with TMB, and TMB with Au@CuO Nc, which did not show any color change (Fig. 4b). This indicates that the oxidation reaction was specifically facilitated by the presence of Au@CuO Nc nanozymes. As the TA concentration rises, there's a notable decrease in the reaction system's absorbance, showcasing the potential for TA detection (Fig. 4c). As the concentration of TA increases, the rate and extent of the rise in absorbance gradually decrease, indicating the inhibitory effect of TA on the oxidation reaction of TMB (Fig. S6†). The color changes seen in the insets of Fig. 4b and c visually confirm the occurrence of the reaction. Furthermore, introducing N2 exerts an inhibitory influence on the color reaction of TMB. This occurs because the injection of N2 lowers the concentration of dissolved oxygen within the reaction system (Fig. S7†). As shown in Fig. S8a and b,† the time-scan curves reveal the POD-mimicking actions of both Cu2O and Au@CuO Nc, highlighting a significant acceleration in reaction speed with rising nanozyme concentrations (10, 15, 20, 25, and 30 μg mL−1). Fig. 5a–d illustrates that the reaction's initial velocity increases with substrate concentration before stabilizing at higher levels. The kinetics of the Au@CuO Nc nanozymes, with a Michaelis constant (Km) of 0.52 mM and a maximum velocity (Vmax) of 51.49 × 10−8 M s−1, shows significant enhancement compared to Cu2O nanozymes (Fig. 5e–h), when TMB serves as the substrate. When H2O2 is the substrate, Au@CuO Nc nanozymes exhibit significantly greater catalytic efficiency than Cu2O nanozymes. The substrate affinity and POD-like catalytic activity of Au@CuO Nc were remarkably boosted (Table S1†).
image file: d4an01013b-f3.tif
Fig. 3 Diagram of the POD-like catalytic reaction based on Au@CuO nanozymes.

image file: d4an01013b-f4.tif
Fig. 4 (a) Schematic representation of the POD-like activity of Au@CuO. (b) UV-vis absorption spectra of different solutions: (I) TMB; (II) TMB + H2O2; (III) TMB + H2O2 + Cu2O; (IV) TMB + H2O2 + Au@CuO. Inset: the color change of the corresponding solution. (c) UV–vis spectra and photographs of different systems: (I) TMB + H2O2 + Au@CuO; (II) TMB + H2O2 + Au@CuO + TA. (d) Schematic representation of the CAT-like activity of Au@CuO. UV-vis absorption spectra of different solutions (e) and 10–30 μg mL−1 Au@CuO (f). (g) Schematic representation of the OXD-like activity of Au@CuO. (h) Michaelis–Menten curves of Au@CuO with TMB as a substrate. (i) The Lineweaver–Burk double-reciprocal diagram of Au@CuO. (j) Schematic representation of the GPx-like activity of Au@CuO. Time-dependent UV–vis spectral changes of NADH over different systems (k) and 10–30 μg mL−1 Au@CuO (l).

image file: d4an01013b-f5.tif
Fig. 5 (a and c) Michaelis–Menten curves of Au@CuO with TMB and H2O2 as substrates. (b and d) Lineweaver–Burk double-reciprocal diagram of Au@CuO. (e and g) Michaelis–Menten curves of Cu2O with TMB and H2O2 as substrates. (f and h) Lineweaver–Burk double-reciprocal diagram of Cu2O.
3.2.2 CAT-like activities. In the presence of Au@CuO Nc ranging from 10 μg mL−1 to 30 μg mL−1, there was a reduction in H2O2 concentration (Fig. 4d and e), suggesting a significant improvement in the CAT-like activity by Au@CuO Nc compared with Cu2O (Fig. 4f).
3.2.3 OXD-like activities. The OXD-like behavior was explored through the real-time observation of changes in the absorption of 3,3,5,5′-tetramethylbenzidine (TMB) at 652 nm (Fig. 4g). The reaction rate notably rose with the increase in TMB concentrations (0.9, 1.75, 2, 2.25, 3, 3.5, 3.75, 5, 7 mM). Michaelis–Menten plots and Lineweaver–Burk graphs are displayed in Fig. 4h and i. The Km and Vmax for the Au@CuO Nc nanozymes were determined to be 4.1 mM and 11.6 × 10−8 M s−1, respectively, demonstrating the strong OXD activity of Au@CuO Nc.
3.2.4 GPx-like activities. Under the influence of GPx, glutathione (GSH) serves as an electron donor and can be oxidized to GSSG, leading to the swift breakdown of the electron acceptor H2O2 into harmless H2O and O2 (Fig. 4j).30 Glutathione reductase (GR) is able to convert GSSG back into GSH with the help of nicotinamide adenine dinucleotide (NADH), enabling the GPx-like function of the Au@CuO Nc to be assessed by observing the alterations in the UV-vis absorption spectrum of NADH at 340 nm. Upon the introduction of Au@CuO Nc, there is a notable reduction in the 340 nm peak over time when compared to other reaction systems (Fig. 4k). Additionally, the rate at which NADH's absorbance decreases can be expedited by raising the concentration of the Au@CuO Nc nanozyme from 10 μg mL−1 to 30 μg mL−1 (Fig. 4l), demonstrating the strong GPx-like capability of the Au@CuO Nc.

3.3 Mechanism for the enzyme-like activity of Au@CuO Nc

To delve deeper into the POD-like activity, the increase in absorbance at 652 nm over time was monitored. As the time extends from one minute to five minutes, the absorbance changes from 0.922 to 2.300. The line chart in the upper right corner shows the variation in the oxidation rates of Au@CuO Nc and Cu2O, indicating that the enzymatic activity of Au@CuO Nc is initially higher than that of Cu2O (Fig. 6a). To investigate the catalytic behavior similar to that of POD involving Au@CuO Nc, we performed an analysis of the fluorescence emission spectra in the H2O2-Au@CuO Nc system, utilizing terephthalic acid (TA) as a probe for fluorescence. TA interacts with ˙OH, forming 2-hydroxy terephthalic acid (TAOH), which fluoresces at 425 nm.31,32 As shown in Fig. 6b, an increase in the concentration of Au@CuO Nc corresponded to a steady increase in the fluorescence emitted by 2-hydroxy terephthalic acid, suggesting an enhanced production of ˙OH in the solution. The intensity of ABTS and OPD increases alongside the concentration of Au@CuO Nc (Fig. 6c, d and Fig. S9†). To verify the formation of ˙OH during the catalytic activity, methylene blue (MB) was used, as illustrated in Fig. 6f. As the concentration of the nanozyme rose, there was a discernible degradation of MB, evident from the significant decrease in its absorption peak at 660 nm.33 The addition of H2O2 and Cu2O to the system did not lead to notable alterations (Fig. 6e). Furthermore, rhodamine B (RhB) acts as a specific scavenger for hydroxyl radicals (˙OH), with its absorbance decreasing after introducing Au@CuO Nc (Fig. 6g and h). This decrease suggests the significant production of ˙OH radicals, which in turn stimulates POD-like activity.
image file: d4an01013b-f6.tif
Fig. 6 (a) UV-vis absorption spectra of TMB solution treated with Au@CuO in the presence of H2O2 (inset: the relative UV-vis absorption intensity at 652 nm treated with Cu2O and Au@CuO). (b) Fluorescence spectra of TA oxidized by ˙OH generated from different concentrations of Au@CuO ranging from 0 μg mL−1 to 30 μg mL−1. UV-vis absorption spectra of different concentrations of ABTS (c) and OPD (d). UV-vis absorption spectra of different MB solutions (e) and 10–30 μg mL−1 Au@CuO (f). UV-vis absorption spectra of different RhB solutions (g) and 10–30 μg mL−1 Au@CuO (h).

To delve into the catalytic behavior of Au@CuO Nc, a variety of techniques were employed to verify the generation of reactive oxygen species (ROS) such as hydroxyl radicals (˙OH), superoxide anion radicals (˙O2−), and singlet oxygen (1O2) during the oxidation process. Initial experiments using UV-vis spectroscopy with isopropyl alcohol (IPA) and p-benzoquinone (PBQ) as scavengers for ˙OH and ˙O2−, respectively, showed a decrease in absorbance at 652 nm upon adding these scavengers to the nanozyme catalytic setup.34,35 This result indicates the formation of ˙OH and ˙O2− radicals through the decomposition of H2O2 by Au@CuO Nc, aiding in the oxidation of TMB. The decrease in the reaction system's catalytic efficiency due to the entrapment of radicals by scavengers lends further credence to the theory (Fig. 7a). 9,10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA), acting as a precise marker for 1O2 capture, shows a reduction in its absorbance spectrum when oxidized by 1O2.36 The effects of H2O2 or Cu2O on ABDA's oxidation are negligible, as depicted in Fig. 7b and c. Nonetheless, introducing Au@CuO Nc results in a noticeable decline in ABDA's absorbance, highlighting the significant production of 1O2 in the catalytic process. Furthermore, the production of ˙OH during the catalytic process was confirmed through EPR analysis, which exhibited a characteristic EPR signal (1[thin space (1/6-em)]:[thin space (1/6-em)]2[thin space (1/6-em)]:[thin space (1/6-em)]2[thin space (1/6-em)]:[thin space (1/6-em)]1). This finding demonstrates the Au@CuO Nc nanozyme's capability to catalyze H2O2, resulting in ˙OH generation (Fig. 7d). What's more, compared to Cu2O, the EPR signal of Au@CuO is stronger, which indicated the more generation of ˙OH. To sum up, under acidic conditions, Au@CuO Nc breaks down H2O2, resulting in the formation of ˙OH, ˙O2−, and 1O2, which efficiently oxidize the substrate to a more elevated oxidation state.


image file: d4an01013b-f7.tif
Fig. 7 (a) The UV-vis absorbance peak at 652 nm of Au@CuO-H2O2-TMB with IPA, PBQ. UV-vis absorption spectra of different ABDA solutions (b) and 0–30 μg mL−1 Au@CuO (c). (d) EPR spectra of ˙OH.

3.4 Optimization of experimental conditions

The catalytic performance of Au@CuO Nc was affected by several factors, including pH, temperature, and the concentrations of substrates such as TMB and H2O2. By adjusting the pH (from 2 to 8) and temperature (from 30 °C to 70 °C), we monitored through UV-visible absorption spectroscopy at the same time. Fig. S8c† demonstrates that at a wavelength of 652 nm, the absorption peak is highest at 50 °C. As the temperature rises from 50 °C to 70 °C, the absorbance diminishes, suggesting that 50 °C is the optimal temperature for the catalytic reaction system. Fig. S8d† indicates that the peroxidase activity of Au@CuO Nc nanozymes initially increases with an increase in pH from 2 to 4. When the peak intensity in the catalytic system reaches a pH of 4, it gradually decreases as the pH continues to rise, signifying that a pH of 4 is optimal for catalysis. Fig. S10† illustrates the color changes of the material under different pH conditions. Furthermore, by altering the concentrations of H2O2 (from 5 mM to 70 mM) and TMB (from 0.2 mM to 0.79 mM), the peak absorption indicating the highest catalytic activity was pinpointed at 0.59 mM for TMB (Fig. S8f†) and 35 mM for H2O2 (Fig. S8e†). From the experimental analysis, it can be concluded that Au@CuO Nc nanozymes exhibit optimal peroxidase-like activity in a solution with 35 mM H2O2 and 0.59 mM TMB at pH 4 and 50 °C. Under these optimized conditions, the catalytic efficiency of Au@CuO Nc was further investigated.

3.5 Colorimetric platform for TA detection

A colorimetric detection approach for measuring TA was developed based on the suppressive impact of TA on the oxidation of TMB molecules (Fig. 8a). Fig. 8b demonstrates that the absorbance at 652 nm consistently decreased as the TA concentration increased from 0 to 75 μM. There was a strong linear correlation between the absorbance and the TA concentrations within the 0.3–2.4 μM range, as shown in Fig. 8c. The detection limit was calculated to be 0.25 μM, using the formula LOD = 3 σ/k (where σ is the standard deviation and k is the slope of the standard curve). This method offers a broader range and a lower detection threshold for TA compared to previous techniques, as detailed in Table S2.† The specificity of this colorimetric assay was also assessed by examining the reaction of the Au@CuO Nc nanozymes to various ions and potential interfering substances.
image file: d4an01013b-f8.tif
Fig. 8 (a) Scheme of the detection mechanism of the sensing system. (b) Images and absorbance spectra of oxTMB obtained by varying the concentration of TA from 0 to 75 μM. (c) Response curve for colorimetric TA determination and linear calibration curve for TA detection in the range of 0.3–2.4 μM. (d) Anti-interference analysis for the Au@CuO colorimetric sensing platform. (e) Long-term storage effects on the POD-like activity of Au@CuO.

Fig. 8d demonstrates that Au@CuO Nc nanozymes show a significant response to TA, as indicated by a substantial shift in absorbance. This reaction is more prominent compared to the response seen with other ions (Ca2+, Na+) and different molecules (ascorbic acid, fructose, glutathione, hyaluronic acid, isoniazid, and uric acid). The intensity of the absorption peak of oxidized TMB at 652 nm remains nearly unchanged and can still be maintained even after the addition of these reducing agents to the Au@CuO-H2O2-TMB system. This indicates that the colorimetric method developed offers selective detection of TA. Regular testing was conducted to evaluate the nanozyme's stability over extended storage periods (Fig. 8e). Remarkably, after being stored under ambient laboratory conditions for 42 days, the nanozyme retained most of its peroxidase activity, demonstrating its stable POD-like behavior over time.

To verify the sensitivity of Au@CuO Nc nanozymes in detecting TA in actual samples, these samples were employed to evaluate the colorimetric method and the system's precision. According to Table S3,† the recovery rate for the actual samples ranged from 96.62% to 100.08%. This successful detection of TA in real samples suggests that the Au@CuO Nc nanozyme holds promise for applications in the field of sensing.

4. Conclusion

To summarize, an easy and effective one-step synthesis approach was introduced for creating the innovative nanozyme Au@CuO Nc, which exhibits strong activity mimicking multiple enzymes, thanks to a core–shell nanostructure. The addition of PVP played a crucial role in directly forming the Au@CuO Nc shell on the Cu2O surface. Due to its densely packed active sites and the highly efficient use of valuable metals, the Au@CuO Nc nanozyme demonstrated an activity similar to that of peroxidase, positioning it as an excellent option for nanozyme development. Importantly, the sensor based on Au@CuO Nc, benefiting from its ability to mimic multiple enzymes, exhibited outstanding catalytic performance in detecting tannic acid (TA), characterized by its extreme sensitivity, broad linear detection range, high selectivity, and exceptional stability. Furthermore, this sensor has been effectively applied to the detection of TA in actual tea samples. The present study proposes a proficient strategy for designing novel nanozymes with high active site density and multi-enzyme mimicking capability, furthering the development of nanomaterials in the fields of biosensing and medical diagnostics.

Data availability

Data for this article, including Supplementary Information, are available at https://doi.org/10.1039/D4AN01013B.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors gratefully acknowledge financial support from the Natural Scientific Foundation of China Project (22174123) and the Jiangsu Outstanding Youth Fund (BK20220062).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4an01013b
This journal is © The Royal Society of Chemistry 2024