Hierarchical Co _x Cu_1–x S Nanoflower as Peroxidase Mimics for Advanced Ascorbic Acid Biosensing

Abstract

Nanozymes, which possess inherent catalytic properties that are akin to those of natural enzymes, have emerged as promising candidates for biomedical innovation. In this work, we successfully synthesize a Co _x Cu_1–x S nanoflower by the solvothermal and soaking method. Fortunately, through cobalt doping and microstructure design, its morphological structure and active sites have been optimized and adjusted, thus bestowing the Co _x Cu_1–x S nanoflower enhanced peroxidase-mimetic activity. When hydrogen peroxide (H₂O₂) is present, the colorless compound 3,3′,5,5′-tetramethylbenzidine (TMB) can be successfully catalyzed to yield the blue oxidized variant of TMB (ox-TMB). The mechanism of its catalytic reaction may be mainly related to the production of hydroxyl radicals (•OH). Leveraging the impressive peroxidase-mimicking properties of the aforementioned Co _x Cu_1–x S nanoflower, a straightforward biosensing mechanism for the visual identification of ascorbic acid (AA) has been established. The colorimetric detection method demonstrates a linear detection range spanning from 50 to 1500 μm, with a limit of detection established at 1.11 μm. Furthermore, it displays commendable resistance to interference and strong selectivity. Ultimately, we effectively employ this technique to identify AA in actual samples, thereby establishing the groundwork for the utilization of the Co _x Cu_1–x S nanoflower within the biomedical domain.

1. Introduction

Ascorbic acid (AA) is regarded as a prevalent antioxidant within the human body and is crucial for numerous physiological and biochemical functions. The typical serum concentration of AA varies between approximately 40 and 120 μM, and the AA content is closely associated with the occurrence and development of some diseases in the human body, including coronary heart disease, hypertension, vascular endothelial dysfunction, atherosclerosis, stroke, and so on. Preserving the relative stability of AA concentrations within the body is critically important for maintaining overall health and proper physiological functions. At the same time, whether the AA content in the body is normal can be used as an important indicator for early diagnosis and prevention of certain diseases. Over the last several decades, conventional techniques utilized for the detection of AA have mainly included high-performance liquid chromatography, electrochemical approaches, and fluorescence methods. Nonetheless, conventional detection techniques frequently encounter several fundamental issues, such as the need for highly trained personnel and specialized equipment, lengthy detection processes, and high costs. Consequently, to tackle the challenges mentioned earlier, it is crucial to create and apply a method that enables the straightforward and sensitive detection of AA levels through visual observation. This development holds considerable importance across multiple domains, including disease diagnosis, health monitoring, and pharmaceutical safety.

Nanozymes, a category of artificially engineered enzymes, address several limitations associated with their natural counterparts. They offer numerous benefits, including reduced production costs, enhanced stability, and the capability to easily adjust their catalytic activities. Therefore, they are widely used in multiple domains, including biomedicine, environmental protection, and others. In recent decades, research on nanozyme materials has increasingly centered on metal particles, transition metal oxides or sulfides, carbon-based nanomaterials, and metal–organic frameworks. Among them, the copper (Cu)-based nanozyme family, such as copper oxide nanoparticles, copper sulfides, and copper-based metal–organic frameworks, has attracted widespread attention due to the adjustability of structural components and morphological diversity. − In addition, a copper-based nanozyme possesses the capability to transform hydrogen peroxide (H₂O₂) into hydroxyl radicals (•OH) through Fenton-like reaction within a certain pH range, so it is regarded as a potential class of peroxidase mimics, which has been extensively used in colorimetric detection of biomolecules. For example, Zheng and colleagues synthesized a polyacrylonitrile copper oxide nanozyme with peroxidase-like activity and then established a biosensing system for detecting AA. Niu’s group prepared a nanocopper carbon fiber particle material by using electrospinning and carbonization methods, which possessed peroxidase-like activity and had been successfully used to detect a variety of antioxidants including AA.

Despite the advancements achieved in the investigation of copper-based metallic materials functioning as peroxidase mimetics, most of the current studies on copper-based metal nanozyme materials are still limited to single metal components. In contrast, the peroxidase-mimicking activity of single metal copper or its compounds is notably inadequate, which severely restricts potential applications. Therefore, it is an immediate necessity to enhance the peroxidase-mimicking functionality of the nanozyme by means of optimized design strategies. A substantial body of research indicates that the catalytic efficacy of a nanozyme is intricately linked to structural attributes and compositional characteristics. Through the strategic design of morphology and the fine-tuning of composition, it is possible to enhance the accessibility of active sites and trigger synergistic effects among components, thereby improving catalytic activity. For example, researchers found that doping with other metal elements was introduced into nanozyme materials to change the elemental composition and electronic structure, thereby changing the crystal structure and charge distribution of nanomaterials. − This synergy effect of bimetallic elements in the active centers could often greatly improve the catalytic performance of the nanozyme. − Furthermore, Liu et al. utilized iron and nitrogen elements to dope into a carbon graphene nanomaterial for a uniform distribution, which not only increased the quantity of available active sites but also changed the structural morphology of the material, thereby increasing its peroxidase-like activity dozens of times compared to before. In addition, Huang et al. synthesized the hybrid nanomaterial by nitrogen doping with copper-based metal nanoparticles, which not only bestowed good structural stability but also enhanced peroxidase-mimicking activity due to the bimetal synergistic interactions. Successfully, it was effectively utilized for colorimetric detection of glucose. Zhang and his colleagues synthesized a kind of platinum–gold nanomaterial according to doping with gold element. Following alterations in the elemental composition and morphological structure of the nanozyme, its peroxidase-like activity was significantly improved on account of exposing more internal catalytic active sites. Eventually, it showed better catalytic activity than horseradish peroxidase (HRP). However, the effective application of element doping and morphological modification strategies to develop innovative hybrid nanomaterials with unique crystal structures and enhanced peroxidase-mimicking activity is still a considerable challenge. This challenge arises from the need to understand the synergistic interactions among different metal elements, which necessitates further in-depth research.

Therefore, based on the insights gained from the above research, we employed the strategy of element doping and morphology design in this study to develop a novel Co _x Cu_1–x S nanoflower material. We had prepared the Co _x Cu_1–x S nanoflower with a unique morphology structure consisting of multiple ultrathin nanoplates interlocked together to form a petal-like structure. This structure greatly increases the surface area of active centers, allowing more reaction active sites to be fully exposed, which is beneficial for maximizing the catalytic reaction. Additionally, it contributes to the rapid penetration and diffusion of reaction solutions, greatly enhancing the catalytic activity of the nanozyme. More importantly, the incorporation of cobalt results in a significant presence of lattice defects within the crystalline structure of the synthesized Co _x Cu_1–x S nanoflower. This phenomenon not only facilitates the exposure and release of a greater number of active sites characterized by unsaturated suspended bonds but also expedites the transfer efficiency between electrons. Following the aforementioned described morphological design and elemental doping process, the Co _x Cu_1–x S nanoflower material demonstrates impressive peroxidase-mimicking activity. These characteristics enable the effective catalysis of hydrogen peroxide (H₂O₂) decomposition, which in turn generate the reactive oxygen species through the Fenton-like reaction. Additionally, it facilitates the oxidative transformation of the uncolored substrate 3,3′,5,5′-tetramethylbenzidine (TMB), leading to the production of the blue oxidized variant of TMB (ox-TMB). Consequently, because of the remarkable peroxidase-like characteristics exhibited by the Co _x Cu_1–x S nanoflower, along with the antioxidant effects of AA, we have successfully developed a straightforward, efficient, and real-time biosensor for the quantification of AA. This advancement facilitates the effective monitoring of AA concentrations within the realm of biomedicine. Additionally, this research establishes a research foundation for the practical utilization of nanozyme materials in associated disciplines.

2. Experiments and Methods

2.1. Regents and Materials

Kolong Chemical (Chengdu, China) supplied us with various reagents or materials, such as 30% hydrogen peroxide (H₂O₂), trisodium acetate trihydrate (CH₃COONa·3H₂O), ethanol (CH₃CH₂OH), acetic acid (CH₃COOH), copper nitrate trihydrate (Cu­(NO₃)₂·3H₂O), and solid sulfur powder (S). Sinopharm Chemical Reagents (Shanghai, China) also supplied us with multiple chemical reagents or materials, including 2-azinobis­(3-ethylbenzothiazoline-6-sulfonate) (ABTS), cobalt nitrate hexahydrate (Co­(NO₃)₂·6H₂O), o-phenylenediamine (OPD), and amino acid molecules in the experiment. The Alfa Aesar Chemical Reagents company (Shanghai, China) provided us with dopamine (DA). In addition, Aladdin Biochemical Technology Company (Shanghai, China) supplied us with 3,3′,5,5′-tetramethylbenzidine (TMB), p-benzoquinone (PBQ), and metal interfering ion compounds in the experiment. McLean Biochemical (Shanghai, China) supplied us with AA and thiourea. The water in the experiment was ultrapure water produced by an automatic distillation system. All of the above chemicals did not need to be purified.

2.2. Preparation and Synthesis of the CuS Nanoflower

The preparation of the CuS nanoflower was accomplished through the solvothermal approach. Initially, anhydrous ethanol (the volume of 16 mL) was precisely measured and added to a beaker. Following this, 0.125 mmol of Cu­(NO₃)₂·3H₂O and 0.5 mmol of sulfur powder were individually introduced into the beaker. The mixture was agitated thoroughly to ensure a uniform blend. Subsequently, the resultant mixture was swiftly moved into a 25 mL autoclave lined with Teflon, where it was kept at a constant temperature of 140 °C for around 6 h. Following the reaction period, the resultant solution underwent centrifugation at a speed of 10,000 rpm/min for multiple cycles. The collected black solid substance underwent a series of washing procedures, which included three consecutive rinses with distilled water, followed by three more rinses with anhydrous ethanol. Finally, the black substance underwent a drying process in the oven maintained at a temperature of 60 °C overnight, resulting in the formation of the CuS nanoflower.

2.3. Preparation and Synthesis of the Co _x Cu_1–x S Nanoflower

100 mg of the CuS nanoflower was weighed and placed in a beaker, and then it was immersed in 20 mL of Co­(NO₃)₂ at a certain concentration of 1 mol/L with continuous stirring for several minutes. Following this step, the sample underwent centrifugation and was washed three times by using distilled water and absolute ethanol. A series of Co _x Cu_1–x S nanoflowers with different proportions were ultimately obtained by drying the resulting mixture in a vacuum oven overnight. The specific experimental parameters are listed in Table S1.

2.4. Preparation and Synthesis of the CoS Nanoflower

The synthesis method of pure CoS nanoflowers is similar to that described in Section , with the only difference being that Cu­(NO₃)₂·3H₂O is replaced by Co­(NO₃)₂·6H₂O during the preparation process.

2.5. Analysis of Peroxidase-like Activity of the Co _x Cu_1–x S Nanoflower

In this work, the peroxidase-mimicking activity of the Co _x Cu_1–x S nanoflower was analyzed by reacting with TMB in the presence of H₂O₂. Specifically, 500 μL of Co _x Cu_1–x S (10 μg/mL), H₂O₂ (10 mM), and HAc-NaAc buffer (pH 5.6) was mixed, and finally, 500 μL of TMB (1.6 mM) was added to form a reaction system with a final volume of 2 mL. After shaking and mixing, the reaction took place at ambient temperature for 10 min. Subsequently, the absorbance of the reaction mixture was measured at a wavelength of 652 nm by utilizing a UV–vis spectrophotometer. Following the aforementioned approach, we also measured the absorbance at 652 nm when we replaced the chromogenic substrate with ABTS or OPD, keeping other reaction conditions unchanged. Simultaneously, to optimize the catalytic reaction, we employed the same methodology to investigate various reaction conditions, which encompassed the concentration of the catalyst, the concentration of TMB, the concentration of H₂O₂, the pH level of buffer, and the duration of the reaction.

Next, in order to evaluate the catalytic efficiency of the Co _x Cu_1–x S nanoflower, we investigated its steady-state kinetics at ambient temperature. First, we assessed the affinity of the catalyst and H₂O₂. To put it simply, 500 μL of Co _x Cu_1–x S (40 μg/mL) and buffer (pH 6.0) was taken and then fully blended with 500 μL of H₂O₂ of different concentrations (1–30 mM). Finally, 500 μL of TMB (2.2 mM) was added, and the mixture was reacted at ambient temperature. The kinetic alterations in the reaction system during the initial 600 s were monitored, with absorbance readings taken at 652 nm being documented. Employing the methodology previously outlined, we explored the interaction between the catalyst and TMB by adjusting the TMB concentration from 0.2 to 3.0 mM. Ultimately, we utilized the Michaelis–Menten equation to ascertain the kinetic parameters associated with the enzyme. ,

$$V_0=V_{\max }[\mathrm{S}]/(K_{\mathrm{m}}+[\mathrm{S}])$$

In this context, V ₀ denotes the starting velocity of the reaction, whereas V _max signifies the peak velocity achievable during the reaction. [S] refers to the concentration of the substance involved in the reaction, and K _m, commonly referred to as the Michaelis constant, represents the substrate concentration at 50 percent of the maximum reaction velocity.

Finally, in order to assess the production of reactive oxygen species during the course of the reaction, we performed a free radical trapping experiment. Specifically, we began by taking 500 μL of Co _x Cu_1–x S (40 μg/mL) and H₂O₂ (20 mM), which were thoroughly mixed with 250 μL of HAc-NaAc buffer (pH 6.0). Following this, we added 250 μL of the free radical quench agent, either PBQ or thiourea at the concentration of 1 mM, ensuring the mixture was well combined. Afterward, 500 μL of TMB (2.2 mM) was added, and the mixture was reacted for 10 min. The absorbance at a wavelength of 652 nm was recorded by utilizing a UV–vis spectrophotometer. To ensure the reliability of the results, each experimental trial was performed in triplicate.

2.6. Stability and Reusability of the Co _x Cu_1–x S Nanoflower

To evaluate the stability of the Co _x Cu_1–x S nanoflower, the reaction solution was thoroughly mixed and allowed to react at ambient temperature. The UV–vis spectrophotometer recorded the absorbance at 652 nm daily for 30 consecutive days. The reusability of the Co _x Cu_1–x S nanoflower was determined by measuring peroxidase-mimicking activity through five successive catalytic cycles. The mass loss observed during the centrifugal process was acceptable experimental tolerances.

2.7. Colorimetric Detection AA

First, 250 μL of AA at varying concentrations (0–1500 μm) was introduced into the mixed solution, comprising 500 μL of Co _x Cu_1–x S (40 μg/mL), 500 μL of H₂O₂ (20 mM), and 250 μL of buffer (pH 6.0). Following this, 500 μL of TMB (2.2 mM) was mixed with the above reaction system solution. After the mixture was fully shaken and mixed, the solution was allowed to sit for 10 min. UV–vis spectrophotometric measurements were performed at 652 nm. To ensure the reliability of the results, each trial was performed in triplicate.

Next, we examined the anti-interference capability of the Co _x Cu_1–x S nanoflower in detecting AA. Simply, 250 μL of metal interference ion solution (30 mM) was introduced into the mixed solution, comprising 500 μL of Co _x Cu_1–x S (40 μg/mL), 250 μL of H₂O₂ (20 mM), 250 μL of buffer (pH 6.0), and 250 μL of AA (1500 μm). After thoroughly mixing this solution, 500 μL of TMB (2.2 mM) was introduced into the above solution to fully react. Then, the solution absorbance at a wavelength of 652 nm was assessed utilizing a UV–vis spectrophotometer. Similarly, it was only necessary to substitute 250 μL of AA with 250 μL of various possible interfering molecular substances (9 mM), thereby assessing the selectivity of the Co _x Cu_1–x S nanoflower in detecting.

2.8. Detection of AA in Real Samples

To detect AA in the real samples, we bought grapefruit juice beverage in the supermarket, and it was diluted 1000-fold to be used as the real sample for testing. In simple terms, 500 μL of Co _x Cu_1–x S (40 μg/mL), 500 μL of H₂O₂ (20 mM), 250 μL of buffer (pH 6.0), 500 μL of TMB (2.2 mM), and 250 μL of the diluted grapefruit juice were fully mixed and shaken. Following the standard addition calibration protocol, AA at varying concentrations were introduced into the above reaction system. After it was placed and reacted for 10 min, we analyzed and computed the recovery rate of AA. To ensure the reliability of the results, each trial was performed in triplicate.

3. Results and Discussion

3.1. Characterization of the Co _x Cu_1–x S Nanoflower

As shown in Figure a–e, the Co _x Cu_1–x S nanoflower was successfully synthesized by the solvothermal-soaking method in this paper, through which the morphology design and doping regulation of transition metal sulfide were realized. The composite is uniform in size and well dispersed without obvious agglomeration (Figure a), indicating the versatility and maturity of the synthesis method. In Figure b, the Co _x Cu_1–x S nanozyme exhibits a nanoflower form assembled from ultrathin nanosheets, which provides a substantial specific surface area to expose the wealth of active sites. Further observation reveals that these nanosheets are cross-linked with each other to construct a large number of channels (Figure c), which contribute to the rapid penetration of the substrate solution and lay the structural foundation for them to exhibit excellent catalytic performance as a simulated enzyme. Similarly, transmission electron microscopy (TEM) images also confirm the nanoflower structure, whose particle size distribution is distributed within the range of 3–4 μm (Figure d,e). The nanosheets appear transparent under the transmission electron microscope (Figure f), which illustrates the ultrathin property of the Co _x Cu_1–x S nanoflower. This characteristic shortens the penetration distance of the electrolyte and accelerates the mass transfer process. In Figure g, the HRTEM image displays a series of regularly arranged lattice stripes, suggesting the nature as a single crystal, which is consisted with the selected area electron diffraction result (inset). After fitting, the crystal face spacing is 2.8 Å, which is identified as the crystal face (103) of the CuS hexagonal phase. Notably, the crystal plane spacing is slightly smaller than that of the CuS (103) crystal plane (2.81 Å), which may be due to the doping of Co atoms with a small atomic radius resulting in slight lattice shrinkage. This is indirect evidence for the introduction of Co atoms into the CuS lattice. Besides, some lattice disorder regions (dotted areas) are also observed in Figure g because the introduction of Co atoms with different atomic radius and electronic structure destroys the local long-range order of the CuS lattice. More importantly, these defect sites tend to possess higher surface energy and unsaturated hanging bonds because they often act as active sites to adsorb substrate molecules and improve catalytic activity.

1.

Synthesis diagram of the Co _x Cu_1–x S nanoflower.

2.

(a) SEM image of the large-scale Co _x Cu_1–x S nanoflower. (b) SEM image of a single Co _x Cu_1–x S nanoflower. (c) SEM image of the Co _x Cu_1–x S nanoflower at higher magnification. (d) TEM image of the large-scale Co _x Cu_1–x S nanoflower. (e) TEM image of a single Co _x Cu_1–x S nanoflower. (f) TEM image of the Co _x Cu_1–x S nanoflower at higher magnification. (g) HRTEM image of the Co _x Cu_1–x S nanoflower (the inset shows the selected electron diffraction). (h) EDS image of the Co _x Cu_1–x S nanoflower (the inset shows the atomic percentage). (i) HAADF-STEM image of the Co _x Cu_1–x S nanoflower. (j–m) Mapping images of different elements in the Co _x Cu_1–x S nanoflower.

The EDS image (Figure h) exhibits that it contains cobalt, copper, sulfur, and oxygen elements, suggesting the successful doping with Co atoms. Among them, the atomic percentage of Co/Cu is close to 1:9, which is consistent with the results of the ICP-OES test (Table S2). The HAADF-STEM image (Figure i) further confirms the microscopic morphology of the Co _x Cu_1–x S nanoflower, and the EDX-mapping images (Figure j–m) show that cobalt, copper, and sulfur elements are uniformly dispersed on the Co _x Cu_1–x S nanoflower without obvious phase separation and impurity phases. It is worth mentioning that Figure m depicts that the Co element is mainly distributed on the surface layer of the nanoflower, which acts as active sites. It is well known that the catalytic reactions mainly take place on the surface of catalysts. The Co _x Cu_1–x S nanoflower is rich in surface active sites and defect sites and is expected to exhibit significant catalytic activity.

Next, in the XRD pattern (Figure a) of the Co _x Cu_1–x S nanoflower, all diffraction peaks match well with CuS of the hexagonal phase (standard card no. 06-0464). No impurity phase is found, which suggests successful synthesis of the pure-phase Co _x Cu_1–x S nanoflower. Compared with the standard card of pure CuS, the diffraction peaks show a slightly positive shift. This phenomenon is attributed to the entry of small radius Co atoms into the lattice of CuS, which is consistent with the results of crystal plane spacing in HRTEM. In addition, the electron paramagnetic resonance (EPR) image (Figure b) shows a clear signal peak at g = 2.003, indicating that Co doping induces a large number of lattice defects, which aligns with the results obtained from HRTEM. As we all know, element doping can introduce numerous lattice defects, thereby adjusting the surface charge distribution of the catalyst and lowering the energy barrier of binding with the reaction substrate, which provides efficient catalytic activity. Therefore, the strategy of element doping imparts excellent simulated enzyme activity to the Co _x Cu_1–x S nanoflower. The Raman images of the Co _x Cu_1–x S nanoflower are exhibited in Figure c. The characteristic peaks at 188 cm^–1 and 465 cm^–1 are, respectively, indexed to the A_1g vibration mode of M–S bonds and the E_g vibration mode of S–S bonds. Moreover, the Raman peak at 511 cm^–1 is ascribed to the F_2g lattice vibration mode of CoS. Based on the above results, the successful synthesis of the Co _x Cu_1–x S nanoflower is adequately proved.

3.

(a) XRD image, (b) EPR image, (c) Raman image, (d) Co 2p XPS spectrum, (e) Cu 2p XPS spectrum (the inset shows the Cu LMM), and (f) S 2p XPS spectrum of the Co _x Cu_1–x S nanoflower.

To further study the surface valence state and composition of the elements in the Co _x Cu_1–x S nanoflower, we carried out XPS tests. In the XPS full spectrum of the Co _x Cu_1–x S nanoflower (Figure S1), it shows the presence of cobalt, copper, sulfur, carbon, and oxygen elements, among which oxygen and carbon elements are, respectively, derived from the unavoidable surface oxidation and the surface adsorption pollutants such as CO₂. Subsequently, further tests were performed on the cobalt, copper, and sulfur elements, and the XPS spectra of each element were collected, which were corrected relative to the peak (284.8 eV) in the C 1s XPS spectra (Figure S2). In the Co 2p spectrum depicted in Figure d, the peaks at 797.9 and 782.7 eV are, respectively, assigned to the spin–orbit splitting characteristics of Co 2p_1/2 and Co 2p_3/2, coupled with the dual satellite peaks on the high-energy side, which suggests that the valence state of cobalt mainly is the Co²⁺ in the Cu_0.4Co_0.6S nanoflower. In addition, the subtle peaks observed at 796.8 and 780.9 eV indicate the existence of Co³⁺, which is ascribed to the oxidation of surface upon exposure to air. The two main peaks of 952.6 eV (Cu 2p_1/2) and 932.5 eV (Cu 2p_3/2) are observed in the Cu 2p spectrum (Figure e), which is related to Cu²⁺ in the CuS material. The satellite peaks also explain the configuration of Cu²⁺ in the ground state d ⁹. Clearly, the Cu LMM Auger peak at 568.6 eV (the inset) further illustrates the presence of Cu­(II). Additionally, the Cu–O bond presents two deconvoluted shoulder peaks at 954.6 eV (Cu 2p_1/2) and 934.8 eV (Cu 2p_3/2) possibly from a small amount of CuSO₄. Toward to the S 2p core-level spectrum (Figure f), the presence of fitting peaks at 168.4 eV (S 2p_1/2) and 161.8 eV (S 2p_3/2) serves as a distinguishing feature of the sulfide, which is assigned to the S–M bond. The fitted peaks at 168.3 eV (S 2p_1/2) and 163.2 eV (S 2p_3/2) are associated with the sulfate, which is derived from the CuS exposed to air oxidation. Therefore, we confirm that the synthesized sample is a Co _x Cu_1–x S nanoflower according to the results above.

3.2. Peroxidase-like Activity of the Co _x Cu_1–x S Nanoflower

In recent years, numerous investigations have confirmed that the copper-based nanozyme exhibits peroxidase-like activity, which can decompose H₂O₂ to produce reactive oxygen species through the Fenton-like reaction, thus giving the copper-based nanozyme a wide range of applications in biosensing, food monitoring, drug safety, and other fields. − It can be seen that evaluating the peroxidase-like activity of the nanozyme is an important basis for further practical application. Therefore, in the presence of H₂O₂, the peroxidase-like activity of the Co _x Cu_1–x S nanoflower is measured by reaction with TMB. As illustrated in Figure a, the absorbance of Co _x Cu_1–x S + TMB + H₂O₂ exhibits a distinct peak at a wavelength of 652 nm, accompanied with the significant color change of the reaction solution. However, in the systems of Co _x Cu_1–x S + TMB, Co _x Cu_1–x S + H₂O₂, or TMB + H₂O₂, the absorbance at 652 nm is almost negligible, and there is no obvious change of solution color. By analyzing the above results, it can be found that the absorbance and color changed in the reaction solution may be related to the free radical cation and charge transfer action during the reaction process, which induces the oxidation of TMB to form ox-TMB. The absorbance change and blue coloration can be observed only in the Co _x Cu_1–x S + TMB + H₂O₂ system, while these phenomena do not occur in individual components (Co _x Cu_1–x S, H₂O₂, or TMB). Therefore, these results confirm that the Co _x Cu_1–x S nanoflower exhibits impressive peroxidase-like activity, capable of decomposing H₂O₂ to produce reactive oxygen species. This process triggers the oxidation of TMB, leading to the production of blue ox-TMB. As for the reasons, we analyze that the above phenomenon is related to the doping of the cobalt element, which alters the electronic structure of the nanozyme, optimizes the oxidation reduction capacity of active sites, creates new catalytic active sites to enhance the surface reactivity, etc. In addition, the anisotropic structure of the Co _x Cu_1–x S nanoflower modulates the direction of electron transport, improves the specific catalytic path, and reveals additional active sites due to the significantly increased specific surface area, thereby markedly enhancing the catalytic activity of Co _x Cu_1–x S. To explore the influence of different components on peroxidase activity, we also synthesized a series of Co _x Cu_1–x S nanoflowers, including CuS, Co _x Cu_1–x S-0.1, Co _x Cu_1–x S-0.3, Co _x Cu_1–x S-0.5, Co _x Cu_1–x S-0.7, Co _x Cu_1–x S-0.9, and pure CoS. The number symbols (0.1, 0.3, 0.5, ...) represent the doping amount, which is an approximate molar ratio of Co/(Co + Cu), determined by ICP-OES testing (Table S3). As shown in Figure S3, all of these materials have typical hexagonal crystal phases. Moreover, as the input ratio of Co increases, due to the increase in lattice defects and disorder, the crystallinity of the materials initially decreases and then increases, eventually gradually transitioning from CuS to CoS. Concurrently, the microstructural features have experienced subtle modifications. In Figure S4, with the increase of Co input, the thickness of the nanosheets gradually increases and tends to form hexagonal nanosheets. It is well known that catalytic reaction is the interfacial reaction. The thinner the nanosheets are, the more conducive it is to the exposure of active sites. It can be seen that the greater the input amount of Co is, not the better. As seen from Figure S5, the absorbance experiment also confirms this idea. Among the series of Co _x Cu_1–x S nanozymes, the absorbance value of Co _x Cu_1–x S-0.1 is the largest. A small and appropriate amount of Co not only provides abundant active sites, but also its appropriate amount of lattice defects avoids the sudden drop in stability caused by structure collapse. Based on the above results, we select the Co _x Cu_1–x S-0.1 nanoflower as the research object for further exploration in our subsequent work. Next, in order to observe whether the Co _x Cu_1–x S nanoflower is specific to a chromogenic substrate, we further select different chromogenic substrates for control study. As illustrated in Figure b, the absorbance spectrum of Co _x Cu_1–x S + H₂O₂ + TMB shows a pronounced peak at 652 nm, accompanied by the appearance of the blue solution. However, the above phenomenon is not observed in the Co _x Cu_1–x S + H₂O₂ + OPD or Co _x Cu_1–x S + H₂O₂ + ABTS group. The above results show that the Co _x Cu_1–x S nanoflower has certain specificity to the chromogenic substrate TMB. The reason may be that doping regulation induces the change of surface charge in the nanoflower active sites, thereby promoting the binding effect between the nanoflower and the electron-donating groups in TMB. In order to substantiate the aforementioned speculation, we proceeded to individually evaluate the zeta potential of the Co _x Cu_1–x S nanoflower in varying proportions by employing a dynamic light scattering apparatus. The zeta potential measurements (Figure S6) demonstrate that the Co _x Cu_1–x S-0.1 nanoflower exhibits the least surface charge (−14.5 mV) in comparison with alternative compositions. A lower zeta potential lessens the electrostatic repulsion between the surface of the nanoflower and the neutral TMB molecule, consequently enhancing the strength of their mutual adsorption. Consequently, the ideal charge equilibrium of Co_0.1Cu_0.9S facilitates its interaction with TMB, aligning with its peak peroxidase-like activity.

4.

(a) UV–visible absorption spectra of different reaction systems. (b) UV–visible absorption spectra of the Co _x Cu_1–x S nanoflower reacting with different chromogenic substrates (TMB, OPD, and ABTS). (c) UV–visible absorption spectra of the Co _x Cu_1–x S nanoflower reacting with different quenchers (PBQ and Thiourea). (d,e) EPR images of the Co _x Cu_1–x S nanoflower.

Subsequently, in order to determine the reactive oxygen species generated in the catalytic reaction process, we select different quenchers to conduct a free radical trapping experiment. The research confirms that quenchers of various reactive oxygen species are different. For example, the common quenchers for •OH and superoxide anion (O₂•^–) are separately thiourea and PBQ. , As shown in Figure c, when no quencher is added, the absorbance of the reaction solution appears as a pronounced peak at 652 nm, accompanied by color change of the reaction solution. Subsequently, after adding quenching agent PBQ or thiourea, we observe the absorbance at 652 nm and color of reaction solution. The experimental results exhibit that when PBQ is added, the peak absorbance at 652 nm is slightly decreased, and the reaction solution also changes from the previous dark blue to light blue. It can be seen that only a small amount of O₂•^– is generated in the catalytic reaction process of the Co _x Cu_1–x S nanoflower. However, when thiourea is added to the reaction system, the absorbance at 652 nm decreases sharply, and the original dark blue color of the reaction solution almost completely fades and disappears, which indicates that a large amount of •OH may be produced during the catalytic reaction process of the Co _x Cu_1–x S nanoflower. To further confirm the reactive oxygen species produced during the reaction, we also utilized EPR spectroscopy, which offered a more immediate explanation. As illustrated in Figure d,e, the four-line characteristic signal (1:2:2:1) representing •OH generation is extremely strong, while the six-line characteristic signal representing O₂•^– generation is weak. Therefore, the above results conclude that the Co _x Cu_1–x S nanoflower decomposes H₂O₂ to produce reactive oxygen species-dominant •OH and infinitesimal O₂•^– by the peroxidase-mimicking activity in the catalytic reaction process. They act as active oxygen to oxidize colorless TMB to blue ox-TMB, undergoing a color reaction.

3.3. Optimization of Catalytic Conditions

In a manner analogous to the natural enzyme, the catalytic efficacy of the nanozyme is additionally influenced by various reaction parameters, such as catalyst concentration, the pH of buffer, and the duration of the reaction, among others. Therefore, we further studied the influence conditions of Co _x Cu_1–x S nanoflower peroxidase-like activity, including catalyst concentration, TMB concentration, H₂O₂ concentration, reaction time, and buffer pH value. First, we studied how different concentrations of H₂O₂ affect the catalytic reaction. As illustrated in Figure S7a,b, we observed that the increase in H₂O₂ concentration resulted in a corresponding rise in absorbance at 652 nm. When the concentration of H₂O₂ reaches 18 mM, the rise rate of the absorbance gradually slows, indicating that the reaction basically reached saturation. We then investigated the influence of buffer pH on the catalytic reaction. As seen from Figure S7c,d, when the pH value is within the range of 4.4–6.0, the reaction activity is favorable, and the catalytic reaction achieves perfect activity when the pH value is 6.0. The results show that a weak acidic environment is more conducive to the catalytic reaction of the Co _x Cu_1–x S nanoflower, which is similar to the natural enzyme under physiological conditions. Consequently, it is also beneficial for the Co _x Cu_1–x S nanoflower to exert the corresponding biological applications under physiological conditions. Similarly, we next studied the influence of catalyst concentration, TMB concentration, and the duration of the catalytic reaction. As illustrated in Figure S7e–j, the reaction activity gradually tends toward saturation with the increase of substance concentration or reaction time, which is similar to the previously obtained results. Ultimately, according to the above experimental results, in order to ensure the accurate progress of the catalytic reaction, we separately selected H₂O₂ (20 mM), HAC-NaAc buffer (pH 6.0), TMB (2.2 mM), Co _x Cu_1–x S (40 μg/mL), and reaction time (10 min) as the optimal conditions for the catalyzed reaction.

3.4. Stability and Reusability of the Co _x Cu_1–x S Nanoflower

Stability and potential for reutilization are important indicators to assess the characteristics of a nanozyme, so we analyzed the stability and reusability of the Co _x Cu_1–x S nanoflower. As illustrated in Figure a, by monitoring the peroxidase-mimicking activity of the Co _x Cu_1–x S nanoflower for 30 consecutive days, it is found that the absorbance at 652 nm does not decrease sharply and basically remains in a relatively stable state, indicating that the Co _x Cu_1–x S nanoflower possesses excellent stability. The reason may be that the active site of the Co _x Cu_1–x S nanoflower is more fully protected after cobalt doping, which effectively improves its stability and corrosion resistance. Additionally, we conducted a more detailed characterization of the samples following the stability assessment. The XRD results (Figure S8) demonstrate that, even after the 30 day stability trial, the diffraction peaks exhibit neither substantial displacement nor alteration. This observation suggests that the crystal structure of the Co _x Cu_1–x S nanoflower remains stable. Meanwhile, the SEM image after the stability test (Figure S9) indicates that despite minor agglomeration, the flower-like structure composed of the nanosheets remains integrity. It robustly validates the structural stability of the material, providing a structural guarantee for its advancement and application in practical scenarios. In addition, the peroxidase-like activity of the Co _x Cu_1–x S nanoflower decreases slightly after 5 reaction cycles, but it can still maintain the activity at more than 85% (Figure b), indicating its outstanding reusability. All in all, the above experiments show that the Co _x Cu_1–x S nanoflower possesses remarkable stability and reusability, which laid a good foundation for biological application.

5.

(a) Stability test of Co _x Cu_1–x S nanoflower peroxidase-like activity for 30 days. (b) Reusability test of the Co _x Cu_1–x S nanoflower after five cycles.

3.5. Steady-State Kinetics of the Co _x Cu_1–x S Nanoflower

To better comprehend the peroxidase-like activity of the Co _x Cu_1–x S nanoflower, we then analyzed and studied the steady-state kinetics. First, we kept the concentration of TMB fixable to evaluate the affinity between the Co _x Cu_1–x S nanoflower and H₂O₂, by adjusting the concentration of H₂O₂ (1–30 mM). Figure S10 illustrates the characteristics curve of the Michaelis–Menten equation, accompanied by relevant double reciprocal plots. , Obviously, during the early phase of the reaction, the rate of the catalytic process escalates swiftly as the concentration of the substrate progressively rises. At the later phase of the reaction, the rate of catalytic reaction gradually stabilizes as the substrate concentration continues to increase, which also suggests that the catalytic reaction of the Co _x Cu_1–x S nanoflower is consistent with the typical Michaelis–Menten equation curve. As illustrated in Figure S10a,c, the K _m and V _max values of the Co _x Cu_1–x S nanoflower with H₂O₂ as a substrate are calculated, which are 3.569 mM and 7.080 × 10^–8 M/s, respectively. Among them, the K _m value signifies the substrate concentration at which the reaction rate attains 50 percent of the maximum velocity. A lower K _m value reflects a preferable interaction between the enzyme and substrate. In the present investigation, the K _m value of the Co _x Cu_1–x S nanoflower (3.569 mM) is lower than HRP (3.7 mM) with H₂O₂ as a substrate, which indicates that the Co _x Cu_1–x S nanoflower possesses better affinity with H₂O₂. For convenient comparison, the K _m values of some nanozymes utilizing H₂O₂ as a substrate are listed in Table S4. The V _max value represents the maximum reaction rate of the catalytic reaction. A larger V _max value reflects stronger reaction activity. In this study, the V _max value of the Co _x Cu_1–x S nanoflower (7.080 × 10^–8 M/s) is higher than majority of reported nanozymes with H₂O₂ as a substrate, indicating that the Co _x Cu_1–x S nanoflower possesses stronger reaction activity. Similarly, the V _max values of some nanozymes with H₂O₂ as a substrate are listed in Table S4 for convenient comparison. Subsequently, by adopting the same method, we kept the concentration of H₂O₂ fixable to evaluate the affinity between the Co _x Cu_1–x S nanoflower and TMB through adjusting the concentration of TMB (0.2–3.0 mM). As shown in Figure S10b,d, the K _m and V _max values of the Co _x Cu_1–x S nanoflower with TMB as a substrate are 0.404 mM and 8.246 × 10^–8 M/s, respectively. The results also show that the Co _x Cu_1–x S nanoflower possesses desirable affinity and catalytic activity with TMB. At the same time, the K _m and V _max values of some nanozymes with TMB as a substrate are also listed in Table S4 for better comparative analysis. Based on the above results, we speculate that introduction of cobalt as a dopant may adjust the electronic state of the active site within the nanoflower, making it more similar to the active center of the natural enzyme, thereby enhancing substrate recognition. In addition, the nanoflower-like structure assembled by ultrathin nanosheets greatly increases the effective contact area between the enzyme and the substrate, enabling the substrate more accessible to the active site. Therefore, all the above reasons may confer the excellent affinity between the Co _x Cu_1–x S nanoflower and substrate.

3.6. Co _x Cu_1–x S Nanoflower Colorimetric Detection of AA

AA is a prevalent antioxidant founded within the organism, mainly ingested from external fruits or vegetables. The stability of the AA concentration is crucial for maintaining numerous physiological and biochemical functions within the organism. When the AA content in the organism is either excessive or insufficient, it will lead to the occurrence and development of various illnesses, including cardiovascular disease, Alzheimer’s disease, neoplastic disease, etc. − Therefore, accurate, sensitive, and rapid detection of AA concentration holds substantial importance for the diagnosis and prevention of disease as well as other fields of biological safety. Leveraging the remarkable peroxidase-mimicking properties of the Co _x Cu_1–x S nanoflower, combined with the antioxidant capability of AA, we constructed a colorimetric assay for the detection of AA utilizing the Co _x Cu_1–x S nanoflower. As illustrated in Figure S11, the introduction of AA into the reaction system demonstrates its ability to compete with the chromogenic substrate TMB, thus inhibiting the peroxidase-catalyzed reaction. It can prevent the formation of the colored product ox-TMB in the Fenton-like reaction, thereby achieving the effect of the antioxidant reaction and enabling the quantitative detection of AA. Figure a demonstrates that as the concentration of AA increases within the Co _x Cu_1–x S nanoflower reaction system, there is a gradual decrease in the absorbance of the reaction mixture at 652 nm. This decline in absorbance is paralleled by a noticeable change in the hue of the reaction solution, which is transitioned from dark blue to a clear and transparent appearance. The absorbance intensity and AA concentration present a robust linear relationship within the range of 50–1500 μm (Figure b). The linear equation derived from this correlation is expressed as ΔA = 0.0012CAA + 0.1946 (R ² = 0.997) (ΔA represents the absorbance difference at 652 nm between the absence and presence of AA). Meanwhile, the limit of detection (LOD) is 1.11 μm by equation 3θ/k (θ represents the standard deviation of the blank sample and k represents the slope of the calibration curve). To better compare the AA detection capabilities of different nanozymes, we summarized the detection range and LOD of some reported nanomaterials in Table S5. By comparing and analyzing Table S5, we could suggest that the detection range and LOD of the Co _x Cu_1–x S nanoflower was superior to the majority of reported nanomaterials. Consequently, the Co _x Cu_1–x S nanoflower exhibits remarkable sensitivity and reliability in the colorimetric detection of AA, which lays a theoretical foundation for further practical application in the biosensing.

6.

(a) UV–visible absorption spectra and color change of the Co _x Cu_1–x S nanoflower sensor system for detecting AA at different concentrations. (b) Linear curve relationship of the Co _x Cu_1–x S nanoflower sensor system for detecting AA at different concentrations. (c) Anti-interference ability of the Co _x Cu_1–x S nanoflower for detecting AA. (d) Selectivity of the Co _x Cu_1–x S nanoflower for detecting AA.

Anti-interference and selectivity are important evaluators of biosensing. Therefore, to better assess the performance of the Co _x Cu_1–x S nanoflower for detecting AA, the anti-interference and selectivity of the Co _x Cu_1–x S nanoflower are further analyzed. As illustrated in Figure c, the addition of various interfering ions to the reaction system containing AA results in minimal changes in the absorbance at 652 nm or the color of the reaction solution. Notably, this observation remains consistent despite the concentration of the interfering ions being more than 20-fold greater than that of AA. It can be concluded that the Co _x Cu_1–x S nanoflower for detecting AA exhibits good anti-interference. Next, we analyze the selectivity of this biosensor system by replacing AA with various possible interfering molecules. As shown in Figure d, significant change of absorbance difference and complete fading of the solution color occur only in the presence of AA. Remarkably, when AA is replaced by various possible interfering molecules, the absorbance difference at 652 nm or the color of reaction solution appear slightly fluctuated or faded in some groups, but the effect is relatively weak. We speculate that the reason for the above phenomenon may be attributed to the limited reducibility of certain interfering molecules, variation in reaction mechanisms, differences in reaction kinetics, and the capabilities of electron transfer. , To sum up, the Co _x Cu_1–x S nanoflower sensor system for detection of AA exhibits excellent anti-interference and selectivity, which is conducive to further practical application of colorimetric detection of AA.

3.7. Co _x Cu_1–x S Nanoflower Colorimetric Detection of AA in the Real Samples

To further investigate the feasibility of the Co _x Cu_1–x S nanoflower sensor system for detecting AA, we detected AA in grapefruit juice beverages. As shown in Table S6, we utilized the standard addition technique to introduce varying concentrations of AA into the diluted grapefruit juice beverage, and then the recovery rate was analyzed to evaluate the performance of the Co _x Cu_1–x S nanoflower sensor system for detecting AA. The experimental results show that the recovery rate ranges from 94.60 to 104.66%, while the relative standard deviation is observed to be between 0.40 and 3.46%. According to the above experimental results, we can suggest that the sensor system of the Co _x Cu_1–x S nanoflower for detecting AA in real samples exhibits satisfactory recovery. Therefore, this sensor system possesses excellent potential for practical application in the future.

4. Conclusion

In conclusion, a novel Co _x Cu_1–x S nanoflower material with excellent peroxidase-mimicking activity is synthesized through element doping and morphology regulation in this study. Subsequently, the morphology characterization and peroxidase-mimicking activity are further confirmed by a series of experiments. Based on the remarkable peroxidase-mimicking activity of the Co _x Cu_1–x S nanoflower, we construct a colorimetric sensor system for quick and sensitive detection of AA by the naked eye, which exhibits satisfactory anti-interference and selectivity. In recent years, it is noteworthy that colorimetric biosensors, facilitated by smartphone technology, have garnered significant interest. Users are now able to effortlessly quantify various substances in samples by observing the colorimetric changes in the reaction solution through a dedicated smartphone application. Therefore, this research opens new avenues for the utilization of nanomaterials in biomedicine or other domains and simultaneously provides a research basis for further in-depth exploration of the translational application of nanomaterials.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c05494.

• XPS full spectrum of the Co _x Cu_1–x S nanoflower; XPS image of C 1S in the Co _x Cu_1–x S nanoflower; XRD image of the Co _x Cu_1–x S nanoflower with different proportions; SEM image of the Co _x Cu_1–x S nanoflower with different proportions; UV–visible absorption spectra of the Co _x Cu_1–x S nanoflower with different proportions; zeta potential measurements of the Co _x Cu_1–x S nanoflower; optimization of various reaction conditions; XRD image of the Co _x Cu_1–x S nanoflower after the stability test; SEM image of the Co _x Cu_1–x S nanoflower after the stability test; steady-state kinetics of the Co _x Cu_1–x S nanoflower; proposed reaction mechanism of the Co _x Cu_1–x S nanoflower for detecting AA; experimental parameters in the synthesis process; elemental contents of the as-prepared samples measured by ICP-OES and elemental analysis; summary of ICP-OES results for Co _x Cu_1–x S nanozymes; comparison of steady-state kinetic parameters between the Co _x Cu_1–x S nanoflower and other reported nanozymes; comparison of the Co _x Cu_1–x S nanoflower and other reported nanozymes for detecting AA; and Co _x Cu_1–x S nanoflower detection of AA in grapefruit juice (n = 3) (PDF)

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Y.L. and Y.C. contributed equally to this paper. Y.L.: data curation, conceptualization, formal analysis, methodology, funding acquisition, writingoriginal draft, and writingreview and editing. Y.C.: data curation, writingoriginal draft, investigation, and resources. J.G.: methodology and formal analysis. C.Z.: data curation and methodology. C.X.: funding acquisition, resources, supervision, and validation. L.L.: formal analysis, data curation, funding acquisition, and writingreview and editing. All authors have read and agreed to the published version of the manuscript.

The authors declare no competing financial interest.