High-performance colorimetric sensor based on PtRu bimetallic nanozyme for xanthine analysis

Abstract

The identification and quantification of xanthine are crucial for assessing the freshness and quality of food products, particularly in the seafood industry. Herein, a new approach was developed, involving the in-situ controllable growth of Pt₉₁Ru₉ nanoparticles on graphitic carbon nitride to yield Pt₉₁Ru₉@C₃N₄ catalytic materials. By integrating Pt₉₁Ru₉@C₃N₄ with the xanthine/xanthine oxidase (XOD) enzyme catalytic system, a nanozyme-enzyme tandem platform was obtained for the quantification analysis of xanthine. Under the catalytic oxidation of xanthine by XOD in the presence O₂, H₂O₂ was generated. Upon the addition of peroxidase-like activity of Pt₉₁Ru₉@C₃N₄, H₂O₂ can be decomposed into •OH and ¹O₂, which can further catalyze the oxidation of TMB to its oxidation product oxTMB with an absorption peak at 652 nm. This smartphone-assisted portable colorimetric sensor for visual monitoring xanthine with a low detection limit of 8.92 nmol L⁻¹, and successfully applied to detect xanthine in grass carp and serum samples.

Highlights

• •Pt₉₁Ru₉@C₃N₄ were prepared through a chelating co-reduction method.
• •The resultant Pt₉₁Ru₉@C₃N₄ exhibited a robust peroxidase-like activity.
• •An enzyme-nanozyme cascade sensor was constructed for xanthine analysis.
• •An ultralow detection limit of 8.92 nmol L⁻¹ was achieved.
• •Offers satisfactory analytical performance of grass carp and serum samples.

1. Introduction

In the food industry, maintaining the freshness of fish meat is paramount for ensuring the production of safe and high-quality products. After the death of fish, adenosine triphosphate (ATP) within their bodies degrades successively into adenosine diphosphate (ADP), adenosine 5′-monophosphate (AMP), inosine 5′-phosphate (IMP), inosine (In), hypoxanthine (HX) and xanthine (Dervisevic et al., 2015) (Fig. S1). The concentration of xanthine increased with the prolonged storage time of fish meat (Tripathi et al., 2022). Monitoring the concentration of xanthine in fish meat is regarded as essential means for assessing its freshness (Xu et al., 2024). Xanthine is a critical intermediate in purine nucleotides and deoxyribonucleotides metabolism, with widespread distribution in the organs and bloodstream of humans and other organisms (Jiang et al., 2024). The abnormal levels of xanthine in the blood and urine are also associated with various diseases, such as hyperuricemia, cerebral ischemia, tumors and renal failure (An et al., 2022; Lu et al., 2020). Thus, the development of sensitive, reliable, and rapid sensors for the monitoring and quantification of xanthine is vital for the clinical diagnosis and industrial production. Several analytic al strategies have so far been developed for xanthine analysis, including colorimetry, voltammetry, and fluorescence techniques (Jesny & Girish Kumar, 2017; Lu et al., 2020; Wang et al., 2022). Among these, the colorimetric approach is more practical due to its rapid response and simplicity. According to previous studies, integrating nanozyme-mediated colorimetric sensing strategies with bio-enzyme could establish enzyme-nanozyme cascade sensing platforms to yield sensing systems with improved sensitivities and broadened determination range (Li, Xie, et al., 2024; Song et al., 2023).

Nanozymes are nanoscale materials with inherent enzyme-mimicking activity, exhibiting both the structural properties of nanomaterials and catalytic functionalities of bio-enzymes (Qiao et al., 2023; Zou et al., 2023). Despite the existence of numerous excellent materials, the development of nanozymes with the characteristics of high stability and robust catalytical performance of is still the focus and hotspot of current research (Geng et al., 2024). To achieve satisfactory catalytical activities, continual adjustments of structural and morphological characteristics of nanozymes are necessary. Among regulatory strategies, elemental doping can effectively modulate the electronic structure and geometry of nanozymes by introducing specific elements into nanomaterials, thereby regulating the catalytic performance of the nanomaterials (Guan et al., 2023; Song et al., 2022). For instance, nanozymes containing metal Pt and Ru as active centers exhibit robust catalytic properties with widespread application in analytical sensing (Liu et al., 2020; Wang et al., 2023). For example, Li et al. (2023) successfully prepared Au–Pt nanoclusters with robust catalytic performance for use as an efficient catalytic label in the development of a colorimetric sensing platform for the highly sensitive analysis of D-dimer. Park et al. (2021) synthesized PVP-stabilized Pt/Ru nanozymes with synergistic enhancement effects for dual-mode colorimetric and fluorescent analysis of glucose in artificial urine and serum samples. Accordingly, the design and development of nanozymes with robust peroxidase-mimic activities for the construction of enzyme-nanozyme cascade colorimetric sensing platforms could achieve sensitive and rapid analysis of xanthine.

Herein, a new approach was developed, involving the in-situ controllable growth of Pt₉₁Ru₉ nanoparticles on graphitic carbon nitride to yield Pt₉₁Ru₉@C₃N₄. The resulting Pt₉₁Ru₉@C₃N₄ exhibited robust peroxidase-mimic activity with a satisfactory affinity toward TMB (0.16 mmol L⁻¹), and the synergistic interplay between Pt and Ru effectively promoted the catalytic activity of Pt₉₁Ru₉@C₃N₄ at pH 6. The combination of the prepared Pt₉₁Ru₉@C₃N₄ with xanthine oxidase (XOD) resulted in a nanozyme-natural nanozyme tandem platform with remarkable sensitivity toward the quantification analysis of xanthine (Scheme 1). With the catalysis of XOD, xanthine was oxidized by O₂ dissolved in the solution to generate H₂O₂ (Fig. S2). H₂O₂ was then decomposed into •OH and ¹O₂ by Fenton-like reaction using the peroxidase-like activity of Pt₉₁Ru₉@C₃N₄. The generated ROS species quickly initiated the chromogenic reaction of 3,3′,5,5′-tetramethylbenzidine (TMB). Changes in absorption signals demonstrated excellent analytical performance of the Pt₉₁Ru₉@C₃N₄-based colorimetric sensing platform for xanthine in real urine and serum samples, with a low detection limit of 8.92 nmol L⁻¹. Meanwhile, the recording of RGB values of color changes at various xanthine concentrations established a relationship between xanthine and color characteristic values for on-site and quantitative analysis of xanthine. In sum, the proposed approach for designing efficient bimetallic nanozymes is potential for future construction of enzyme-nanozyme cascade sensing systems for visual analysis of relevant analytes in food and biological systems.

Scheme 1.

Illustration of constructing Pt₉₁Ru₉@C₃N₄-based biosensor for xanthine assay.

2. Experimental

2.1. Chemicals and apparatus

Ruthenium (III) chloride trihydrate (RuCl₃·3H₂O), chloroplatinic acid hexahydrate (H₂PtCl₆·6H₂O), 3,3′,5,5′-tetramethylbenzidine (TMB), and sodium borohydride (NaBH₄) were bought from Sigma-Aldrich (Shanghai, China). Melamine, isopropyl alcohol (IPA), p-benzoquinone (p-BQ), sodium azide (NaN₃), sodium acetate (NaAc), acetic acid (HAc), hydrogen peroxide (H₂O₂), and sodium citrate dihydrate (SCD) were purchased from Beijing Chemical Works. Xanthine oxidase (XOD), xanthine, uric acid, and urea were bought from Yuanye Biotechnology Co., Ltd. (Shanghai, China). All individual solutions were prepared with 18 MΩ deionized water.

UV–vis measurements were recorded on a SHIMADZU UV-2700 UV–visible spectrophotometer. The surface element compositions and valence states of Pt₉₁Ru₉@C₃N₄ was analyzed using an X-ray photoelectron spectrometer (XPS) (ThermoFischer，ESCALAB Xi+). Field-emission scanning electron microscopy (SEM, FEI Nova Nano-450), transmission electron microscopy (TEM, Talos F200X) and elemental mapping images were utilized for the surface morphology and structural characterizations. Zeta potential of C₃N₄ and Pt₉₁Ru₉@C₃N₄ were carried out on a Zeta-sizer (Nano-S90, Malvern, UK). X-ray diffraction (XRD) analysis was achieved on a SmartLab SE (Japan Rigaku Co.).

2.2. Synthesis of C₃N₄ nanosheets

Firstly, bulk C₃N₄ was prepared using a previously reported method (Wang et al., 2020). 20 g of melamine was calcined at 550 °C for 4 h in a tube furnace. After cooling down to 25 °C, a light-yellow C₃N₄ solid was obtained. To prepare C₃N₄ nanosheets, 1 g of bulk C₃N₄ powder was dispersed in 120 mL of 5 mol·L⁻¹ HNO₃ and refluxed for 24 h. The resulting product underwent several washes with deionized water until achieving a nearly neutral pH. Finally, the C₃N₄ suspension was sonicated and centrifuged at 8000 rpm for 12 min to remove unexfoliated nanosheets, followed by freeze-drying to obtain monodisperse C₃N₄ nanosheets.

2.3. Synthesis of Pt₃₉Ru₆₁@C₃N₄(39:61), Pt₈₅Ru₁₅@C₃N₄(85:15), Pt₉₁Ru₉@C₃N₄(91:9), and Pt₉₃Ru₇@C₃N₄(93:7)

Pt_xAu_y@C₃N₄ catalysts with variable Pt/Au mass ratios were prepared through a facile co-reduction process using sodium borohydride (NaBH₄) and sodium citrate tribasic dihydrate (C₆H₅Na₃O₇·2H₂O) at room temperature. The synthesis process of Pt₉₁Ru₉@C₃N₄ consisted of first mixing 0.33 mL of RuCl₃·3H₂O (38.60 mmol L⁻¹) with 1.67 mL of H₂PtCl₆·6H₂O (38.60 mmol L⁻¹). Subsequently, 16 mL of an aqueous solution containing C₃N₄ (1.25 mg mL⁻¹) was added to the mixture, and the entire solution was stirred for 10 min. After stirring well, 4 mL of C₆H₅Na₃O₇·2H₂O (6 mg mL⁻¹) was added dropwise to the suspension, followed by 20 mL of NaBH₄ (1.25 mg mL⁻¹), and the co-reduction reaction continued for 12 h (Jiang et al., 2022). The resulting crude product was filtered, washed by alternately ethanol and H₂O, and dried at 65 °C for 15 h to obtain the pure product Pt₉₁Ru₉@C₃N₄. The synthesis of Pt₃₉Ru₆₁@C₃N₄, Pt₈₅Ru₁₅@C₃N₄ and Pt₉₃Ru₇@C₃N₄ was carried out by the same methodology employed for Pt₉₁Ru₉@C₃N₄, with variations in the quantities of added metal precursor solutions. The two control samples, Pt@C₃N₄ and Ru@C₃N₄ were prepared by an identical method, omitting the addition of RuCl₃·3H₂O-water solution and H₂PtCl₆·6H₂O-water solution, respectively.

2.4. Kinetics assays of Pt₉₁Ru₉@C₃N₄

The kinetic assays of Pt₉₁Ru₉@C₃N₄ were assessed using TMB and H₂O₂ as substrates. TMB is widely preferred as a chromogenic substrate for both enzyme-catalyzed reactions and nanomaterial catalytic systems due to its high sensitivity, excellent color purity, and the stability of oxidation products (Wu et al., 2019; Zhang et al., 2020). To achieve optimal catalytical performance, various factors including temperature, reaction time, and contents of H₂O₂ and Pt₉₁Ru₉@C₃N₄, were systematically optimized. Under the optimal conditions, the steady-state kinetics of Pt₉₁Ru₉@C₃N₄ were conducted in 0.01 mol L⁻¹ HAc-NaAc buffer (pH 5) containing 2.5 μg mL⁻¹ Pt₉₁Ru₉@C₃N₄. For H₂O₂ concentration maintained at 0.5 mmol L⁻¹, TMB was varied from 0.05 to 0.3 mmol L⁻¹ to yield final concentrations of 0.05, 0.075, 0.1, 0.125, 0.175, 0.2, 0.25, and 0.3 mmol L⁻¹. Likewise, H₂O₂ was adjusted in the range of 0.1–0.8 mmol L⁻¹ to yield final concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.8 mmol L⁻¹ at a constant TMB concentration of 0.2 mmol L⁻¹.

The UV–vis absorbances at 652 nm were measured 30 s post-reaction. Finally, the K_m and V_max values were obtained through double-reciprocal Lineweaver-Burk plots using the following equation (Chai et al., 2023; Yang, Yuan, et al., 2024):

$$\frac{1}{V_0}=\frac{K_{\mathrm{m}}}{V_{\max }}\left(\frac{1}{\left[S\right]}+\frac{1}{K_{\mathrm{m}}}\right)$$

where V₀ denotes the initial rate, V_max is the maximal reaction velocity, [S] refers to the substrate concentration, and K_m denotes the Michaelis constant.

2.5. Xanthine analysis

The process consisted of introducing 200 μL of xanthine concentration (0–300 μmol L⁻¹) and 40 μL XOD (20 U mL⁻¹) into 50 μL phosphate buffer solution (PBS, 0.1 mol L⁻¹, pH 8) at 37 °C. After 25 min of incubation, 50 μL Pt₉₁Ru₉@C₃N₄ (0.6 mg mL⁻¹), 300 μL PBS (pH 5, mol L⁻¹) and 200 μL TMB (2 mmol L⁻¹) were introduced, and the mixture was incubated at 45 °C for an additional 10 min. Afterward, 1160 μL H₂O were added to the mixture and the absorbance-concentration curves were recorded in the range of 500–800 nm.

2.6. Xanthine analysis with a smartphone

To achieve portable xanthine analysis, the RGB color signal was directly recorded and analyzed by a smartphone application (Chen et al., 2023). The samples to be tested were prepared by the same procedure as described in the xanthine colorimetric assay of Section 2.5, followed by transfer to a 24-well microplate. Under consistent lighting conditions, the smartphone captured a colorimetric image of the solution, followed by subsequent analysis using ImageJ to obtain the grayscale value.

2.7. Analysis of xanthine in real sample

The practicality of the Pt₉₁Ru₉@C₃N₄/TMB/XOD-based colorimetric sensor was validated in grass carp and blood samples. Blood samples were obtained from the Third Bethune Hospital of Jilin University, and the grass carp were purchased from a local seafood market. The serum sample processing method can be found in our previous work (Zhang et al., 2019). The serum samples were filtered using a 0.22 μm filter membrane, followed by centrifugation at 11,000 rpm for 10 min. The supernatant was collected and neutralized with NaOH to a neutral pH. Then, the neutral serum sample was diluted 50-fold with deionized water, and the xanthine solutions were added to achieve the final xanthine concentrations of 0, 20, 60, and 100 μmol L⁻¹. Subsequently, the analysis of xanthine in serum samples was conducted as described in Section 2.5.

The live grass carp were purchased and then euthanized using the electrical stunning method by the skilled seller. Percussive stunning was performed by trained staff to minimize stress reactions. Following euthanasia, the fish were immediately immersed in an ice slurry within a styrofoam box. This box, containing the euthanized fish, was promptly transported to the laboratory and processed under sterile conditions to prepare fish meat samples. In the laboratory, the killed fish was scaled, gutted, decapitated, and rinsed in flowing tap water to remove the blood and viscera (Xu et al., 2024; Zhang et al., 2018). This process strictly adhered to the guidelines recommended by the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Subsequently, the fresh fish meat was ground into a homogenate, then the resulting homogenate was combined with ultra-pure water at a ratio of 1:3 (w/w). After filtering through a filter paper, the fish homogenate was mixed with varying contents of xanthine to create spiked samples with final concentrations of 0, 20, 60, and 100 μmol L⁻¹. The correlation between the increase in xanthine concentration and the degree of fish spoilage (Dervisevic et al., 2015) prompted us to conduct xanthine analysis on grass carp samples stored at 4 °C for different durations (0, 96, and 168 h). Finally, the analysis of xanthine in serum and grass carp samples was conducted following the above-outlined procedure (Section 2.5).

3. Results and discussions

3.1. Fabrication and characterization of Pt₉₁Ru₉@C₃N₄

In this study, PtRu@C₃N₄ was prepared through the mild co-reduction process shown in Fig. 1a. The experiments revealed a platinum-to‑ruthenium mass ratio of 91:9 as optimal to yield exceptional catalytical performance of PtRu nanoparticles, thereby used in subsequent experiments (Fig. S3). As illustrated in Fig. S4, the zeta potential of Pt₉₁Ru₉@C₃N₄ was estimated to be −20.75 ± 0.77 mV, a value slightly more positive than that of C₃N₄ (−21.65 ± 0.35 mV). The notable change in surface charge was probably due to the presence of positively charged metal ions in Pt₉₁Ru₉@C₃N₄ (Shen et al., 2023). Pt₉₁Ru₉@C₃N₄ with negatively charged surface, exhibits favorable affinity for TMB (Zhou, Chen, et al., 2022), which contains two amine groups, through electrostatic attraction. The robust affinity between the catalyst and its substrate contributes to the improved catalytic efficiency of the catalyst. The microstructure of Pt₉₁Ru₉@C₃N₄ was characterized by transmission electron microscopy (TEM). As displayed in Fig. 1b-e, uniformly distributed PtRu nanoparticles with an average size of approximately 4.14 nm were formed on the entire surface of C₃N₄. The lattice distance of Pt₉₁Ru₉@C₃N₄ was determined to be approximately 0.22 nm, implying successfully prepared catalytic material (Li et al., 2023). From Fig. 1f, the Pt/Ru atomic fraction ratio in Pt₉₁Ru₉@C₃N₄ estimated by energy dispersive spectroscopy (EDS) to be 4.23:1. The selected area electron diffraction (SAED) patterns in Fig. 1g revealed Pt₉₁Ru₉@C₃N₄ featuring a face-centered cubic lattice structure with four rings corresponding to (111), (200), (220) and (311) crystal planes (from inner to outer rings). The images captured by EDS mapping in Fig. 1h showed the elements Pt, Ru, and N were uniformly distributed in the Pt₉₁Ru₉@C₃N₄ nanozyme, which demonstrated that PtRu was evenly modified on C₃N₄ nanosheets.

Fig. 1.

(a) Illustration of Pt₉₁Ru₉@C₃N₄ synthesis. (b-d) TEM images of Pt₉₁Ru₉@C₃N₄. (e) Size distribution of Pt₉₁Ru₉@C₃N₄. (f) EDS analysis of Pt₉₁Ru₉@C₃N₄. The inset shows the atomic and mass proportions of Pt and Ru. (g) SAED pattern and (h) element mapping of Pt₉₁Ru₉@C₃N₄.

The X-ray diffraction (XRD) patterns of various catalysts, including Pt@C₃N₄, Pt₃₉Ru₆₁@C₃N₄, Pt₈₅Ru₁₅@C₃N₄, Pt₉₁Ru₉@C₃N₄, Pt₉₃Ru₇@C₃N₄, and Ru@C₃N₄ are presented in Fig. 2a. The peaks at 2θ values of 39.7°, 46.3°, 67.4°, and 80.3° corresponded to the (111), (200), (220) and (311) planes, respectively. No obvious Ru or related oxide diffraction peaks were observed, indicating no phase separation in the Pt_xRu_y@C₃N₄. In Fig. 2b, the broad XRD peaks of all samples were observed in the 2θ angle range between 36.5° and 48°. As RuCl₃·3H₂O content rose, the diffraction peak of the (111) plane gradually sharpened, suggesting variation in the Pt–Ru ratio within the Pt–Ru alloy nanostructure. Thus, the alloying degree of PtRu@C₃N₄ can be effectively controlled by varying the quantity of Pt–Ru metal precursor, consistent with the SAED analysis observed in Fig. 1g.

Fig. 2.

(a) XRD patterns of the Pt@C₃N₄, Pt₉₃Ru₇@C₃N₄, Pt₉₁Ru₉@C₃N₄, Pt₈₅Ru₁₅@C₃N₄, Pt₃₉Ru₆₁@C₃N₄ catalysts. (b) Magnified XRD profiles of all samples in the 2θ angle range between 36.5 and 48°. (c) XPS spectra of Pt₉₁Ru₉@C₃N₄. (d-f) Pt 4f, Ru 3p and N 1 s XPS high-resolution spectra of Pt₉₁Ru₉@C₃N₄.

The elemental composition and valence state of Pt₉₁Ru₉@C₃N₄ were explored by X-ray photoelectron spectroscopy (XPS). In Fig. 2c, elements like Pt, Ru, N, and O were presented in the wide-scan XPS of Pt₉₁Ru₉@C₃N₄. In Fig. 2d, the Pt 4f segment showed the existence of Pt species in the Pt⁰/ Pt²⁺ state (Liu et al., 2020). In Fig. 2e, the peaks at 462.04 and 465.09 eV corresponded to Ru⁰ and Ru⁴⁺ species, respectively, based on the spin-orbit splitting of Ru 3p_3/2. The others at 484.48 and 487.14 eV were ascribed to Ru⁰ and Ru⁴⁺ of Ru 3p_5/2, respectively. Besides, Pt₉₁Ru₉@C₃N₄ contained Pt/Ru at an atomic ratio of 6.63:1, suggesting a strong alloy effect (Table S1). The N 1 s spectrum in Pt₉₁Ru₉@C₃N₄ (Fig. 2f) can be fitted into five nitrogen species, corresponding to pyridinic N (398.47 eV), M-N_x (399.02 eV), pyrrolic N (400.32 eV), graphic N (401.39 eV) and oxidized N (404.51 eV) (Lyu et al., 2024). The presence of M-N_x peaks signified an effective association between metal atoms and N atoms. Such interactions enhanced the stability of metal atoms on the support owing to the superior adsorption ability of pyridine nitrogen in forming coordination bonds with metal atoms (Wang et al., 2021).

3.2. Peroxidase activity of Pt₉₁Ru₉@C₃N₄

The peroxidase activity of Pt₉₁Ru₉@C₃N₄ was systematically explored. As illustrated in Fig. 3a, the presence of H₂O₂ and Pt₉₁Ru₉@C₃N₄ resulted in TMB oxidation to oxTMB to yield a deep blue color. By comparison, no significant absorption peak was observed in the presence of only Pt₉₁Ru₉@C₃N₄, C₃N₄, or H₂O₂. The absorbance value at 652 nm with Pt₉₁Ru₉@C₃N₄ was higher than those observed with Pt@C₃N₄ and Ru@C₃N₄ (Fig. 3b), indicating superior catalytic activity of Pt₉₁Ru₉@C₃N₄. These features elucidated the presence of a synergistic effect between the metallic Pt and Ru, leading to the superior catalytic performance of Pt₉₁Ru₉@C₃N₄. The TMB oxidation reactions at varied Pt₉₁Ru₉@C₃N₄ contents were determined by recording the time-dependent changes in UV–vis absorbance at 652 nm. As depicted in Fig. 3c, the rate of TMB oxidation increased as a function of the Pt₉₁Ru₉@C₃N₄ concentration from 5 to 25 μg mL⁻¹, with a plateau observed at 15 μg mL⁻¹. The catalytic conditions of Pt₉₁Ru₉@C₃N₄ were also estimated under variable pH and temperature conditions. In Fig. S5a and b, pH 5 HAc-NaAc buffer and 45 °C were determined as the optimal conditions for the Pt₉₁Ru₉@C₃N₄/TMB/ H₂O₂ colorimetric system.

Fig. 3.

(a) UV–vis spectra in various reaction systems (blue line: Pt@C₃N₄/TMB/H₂O₂, green line: Ru@C₃N₄/TMB/H₂O₂, fuchsia line: Pt₉₁Ru₉@C₃N₄/TMB). (b) UV–vis spectra of TMB/H₂O₂, C₃N₄/TMB/H₂O₂, Pt₉₁Ru₉@C₃N₄/TMB, Pt₉₁Ru₉@C₃N₄/TMB/H₂O₂. (c) The time-dependent absorbance curves for varying concentrations of Pt₉₁Ru₉@C₃N₄ in the TMB/H₂O₂ system (Pt₉₁Ru₉@C₃N₄ concentration from down to top: 5, 10, 15, 20 and 25 μg mL⁻¹). ESR spectra for trapping (d) ·OH and (e) ¹O₂ in the DMPO/Pt₉₁Ru₉@C₃N₄ aqueous solution and TEMP/Pt₉₁Ru₉@C₃N₄ aqueous solution, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Radical scavenging trials were then conducted to confirm the species of ROS. As shown in Fig. S6a, the absorbance of Pt₉₁Ru₉@C₃N₄/TMB/H₂O₂ remained nearly unchanged upon the addition of sodium azide (NaN₃), while a noticeable decline was observed after the introduction of p-benzoquinone (p-BQ), indicating superoxide radical (¹O₂) as the active intermediate in Pt₉₁Ru₉@C₃N₄/H₂O₂ (Yang, Bi, et al., 2024; Yang, Yuan, et al., 2024). Terephthalic acid (TA) was then utilized for additional confirmation of the presence of hydroxyl radicals (·OH) (Zhou, Chen, et al., 2022; Zhou, Wang, et al., 2022). As proved by Fig. S6b, the enhanced fluorescence signals affirmed the existence of ·OH in the Pt₉₁Ru₉@C₃N₄/H₂O₂ system. Therefore, Pt₉₁Ru₉@C₃N₄ contributed to the generation of the radical species of •OH and ¹O₂. Electron spin resonance (ESR) spectroscopy was conducted to further examine the active substances generated during the reaction. Using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the scavenger, EPR detected a significant •OH signal with an intensity ratio of 1:2:2:1 (Wang et al., 2024), as shown in Fig. 3d. Similarly, when 2,2,6,6-tetramethylpiperidine (TEMP) was employed, characteristic peaks of ¹O₂ were observed with an intensity ratio of 1:1:1 (Fig. 3e). These findings indicated that both •OH and ¹O₂ participated in the catalytic reaction, which was consistent with the results of free radical scavenging experiments (Fig. S6a and b).

The steady-state kinetic analysis of Pt₉₁Ru₉@C₃N₄ was conducted using the double-fitting method (Li, Chen, et al., 2024; Li, Xie, et al., 2024). The calculated kinetic constants K_m and V_max of Pt₉₁Ru₉@C₃N₄ from Fig. 4a-d were (0.26 mmol L⁻¹, 4.93 × 10⁻⁷ mol L⁻¹ s⁻¹) for TMB and (1.01 mmol L⁻¹, 7.19 × 10⁻⁷ mol L⁻¹ s⁻¹) for H₂O₂, respectively. Hence, Pt₉₁Ru₉@C₃N₄ exhibited excellent enzymic kinetic properties, comparable to other reported nanozymes (Table S2). Moreover, the absorbance intensity of Pt₉₁Ru₉@C₃N₄/TMB gradually increased with H₂O₂ concentration (Fig. 4e) and maintained an excellent linear relationship with xanthine concentration from 1 to 200 μmol L⁻¹ (Fig. 4f) (Y = 0.0058[H₂O₂] + 0.18, μmol L⁻¹, R² = 0.999). An LOD of 0.39 μmol L⁻¹ was obtained, indicating superior sensitivity of Pt₉₁Ru₉@C₃N₄ when compared to other catalytic materials (Table S3). The reliable sensing performance of Pt₉₁Ru₉@C₃N₄ toward H₂O₂ revealed promising distinctive colorimetric biosensor for the analysis of H₂O₂ and substances capable of generating H₂O₂.

Fig. 4.

(a) The influence curves of the H₂O₂ (d, e) and TMB (f, g) concentrations on the catalytic rate of peroxidase, respectively. (h) UV–vis spectra of Pt₉₁Ru₉@C₃N₄/TMB following addition of varied contents of H₂O₂. Inset: corresponding snapshots of the test solution. (i) Linear calibration plot of absorption values with varied contents of H₂O₂.

3.3. Colorimetric sensor for xanthine analysis

The robust peroxidase-mimicking activity of Pt₉₁Ru₉@C₃N₄ was used to construct a biosensing platform for the analysis of xanthine. The feasibility of the proposed Pt₉₁Ru₉@C₃N₄-based colorimetric sensing strategy was then evaluated. As depicted in Fig. 5a, faint color changes and weak absorbance signals were observed using Pt₉₁Ru₉@C₃N₄/TMB, Pt₉₁Ru₉@C₃N₄/TMB/xanthine, and Pt₉₁Ru₉@C₃N₄/TMB/XOD, separately. As expected, the addition of xanthine and XOD into the Pt₉₁Ru₉@C₃N₄/TMB system resulted in increased absorbance at 652 nm, confirming feasible Pt₉₁Ru₉@C₃N₄-based colorimetric sensor for the determination of xanthine.

Fig. 5.

(a) UV–vis spectra of Pt₉₁Ru₉@C₃N₄/TMB, Pt₉₁Ru₉@C₃N₄/TMB/xanthine, Pt₉₁Ru₉@C₃N₄/TMB/XOD and Pt₉₁Ru₉@C₃N₄/TMB/XOD/xanthine (Inset: corresponding color changes). (b) UV–vis spectra of Pt₉₁Ru₉@C₃N₄/TMB/XOD following addition of varied contents of xanthine. Inset: corresponding snapshots of the test solution. (c) Linear calibration plot of absorption values with varied contents of xanthine. (d) The Pt₉₁Ru₉@C₃N₄/TMB/XOD-based sensing platform based on a smartphone for xanthine analysis and corresponding images of the colorimetric system toward varied xanthine concentrations. (f) Selectivity investigation of the Pt₉₁Ru₉@C₃N₄-based colorimetric probe for xanthine analysis. [Alanine] = [Proline] = [Serine] = [Threonine] = [Uric acid] = [Urea] = 1.20 mmol L⁻¹; [ALP] = 20 μg mL⁻¹; [Xanthine] = 0.20 mmol L⁻¹; [Cu²⁺] = [K⁺] = [NO₃⁻] = 1 mmol L⁻¹.

Before xanthine analysis, the reaction conditions of xanthine were optimized to promote the sensitivity of the proposed method, and 0.40 U L⁻¹ XOD was selected for xanthine analysis (Fig. S7). Under pH 8 and 37 °C (Wang et al., 2022), the absorbance signal of Pt₉₁Ru₉@C₃N₄/TMB/XOD rose as a function of xanthine content (0–300 μmol L⁻¹) (Fig. 5b). The absorbance signal showed a correlation with the xanthine content with satisfactory linearity from 0.05 to 150 μmol L⁻¹ (Y = 0.0024[xanthine] + 0.34, μmol L⁻¹, R² = 0.991, inset in Fig. 5b) and a LOD of 8.92 nmol L⁻¹ (Fig. 5c). In addition, results of xanthine analysis compared well with previously reported methods for xanthine measurement (Table S4), verifying the usefulness of Pt₉₁Ru₉@C₃N₄/TMB/XOD-based colorimetric sensor for xanthine analysis.

Based on the above colorimetric sensing strategy, a proof-of-concept smartphone-assisted analysis was developed. As illustrated in Fig. 5d, the color gradient changed from light blue to deep blue as the xanthine concentration increased. A linear correlation was observed from 0.05 to 150 μmol L⁻¹ between RGB values and xanthine concentration, with I = 0.0024[xanthine] + 1.01 (μmol L⁻¹, R² = 0.997), and a LOD of 17.51 nmol L⁻¹ (Fig. 5e). Thus, the smartphone-readout analysis provided a visual and equipment-free ease-operating on-site quantitative analysis of xanthine.

3.4. Selectivity and spiked sample analysis

Selectivity is crucial for evaluating sensor performance. Thus, the absorbance signals of Pt₉₁Ru₉@C₃N₄/TMB/XOD system toward various interference substances were investigated and the results are compared in Fig. 5f. Obviously, the influence of alanine, proline, serine, threonine, uric acid, urea, ALP and common ions (Cu²⁺, K⁺, and NO₃⁻) on the absorbance intensity of Pt₉₁Ru₉@C₃N₄/TMB/XOD can be disregarded, while xanthine showed significantly enhanced absorbance intensity. These results confirmed the satisfactory selectivity of the Pt₉₁Ru₉@C₃N₄-based colorimetric probe for xanthine determination.

The practicality of the Pt₉₁Ru₉@C₃N₄-based sensor for colorimetric analysis of xanthine was accessed through spiked recovery experiments. The detailed analysis data of serum and grass carp samples exhibited recoveries between 93.95% and 116.50%, with relative standard deviations of less than 3.78% (Table S5 and Table S6), indicating the suitability of Pt₉₁Ru₉@C₃N₄-based colorimetric sensor for analysis of xanthine with reliable performance in real samples. Further, a reported analytical standard method was employed for xanthine determination and compared with our colorimetric sensor. In the sensing strategy (Zhang et al., 2022), Fe/N-CDs was employed as a fluorescence indicator and catalyst for the oxidation reaction between OPD and H₂O₂. And a fluorescence/colorimetric dual-mode sensing platform was developed for xanthine analysis based on the FRET between the oxidation product of oxOPD and Fe/N-CDs. As shown in Fig. S8, the results of xanthine analysis using the Pt₉₁Ru₉@C₃N₄-based colorimetric sensor were very close to the values obtained with the Fe/N-CDs-based method (Table S5 and 6), indicating the satisfactory accuracy and reliability of the Pt₉₁Ru₉@C₃N₄-based method.

4. Conclusions

In summary, the Pt₉₁Ru₉@C₃N₄ with satisfactory catalytic performance was successfully synthesized through a room-temperature co-reduction strategy. By combining the peroxidase-mimicking activity of Pt₉₁Ru₉@C₃N₄ and the xanthine/XOD enzyme catalytic system, an efficient, sensitive, and reliable nanozyme-enzyme tandem platform was designed for xanthine analysis. With the assistance of a smartphone app, the Pt₉₁Ru₉@C₃N₄/TMB/XOD-based colorimetric sensor achieved convenient and quantitative visual analysis of xanthine without using sophisticated instruments. The proposed colorimetric sensor had been successfully utilized for xanthine analysis in grass carp and serum samples. Overall, a new synthetic approach for the rational design of bimetallic and lamellar nanozymes with efficient catalytical performance was proposed along with insights into the construction of enzyme-nanozyme cascade sensors with high response to target analytes.

CRediT authorship contribution statement

Mengjun Wang: Supervision, Funding acquisition, Conceptualization. Minghang Jiang: Data curation. Xiaojun Luo: Formal analysis. Liyun Zhang: Methodology. Yi He: Methodology, Investigation. Fanjie Xue: Software, Resources. Xingguang Su: Supervision, Funding acquisition.

Declaration of competing 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.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.