A BiVO4 sensor array and its application in the detection of anthrax biomarkers and analogues

Abstract

This invention relates to the field of bioassay materials technology, and more particularly to a BiVO4 sensor array and its application in the detection of anthrax biomarkers and their analogues. This invention utilizes vacancy-regulated bismuth vanadate nanomaterials to construct a BiVO4 sensor array, achieving efficient differentiation and detection of anthrax biomarkers and their structural analogues, and highly selective identification of DPA and its analogues. This provides a reliable and flexible method platform for the rapid detection of anthrax biomarkers, without relying on complex instruments or cumbersome sample pretreatment steps, and shows great application potential.

Description

Technical Field

[0001] This invention relates to the field of bioassay materials technology, and in particular to a BiVO4 sensor array and its application in the detection of anthrax biomarkers and analogues. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In current anthrax detection practices, gene sequencing, PCR amplification, and ELISA immunoassay are the mainstream clinical diagnostic methods. However, these methods generally have limitations such as long operation cycles, reliance on specialized equipment, and high-cost reagents. Pyridine-2,6-dicarboxylic acid (DPA) is a major component of Bacillus anthracis spores, and other common spores do not contain DPA. Therefore, DPA can serve as a biomarker for Bacillus anthracis.

[0004] Currently established DPA analysis techniques encompass various approaches, including mass spectrometry, colorimetry, electrochemical sensing, surface-enhanced Raman spectroscopy, and fluorescence detection. Among these, colorimetric sensing strategies based on color changes have gained widespread attention due to their intuitive operation and ease of implementation. However, conventional colorimetric sensors are susceptible to matrix interference in complex samples, especially when substances with similar molecular structures to DPA are present, often affecting selectivity. Summary of the Invention

[0005] In view of this, the present invention provides a BiVO4 sensor array and its application in the detection of anthrax biomarkers and analogues.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a BiVO4 sensor array, the BiVO4 sensor array comprising three sensing elements, the three sensing elements being r-BiVO4, m-BiVO4 and p-BiVO4 respectively.

[0007] Among various vanadium-based nanomaterials, two-dimensional bismuth vanadate nanosheets possess advantages such as atomic-level thickness, high specific surface area, and tunable electronic properties. However, the catalytic efficiency of original two-dimensional nanosheets in practical applications still has room for improvement. By constructing cation vacancies in materials through defect engineering, these vacancy structures can not only enhance the adsorption and activation efficiency of reactant molecules but also adjust the local charge distribution of the material, strengthening the electron transfer efficiency of active sites and thus improving the overall redox reaction activity. Colorimetric sensor array technology differs from traditional colorimetric methods based on a "one-to-one" specific recognition mechanism. This technology constructs a multi-element cross-response system, integrating multiple sensitive units and pattern recognition algorithms (such as linear discriminant analysis and hierarchical clustering analysis), thereby achieving precise identification of structurally highly similar components in complex samples. This design strategy significantly improves the sensor array's ability to distinguish similar structural interferences and enables simultaneous identification of multiple targets.

[0008] This invention prepares various BiVO4 materials with tunable cation vacancy concentrations. These vacancies not only modulate their peroxidase-like catalytic activity but also endow them with differentiated binding capabilities to analytes with different structures. By detecting the specific response patterns of different pyridine carboxylic acids on nanozymes with surface defects, this system can generate distinctive "fingerprint" signals for each analyte. This vacancy-engineered multichannel sensing strategy can achieve highly selective recognition of DPA and its analogues in complex real-world samples, thus providing a reliable and flexible method platform for the rapid detection of anthrax biomarkers without relying on complex instruments or cumbersome sample pretreatment steps.

[0009] Furthermore, the BiVO4 sensor array can be used to detect pyridine carboxylic acid compounds (PAs); the PAs include DPA, 2,3-pyridinedicarboxylic acid (2,3-PA), 3,4-pyridinedicarboxylic acid (3,4-PA), 2,4-pyridinedicarboxylic acid (2,4-PA), 2-pyridinecarboxylic acid (2-PA), 3-pyridinecarboxylic acid (3-PA), and 4-pyridinecarboxylic acid (4-PA).

[0010] Further, the preparation method of the r-BiVO4 is as follows: bismuth chloride, hexadecyltrimethylammonium bromide and ethylene glycol are dissolved in proportion, sodium dodecyl vanadate solution is added under stirring, and hydrothermal reaction is carried out at 155-165 °C for 2.9-3.1 hours. After the reaction is completed, the mixture is cooled, centrifuged, washed and dried to obtain the product.

[0011] Further, the preparation method of m-BiVO4 is as follows: bismuth chloride, hexadecyltrimethylammonium bromide and ethylene glycol are dissolved in proportion, sodium dodecyl vanadate solution is added under stirring, and the reaction is carried out at 135-145 °C for 4.9-5.1 hours in a solvothermal manner. After the reaction is completed, the mixture is cooled, centrifuged, washed and dried to obtain the product.

[0012] Further, the preparation method of the p-BiVO4 is as follows: bismuth chloride, hexadecyltrimethylammonium bromide and ethylene glycol are dissolved in proportion, sodium dodecyl vanadate solution is added under stirring, and hydrothermal reaction is carried out at 115-125 °C for 8.9-9.1 hours. After the reaction is completed, the mixture is cooled, centrifuged, washed and dried to obtain the p-BiVO4.

[0013] Furthermore, the ratio of bismuth chloride, hexadecyltrimethylammonium bromide to ethylene glycol is 2.21 g : 1.05 g : 55-65 mL.

[0014] Furthermore, the mass ratio of bismuth chloride to sodium dodecyl vanadate is 2.21:2.8; the molar ratio of bismuth chloride to sodium dodecyl vanadate is 1:1.

[0015] Furthermore, the washing process involves washing with deionized water and anhydrous ethanol 2-4 times in sequence.

[0016] By adjusting the temperature and time conditions of the solvothermal reaction, BiVO4 with different vacancy concentrations can be prepared.

[0017] Secondly, the present invention provides the application of the BiVO4 sensor array described in the first aspect in the detection of anthrax biomarkers and analogues.

[0018] Furthermore, the anthrax biomarker and its analogues are at least one of DPA, 2,3-PA, 3,4-PA, 2,4-PA, 2-PA, 3-PA, and 4-PA.

[0019] Furthermore, the detection concentration of anthrax biomarkers and analogues is 10-100 µM.

[0020] Thirdly, the present invention provides a method for detecting anthrax biomarkers and analogues, comprising the following steps: r-BiVO4, m-BiVO4 and p-BiVO4 were added to sodium acetate-acetic acid buffer solution, followed by the introduction of anthrax biomarker and analogue solution, and co-incubation. After incubation, TMB solution was added first, followed by hydrogen peroxide solution, and then incubated again to obtain a mixed system. The absorbance value of the mixed system was tested, and the absorbance change was calculated using a sample without analyte as a blank control. r-BiVO4, m-BiVO4 and p-BiVO4 were used as independent sensing channels to detect the analyte solution, and five parallel experiments were performed for each concentration. By integrating test data, a three-dimensional data matrix containing sensor channels, analyte concentrations, and the number of parallel experiments is constructed. This data matrix is ​​then processed to establish a recognition model. Based on the analysis of the detection data of the sample under test using the recognition model, the qualitative and quantitative identification of analytes in the sample under test can be realized.

[0021] Furthermore, the anthrax biomarker and its analogues are at least one of DPA, 2,3-PA, 3,4-PA, 2,4-PA, 2-PA, 3-PA, and 4-PA.

[0022] Furthermore, the concentrations of r-BiVO4, m-BiVO4, and p-BiVO4 were 0.095–0.105 mg / mL.

[0023] Furthermore, the co-incubation time is 8-12 minutes; the subsequent incubation time is 48-52 seconds.

[0024] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention constructs a BiVO4 sensor array by fusing vacancy-regulated bismuth vanadate nanomaterials with a sensor array, enabling efficient differentiation and detection of anthrax biomarkers and their structural analogs. The vacancy-regulated bismuth vanadate nanomaterials not only modulate their peroxidase-like catalytic activity but also endow them with differentiated binding capabilities to analytes with different structures, achieving highly selective recognition of DPA and its analogs. This provides a reliable and flexible method platform for the rapid detection of anthrax biomarkers, eliminating the need for complex instruments or cumbersome sample pretreatment steps. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 These are scanning electron microscope images of BiVO4 samples prepared according to embodiments of the present invention; wherein, (A) is r-BiVO4; (B) is m-BiVO4; and (C) is p-BiVO4. Figure 2 These are transmission electron microscope (TEM) images of BiVO4 samples prepared according to embodiments of the present invention; wherein, (A) is r-BiVO4; (B) is m-BiVO4; and (C) is p-BiVO4. Figure 3 This is an X-ray diffraction pattern of the BiVO4 sample prepared in an embodiment of the present invention; Figure 4 This is an X-ray photoelectron spectrum of the BiVO4 sample prepared in the embodiments of the present invention; wherein, (A) is r-BiVO4; (B) is m-BiVO4; and (C) is p-BiVO4. Figure 5 This is a peroxidase-like activity characterization diagram of the BiVO4 sample prepared in the embodiments of the present invention; wherein, (A) is the activity characterization diagram of the sample at pH 1. Characteristic absorption curves of TMB, TMB with H2O2, TMB with nanozyme, and TMB with H2O2 and nanozyme in different reaction systems after catalytic oxidation in the NaAc / HAc buffer system of 4.0. (B) is the reaction mechanism diagram. (C) is the Michaelis-Menten kinetic curve of r-BiVO4 with substrate H2O2 and the corresponding double reciprocal plot. (D) is the Michaelis-Menten kinetic curve of m-BiVO4 with substrate H2O2 and the corresponding double reciprocal plot. (E) is the Michaelis-Menten kinetic curve of p-BiVO4 with substrate H2O2 and the corresponding double reciprocal plot. (F) is the Michaelis-Menten kinetic curve of r-BiVO4 with substrate TMB and the corresponding double reciprocal plot. (G) is the Michaelis-Menten kinetic curve of m-BiVO4 with substrate TMB and the corresponding double reciprocal plot. (H) is the Michaelis-Menten kinetic curve of p-BiVO4 with substrate TMB and the corresponding double reciprocal plot. Figure 6 This is a catalytic effect diagram of the BiVO4 sample prepared in the embodiments of the present invention; wherein, (A) is a graph showing the change trend of fluorescence intensity at 450 nm wavelength with the increase of reaction time in the reaction system composed of TPA, H2O2 and r-BiVO4; (B) is a graph showing the UV-Vis absorption spectrum of the coexistence system of NBT, H2O2, NADH and r-BiVO4 at different reaction times; and (C) is a graph showing the relationship between r-BiVO4 concentration and UV-Vis absorption spectrum in the DPBF and H2O2 system. Figure 7 This is a feasibility diagram of the colorimetric determination method for BiVO4 samples prepared in the embodiments of the present invention; wherein, (A) a comparison of the typical absorption spectrum of r-BiVO4 and the absorption spectrum of the oxidation reaction of TMB and H2O2 catalyzed by r-BiVO4 and DPA after co-incubation for 10 minutes; (B) a comparison of the typical absorption spectrum of m-BiVO4 and the absorption spectrum of the oxidation reaction of TMB and H2O2 catalyzed by m-BiVO4 and DPA after co-incubation for 10 minutes; (C) a comparison of the typical absorption spectrum of p-BiVO4 and the absorption spectrum of the oxidation reaction of TMB and H2O2 catalyzed by p-BiVO4 and DPA after co-incubation for 10 minutes; and (D) the absorption spectra of the reaction of DPA and its structural analogs with TMB and H2O2 in the presence of m-BiVO4. Figure 8This invention relates to the identification of various pyridinecarboxylic acids by a BiVO4 sensor array prepared in this embodiment. Specifically, (A1) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 10 μM; (A2) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 25 μM; (A3) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 40 μM; (A4) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 55 μM; (A5) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 70 μM; (A6) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 85 μM; (A7) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 100 μM; and (B1) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 10 μM. (B2) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 25 μM for the BiVO4 sensor array prepared in this embodiment of the invention; (B3) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 40 μM for the BiVO4 sensor array prepared in this embodiment of the invention; (B4) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 55 μM for the BiVO4 sensor array prepared in this embodiment of the invention; (B5) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 70 μM for the BiVO4 sensor array prepared in this embodiment of the invention; (B6) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 85 μM for the BiVO4 sensor array prepared in this embodiment of the invention; (B7) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 100 μM for the BiVO4 sensor array prepared in this embodiment of the invention. Radar graphs of colorimetric sensor array responses to different PAs at μM concentrations: (C1) Linear relationship between DPA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention; (C2) Linear relationship between 4-PA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention; (C3) Linear relationship between 3-PA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention; (C4) Linear relationship between 3,4-PA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention; (C5) Linear relationship between 2-PA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention; (C6) Linear relationship between 2,4-PA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention.(C7) Linear relationship between 4-PA concentration and the first discriminant factor score; Linear relationship between 2,3-PA concentration and the first discriminant factor score of the BiVO4 sensor array prepared in this embodiment of the invention; Figure 9 This is a diagram showing the identification of a mixture of pyridine carboxylic acids by the BiVO4 sensor array prepared in an embodiment of the present invention. Detailed Implementation

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0029] Example 1 Synthesis of r-BiVO4, m-BiVO4, and p-BiVO4 Bismuth chloride (2.21 g) and hexadecyltrimethylammonium bromide (1.05 g) were dissolved in ethylene glycol (60 mL). Sodium orthovanadate dodecahydrate (2.8 g) was slowly added with stirring. After complete mixing, the mixture was transferred to a sealed reactor and reacted at 160 °C for 3 hours. After the reaction was completed and cooled, the mixture was centrifuged and washed three times successively with deionized water and anhydrous ethanol. Finally, it was vacuum dried at 60 °C to obtain r-BiVO4 powder.

[0030] Bismuth chloride (2.21 g) and hexadecyltrimethylammonium bromide (1.05 g) were dissolved in ethylene glycol (60 mL). Sodium dodecapate dodecahydrate (2.8 g) was slowly added with stirring. After complete mixing, the mixture was transferred to a sealed reaction vessel and reacted at 140 °C for 5 hours. After the reaction was completed and cooled, the mixture was centrifuged and washed three times successively with deionized water and anhydrous ethanol. Finally, it was vacuum dried at 60 °C to obtain m-BiVO4 powder.

[0031] Bismuth chloride (2.21 g) and hexadecyltrimethylammonium bromide (1.05 g) were dissolved in ethylene glycol (60 mL). Sodium orthovanadate dodecahydrate (2.8 g) was slowly added with stirring. After complete mixing, the mixture was transferred to a sealed reactor and reacted at 120 °C for 9 hours. After the reaction was completed and cooled, the mixture was centrifuged and washed three times successively with deionized water and anhydrous ethanol. Finally, the mixture was vacuum dried at 60 °C to obtain p-BiVO4 powder.

[0032] Example 2 This embodiment provides a BiVO4 sensor array and its detection method. The BiVO4 sensor array includes three sensing elements: r-BiVO4, m-BiVO4, and p-BiVO4.

[0033] In a typical anthrax biomarker detection experiment, 200 μL of r-BiVO4, m-BiVO4, or p-BiVO4 suspension (1 mg / mL) was added to 1.3 mL of sodium acetate / acetic acid buffer solution at pH 4.0. Then, 100 μL of DPA or PA solutions of different concentrations (concentration gradient: 10, 25, 40, 55, 70, 85, 100 μM / L) were introduced, and the mixtures were incubated for 10 minutes. After incubation, 200 μL of 5 mM / L TMB solution was added, followed immediately by 200 μL of 1 mM / L hydrogen peroxide solution, and incubation was continued for 50 seconds. To quantify the analyte response, the change in absorbance difference (ΔA) at 652 nm was measured between the BiVO4 nanozyme-TMB colorimetric system containing different concentrations of DPA / PA and the blank control. The signal output was calculated as the difference absorbance, ΔA = A. sample - A blank Each BiVO4 nanozyme can constitute an independent sensing channel (channels 1, 2, and 3). Five replicate experiments were performed for each DPA or PA concentration to obtain multidimensional detection data and ensure data accuracy. A three-dimensional data matrix (3 channels × 7 analyte concentrations × 5 replicates) was constructed, and linear discriminant analysis (LDA) was performed using SPSS statistical software. Finally, concentration-dependent curves for DPA or PA were plotted based on the scores obtained from the first canonical discriminant function in the LDA model.

[0034] Results Characterization and Performance Testing (1) Morphological characterization of BiVO4 nanosheets Figure 1 These are scanning electron microscope images of BiVO4 samples prepared according to embodiments of the present invention; wherein, (a) is r-BiVO4; (b) is m-BiVO4; and (c) is p-BiVO4. Figure 2 These are transmission electron microscope (TEM) images of BiVO4 samples prepared according to embodiments of the present invention; wherein, (a) is r-BiVO4; (b) is m-BiVO4; and (c) is p-BiVO4. Figure 1 and Figure 2 The morphological characterization results of the prepared bismuth vanadate nanosheets are presented. Scanning electron microscopy images show that all three samples (r-BiVO4, m-BiVO4, and p-BiVO4) exhibit typical two-dimensional thin-layer structures with smooth surfaces and clear edges. Transmission electron microscopy images further reveal their ultrathin characteristics; the samples have good overall light transmittance and uniform thickness, presenting a typical single-layer or few-layer two-dimensional nanosheet morphology.

[0035] (2) XRD characterization of BiVO4 nanosheets Figure 3 This is an X-ray diffraction pattern of the BiVO4 sample prepared in an embodiment of the present invention. The crystal structure and phase purity of the three BiVO4 samples were analyzed using PXRD. Figure 3 It can be seen that all samples exhibit characteristic diffraction peaks at positions such as 2θ ≈ 25.40°, 28.43°, 29.73°, 33.12°, 46.98°, 54.55°, and 55.41°, which correspond to the crystal planes (111), (013), (004), (113), (024), (215), (311), and (323) in the standard card (JCPDS 85-1730) of the orthorhombic phase BiVO4, confirming that the three materials have the same crystal structure and space group. It is worth noting that, unlike the sharp diffraction peaks that usually appear, the diffraction peaks of these three samples all exhibit a certain degree of broadening. This broadening phenomenon mainly originates from lattice defects and microstrain within the materials, indicating that there are different degrees of crystal structure defects in different samples caused by differences in the synthesis process.

[0036] (3) XPS characterization of BiVO4 nanosheets Figure 4 These are X-ray photoelectron spectra of the BiVO4 samples prepared in this embodiment of the invention; where (a) represents r-BiVO4; (b) represents m-BiVO4; and (c) represents p-BiVO4. The elemental composition and chemical valence states of the materials were systematically analyzed using X-ray photoelectron spectroscopy (XPS), such as... Figure 4 As shown, only bismuth (Bi), oxygen (O) and vanadium (V) signal peaks were detected in all samples, and no characteristic peaks of impurity elements such as chlorine (Cl) were observed, indicating that high-purity BiVO4 phase was successfully synthesized.

[0037] (4) Peroxidase-like activity test of BiVO4 nanosheets Figure 5These are peroxidase-like activity characterization diagrams of BiVO4 samples prepared in the embodiments of the present invention; wherein, (A) are characteristic absorption curves after catalytic oxidation of different reaction systems in a NaAc / HAc buffer system at pH 4.0, including TMB, TMB and H2O2, TMB and nanozyme, and TMB and H2O2 and nanozyme; (B) is a reaction mechanism diagram; (C) is the Michaelis-Menten kinetic curve of r-BiVO4 on substrates H2O2 and TMB; (D) is the Michaelis-Menten kinetic curve of m-BiVO4 on substrates H2O2 and TMB; (E) is the Michaelis-Menten kinetic curve of p-BiVO4 on substrates H2O2 and TMB; (F) is the double reciprocal plot corresponding to the Michaelis-Menten kinetic curve of r-BiVO4; (G) is the double reciprocal plot corresponding to the Michaelis-Menten kinetic curve of m-BiVO4; and (H) is the double reciprocal plot corresponding to the Michaelis-Menten kinetic curve of p-BiVO4.

[0038] Based on the structural characteristics of bismuth vanadate, the peroxidase-like activity of three BiVO4 materials was evaluated by catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. The TMB oxidation product (ox-TMB) exhibited a characteristic blue absorption peak at 652 nm. Figure 5 As shown in (A), under acidic conditions, the solutions all exhibited a distinct blue color when r-BiVO4, m-BiVO4, or p-BiVO4 were added alone to the H2O2-TMB system; in contrast, no significant color change was observed in the control groups (r-BiVO4, m-BiVO4, p-BiVO4 with TMB alone, or H2O2 with TMB only). Notably, the absorbance of the m-BiVO4 catalytic system was higher than that of the p-BiVO4 system, while the r-BiVO4 system exhibited the highest absorbance, indicating that BiVO4 materials possess excellent peroxidase-like activity. Figure 5 (B) Furthermore, the introduction of vanadium vacancies can regulate its catalytic activity, which provides favorable conditions for constructing a colorimetric sensor array.

[0039] Michaelis constant reflects the affinity between enzyme and substrate, and the catalytic performance of the three materials was evaluated through kinetic analysis. Figure 5The initial reaction rate in (C)-(H) followed typical Michaelis-Menten curve characteristics with respect to substrate (TMB, H2O2) concentrations. The Michaelis constant (Km) and maximum reaction rate (Vm) were calculated using the Lineweaver-Burk equation. The results showed that the Km values ​​for r-BiVO4, m-BiVO4, and p-BiVO4 with respect to TMB were 0.11 mM, 0.27 mM, and 0.69 mM, respectively, and the Km values ​​for H2O2 were 0.096 mM, 0.11 mM, and 0.47 mM, respectively. The significant differences in Km values ​​indicate different enzyme-like activities among the three materials. Notably, r-BiVO4, with its higher vanadium vacancy concentration, exhibited the lowest Km values ​​for both substrates, indicating a positive correlation between catalytic activity and vanadium vacancy concentration.

[0040] (5) Catalytic mechanism of BiVO4 nanosheets To clarify the catalytic and sensing mechanisms of BiVO4 nanozymes, this study systematically investigated the types of reactive oxygen species generated during the catalytic process. Previous reports indicate that peroxidase-like activity originates from the nanozyme's decomposition of H₂O₂ into reactive oxygen species such as hydroxyl radicals, superoxide radicals, and singlet oxygen, which then oxidize colorless TMB to the blue oxidation product ox-TMB. To identify the main reactive oxygen species involved in this process, we selected terephthalic acid, nitroblue tetrazolium, and 1,3-diphenylisobenzofuran as probe molecules specifically for detecting hydroxyl radicals, superoxide radicals, and singlet oxygen. Figure 6 This is a catalytic effect diagram of the BiVO4 sample prepared in the embodiments of the present invention; wherein, (A) is a graph showing the change trend of fluorescence intensity at 450 nm wavelength with increasing reaction time in the reaction system composed of TPA, H2O2 and r-BiVO4; (B) is a graph showing the UV-Vis absorption spectrum of the coexisting system of NBT, H2O2, NADH and r-BiVO4 at different reaction times; and (C) is a graph showing the relationship between r-BiVO4 concentration and UV-Vis absorption spectrum in the DPBF and H2O2 system. Figure 6 As shown in (B)-(C), in the reaction systems containing NBT, NADH, H2O2, and BiVO4, and those containing DPBF, H2O2, and BiVO4, the absorbance changes during the reaction were not significant, indicating that superoxide radicals and singlet oxygen contributed very little to the catalytic process. In contrast, the system containing TPA, H2O2, and BiVO4 exhibited a significant fluorescence enhancement phenomenon. Figure 6 (A) proves that the hydroxyl radical is the main active intermediate driving the redox reaction.

[0041] (6) Feasibility of BiVO4 nanosheet colorimetric determination method Pyridine carboxylic acids (PAs) can act as bidentate ligands to bind to bismuth ions on the surface of BiVO4, thereby covering the active sites required for catalytic reactions. This strong coordination inhibits the catalytic activity of BiVO4. Although it limits sustained catalytic performance, it also offers the possibility of developing colorimetric sensors based on the activity inhibition effect. To explore this potential, DPA was added to solutions containing r-BiVO4, m-BiVO4, or p-BiVO4, and after incubation for 10 minutes, H2O2 and TMB were added. Figure 7 This is a feasibility diagram of the colorimetric determination method for BiVO4 samples prepared in the embodiments of the present invention; wherein, (A) a comparison of the typical absorption spectrum of r-BiVO4 and the absorption spectrum of the oxidation reaction of TMB and H2O2 catalyzed by r-BiVO4 and DPA after co-incubation for 10 minutes; (B) a comparison of the typical absorption spectrum of m-BiVO4 and the absorption spectrum of the oxidation reaction of TMB and H2O2 catalyzed by m-BiVO4 and DPA after co-incubation for 10 minutes; (C) a comparison of the typical absorption spectrum of p-BiVO4 and the absorption spectrum of the oxidation reaction of TMB and H2O2 catalyzed by p-BiVO4 and DPA after co-incubation for 10 minutes; and (D) the absorption spectra of the reaction of DPA and its structural analogs with TMB and H2O2 in the presence of m-BiVO4.

[0042] like Figure 7 As shown in (A)-(C), all three systems exhibited significant color fading, but the degree of absorbance decrease differed significantly, indicating that DPA had different inhibitory effects on different BiVO4 materials. Furthermore, seven different PAs (all at a concentration of 25 μmol / L) were added to the m-BiVO4-H2O2-TMB system, and the changes in their UV-Vis absorption spectra were investigated. Figure 7 (D) The results showed that, under the same conditions, all PAs caused varying degrees of reduction in the blue intensity of the system. Among them, the system with added 2-PA had the highest absorbance, while 2,4-PA had the strongest inhibitory effect on the absorbance of the system. This result indicates that the coordination ability of different PAs with BiVO4 materials varies significantly.

[0043] (7) Identification of various pyridine carboxylic acids by BiVO4 sensor array Figure 8This invention relates to the identification of various pyridinecarboxylic acids by a BiVO4 sensor array prepared in this embodiment. Specifically, (A1) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 10 μM; (A2) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 25 μM; (A3) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 40 μM; (A4) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 55 μM; (A5) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 70 μM; (A6) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 85 μM; (A7) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 100 μM; and (B1) is the LDA three-dimensional score diagram of PAs by the BiVO4 sensor array prepared in this embodiment at a concentration of 10 μM. (B2) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 25 μM for the BiVO4 sensor array prepared in this embodiment of the invention; (B3) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 40 μM for the BiVO4 sensor array prepared in this embodiment of the invention; (B4) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 55 μM for the BiVO4 sensor array prepared in this embodiment of the invention; (B5) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 70 μM for the BiVO4 sensor array prepared in this embodiment of the invention; (B6) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 85 μM for the BiVO4 sensor array prepared in this embodiment of the invention; (B7) Response radar diagrams of colorimetric sensor arrays of different PAs at a concentration of 100 μM for the BiVO4 sensor array prepared in this embodiment of the invention. Radar graphs of colorimetric sensor array responses to different PAs at μM concentrations: (C1) Linear relationship between DPA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention; (C2) Linear relationship between 4-PA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention; (C3) Linear relationship between 3-PA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention; (C4) Linear relationship between 3,4-PA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention; (C5) Linear relationship between 2-PA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention; (C6) Linear relationship between 2,4-PA concentration and first discriminant factor score for the BiVO4 sensor array prepared in this embodiment of the invention.The linear relationship between 4-PA concentration and the first discriminant factor score is shown in Figure (C7). The linear relationship between the BiVO4 sensor array prepared in this embodiment of the invention and the 2,3-PA concentration and the first discriminant factor score is also shown. Based on the fact that vanadium vacancies can regulate the activity of BiVO4-type peroxidases, and that different BiVO4 materials exhibit significant differences in their catalytic inhibition responses to DPA and its analogues, this invention constructs a sensor array containing 3 BiVO4 materials × 7 PAs × 5 repeated tests. To achieve high-precision pattern recognition of multidimensional response signals, linear discriminant analysis is used to process the data. For example, Figure 8 As shown in (A1), at a concentration of 10 µM, the response data of the seven PAs formed seven completely separate clusters in the LDA two-dimensional plot, achieving a classification accuracy of 100%. The array performance was examined within the concentration range of 10-100 µM, and the results showed that each PA still exhibited a unique response pattern at different concentrations. Figure 8 (B1)-(B7)), the categories in the LDA analysis still maintained clear separation. Figure 8 (A1)-(A7) indicates that the array has good concentration stability. Analysis of the relationship between PA concentration and response signal revealed that the first discriminant component of LDA (contribution rate > 60%) shows a regular change with PA concentration. Based on this, quantitative fitting curves for each PA were established ( Figure 8 (C1)-(C7)). Compared with existing DPA detection methods, this sensor array not only has high sensitivity and wide linear range for DPA, but can also effectively distinguish its structural analogs, demonstrating excellent identification and detection capabilities.

[0044] (8) Identification of pyridine carboxylic acid mixtures by BiVO4 sensor array Figure 9 This is a discrimination diagram of a mixture of pyridine carboxylic acids by the BiVO4 sensor array prepared in this embodiment of the invention. To examine the discrimination performance of the constructed sensor array on multi-component mixed systems, various pyridine carboxylic acid mixtures with different compositions were tested in the experiment.

[0045] The samples included binary mixtures (DPA and 2-PA), ternary mixtures (DPA, 4-PA, and 3-PA), quaternary mixtures (one group consisting of DPA, 2,3-PA, 2,4-PA, and 3,4-PA; another group consisting of DPA, 2-PA, 3-PA, and 4-PA), and complex systems with more components (e.g., mixtures of DPA, 2,3-PA, 2,4-PA, 3-PA, and 4-PA; mixtures of DPA, 2-PA, 2,3-PA, 2,4-PA, 4-PA, and 3,4-PA; and mixtures of all seven PAs), with varying molar ratios of components in each mixture. The total concentration for all samples was uniformly set at 100 μM, and each mixture was tested five times, for a total of 35 samples. Figure 9In the diagram, A represents a 1:1 mixture of DPA and 2-PA; B represents a 3:3:4 mixture of DPA, 4-PA, and 3-PA; C represents a 2:3:3:2 mixture of DPA, 2,3-PA, 2,4-PA, and 3,4-PA; D represents a 1:2:3:4 mixture of DPA, 2-PA, 3-PA, and 4-PA; E represents a 1:2:1:3:3 mixture of DPA, 2,3-PA, 2,4-PA, 3-PA, and 4-PA; and F represents an equimolar mixture of seven PAs (DPA, 2,3-PA, 3,4-PA, 2,4-PA, 2-PA, 3-PA, and 4-PA) in a 1:1:1:1:1:1:1 ratio. The results are as follows: Figure 9 As shown, mixtures with different compositions and ratios form distinct, non-overlapping independent clusters in the pattern recognition diagram, achieving 100% accurate differentiation. This result demonstrates that the sensor array constructed in this study possesses high discrimination capability for complex PA mixture systems.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A BiVO4 sensor array, characterized in that... The BiVO4 sensor array includes three sensing elements, namely r-BiVO4, m-BiVO4 and p-BiVO4.

2. The BiVO4 sensor array as described in claim 1, characterized in that, The BiVO4 sensor array can be used to detect pyridine carboxylic acid compounds, including DPA, 2,3-PA, 3,4-PA, 2,4-PA, 2-PA, 3-PA, and 4-PA.

3. The BiVO4 sensor array as described in claim 1, characterized in that, The preparation method of r-BiVO4 is as follows: Bismuth chloride, hexadecyltrimethylammonium bromide and ethylene glycol are dissolved in proportion, sodium dodecyl vanadate solution is added under stirring, and hydrothermal reaction is carried out at 155-165 °C for 2.9-3.1 hours. After the reaction is completed, the mixture is cooled, centrifuged, washed and dried to obtain the product.

4. The BiVO4 sensor array as described in claim 1, characterized in that, The preparation method of m-BiVO4 is as follows: Bismuth chloride, hexadecyltrimethylammonium bromide and ethylene glycol are dissolved in proportion, sodium dodecyl vanadate solution is added under stirring, and hydrothermal reaction is carried out at 135-145 °C for 4.9-5.1 hours. After the reaction is completed, the mixture is cooled, centrifuged, washed and dried to obtain the product.

5. The BiVO4 sensor array as described in claim 1, characterized in that, The preparation method of p-BiVO4 is as follows: Bismuth chloride, hexadecyltrimethylammonium bromide and ethylene glycol are dissolved in proportion, sodium dodecyl vanadate solution is added under stirring, and hydrothermal reaction is carried out at 115-125 °C for 8.9-9.1 hours. After the reaction is completed, the mixture is cooled, centrifuged, washed and dried to obtain the p-BiVO4.

6. The BiVO4 sensor array as described in any one of claims 2-4, characterized in that, The ratio of bismuth chloride, hexadecyltrimethylammonium bromide, and ethylene glycol is 2.21 g : 1.05 g : 55-65 mL; Alternatively, the mass ratio of bismuth chloride to sodium orthovanadate dodecahydrate is 2.21:2.8; Alternatively, the washing process may involve washing with deionized water and anhydrous ethanol 2-4 times sequentially.

7. The application of a BiVO4 sensor array as described in claim 1 in the detection of anthrax biomarkers and analogues, characterized in that, The anthrax biomarker and its analogues are at least one of DPA, 2,3-PA, 3,4-PA, 2,4-PA, 2-PA, 3-PA, and 4-PA.

8. The application as described in claim 7, characterized in that, The detection concentration of anthrax biomarkers and analogues is 10-100 µM.

9. A method for detecting anthrax biomarkers and analogues, characterized in that, Includes the following steps: r-BiVO4, m-BiVO4 and p-BiVO4 were added to sodium acetate-acetic acid buffer solution, followed by the introduction of anthrax biomarker and analogue solution, and co-incubation. After incubation, TMB solution was added first, followed by hydrogen peroxide solution, and then incubated again to obtain a mixed system. The absorbance value of the mixed system was tested, and the absorbance change was calculated using a sample without analyte as a blank control. r-BiVO4, m-BiVO4 and p-BiVO4 were used as independent sensing channels to detect the analyte solution, and five parallel experiments were performed for each concentration. By integrating test data, a three-dimensional data matrix containing sensor channels, analyte concentrations, and the number of parallel experiments is constructed. This data matrix is ​​then processed to establish a recognition model. Based on the analysis of the detection data of the sample under test using the recognition model, the qualitative and quantitative identification of analytes in the sample under test can be realized.

10. The detection method as described in claim 9, characterized in that, Anthrax biomarkers and their analogues include at least one of DPA, 2,3-PA, 3,4-PA, 2,4-PA, 2-PA, 3-PA, and 4-PA. Alternatively, the concentrations of r-BiVO4, m-BiVO4, and p-BiVO4 are 0.095–0.105 mg / mL. Alternatively, the co-incubation time is 8-12 minutes; the subsequent incubation time is 48-52 seconds.

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