Three-channel mn02 nanoszyme colorimetric array and its application in functional citrus product identification

Abstract

The application belongs to the technical field of colorimetric sensing, and particularly relates to a three-channel MnO2 nanoscale enzyme colorimetric array and application thereof in functional citrus product identification. The application constructs a colorimetric sensing array composed of three different morphologies of MnO2 nanoscale enzymes, which is used for instant identification of representative citrus flavonoids and functional citrus products. By integrating three wavelengths / RBG signals with a three-channel sensor, a highly cross-reactive fingerprint database is generated. In combination with hierarchical cluster analysis, principal component analysis and linear discriminant analysis, 100% accuracy, high sensitivity and anti-interference identification of flavone isomers are realized. A further constructed paper-based platform also realizes 100% accuracy of instant detection of related citrus samples. The application provides theoretical guidance for new sensor array design for highly similar analytes, and provides a reliable instant detection tool for food adulteration prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of colorimetric sensing technology, specifically involving a three-channel MnO2 nanozyme colorimetric array and its application in the identification of functional citrus products. Background Technology

[0002] Citrus products, due to their excellent antioxidant, blood sugar-lowering, blood lipid-lowering, and health-promoting functions, have been widely used in food (such as the flowers, fruits, and peels of Changshan pomelo) and the pharmaceutical industry (such as bitter orange peel, dried bitter orange fruit, and tangerine peel). The components of functional citrus plants are closely related, with similar structures but different content levels. This leads to widespread adulteration and misidentification in commercial products, damaging market integrity and consumer trust, and hindering the healthy development of the industry.

[0003] Flavonoids are considered the main active ingredients in citrus plants with both edible and medicinal functions. Notably, naringin (Nari) and neohesperidin (NH) have been designated as quality markers in the Chinese Pharmacopoeia (2025 edition). Other bioactive flavonoids, including erucic acid (Eri), neo-northern erucic acid (Neo), rutin (Nar), and hesperidin (Hes), are also closely related to the efficacy of citrus medicinal materials and are frequently used in comprehensive quality evaluation. However, the similar morphological characteristics, similar structures, and compositions of flavonoid components are a major obstacle to rapidly and accurately distinguishing citrus plant sources. Therefore, there is an urgent need to develop efficient identification methods.

[0004] Currently, the identification of citrus species and their parts mainly relies on spectroscopic techniques (such as UV-Vis spectrophotometry and fluorescence spectroscopy) and chromatographic techniques (such as high-performance liquid chromatography-mass spectrometry and HPLC-MS). Among these, spectroscopic techniques have the advantages of rapid, simple, and high-throughput detection, but their ability to identify and distinguish complex analytes remains limited. In contrast, chromatographic methods have strong separation capabilities and high sensitivity for complex samples, but involve expensive instruments and require specialized operating skills. By combining sensor array strategies with spectroscopic techniques, rapid, high-throughput, and low-cost on-site detection can be achieved in complex systems, while capturing richer data to improve the ability to distinguish target analytes. Currently, the sensor arrays reported for distinguishing flavonoid components mainly rely on two strategies: (1) constructing multiple sensors with a single response mechanism or coordination ability; and (2) changing the reaction conditions of specific sensors, such as response time, pH, or substrate. These strategies, combined with common chemometric data processing techniques such as hierarchical cluster analysis (HCA), principal component analysis (PCA), linear discriminant analysis (LDA), and random forest (RF), can efficiently identify multiple active ingredients in a single step. However, a single response pattern results in limited data cross-referencing, making it difficult to accurately distinguish structurally similar flavonoid isomers or citrus species and their closely related species with extremely similar compositions. It is known that more diverse sensing patterns are beneficial for generating cross-referencing datasets, and analysis using advanced algorithms such as radio frequency (RF), support vector machines (SVM), and artificial neural networks can effectively improve recognition accuracy. However, complex operations, redundant information, or overfitting problems must also be considered. Conversely, when the sensing unit is too simple or the amount of data is limited, the accuracy and precision of recognition will be constrained. Therefore, balancing signal dimensionality, data volume, and ease of preparation is key to developing effective sensing arrays, and exploring sensor arrays regulated by multiple synergistic mechanisms is crucial for distinguishing active flavonoid isomers and highly similar or closely related citrus products. Summary of the Invention

[0005] The purpose of this invention is to provide a three-channel MnO2 nanozyme colorimetric array and its application in the identification of functional citrus products.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a three-channel MnO2 nanozyme colorimetric sensing array, comprising three detection channels, each channel comprising an MnO2 nanozyme with oxidase-like activity; The first channel includes an α-MnO2 nanozyme with a nanorod morphology; The second channel includes δ-MnO2 nanozymes with a mixed morphology of flower-like and nanotube structures; The third channel includes δ-MnO2 nanozymes with a flower-like lamellar morphology; The MnO2 nanozyme exhibits specific color responses at 370nm, 450nm, and 652nm in the TMB chromogenic system, forming a cross-reactive fingerprint spectrum.

[0007] Preferably, the α-MnO2 nanozyme with a nanorod morphology has a diameter of 25~45nm, a length of 350~820nm, and a lattice fringe spacing of 2.4Å. The lattice fringe spacing of the δ-MnO2 nanozyme with a mixed morphology of flower-like and nanotube is 2.4 Å. The δ-MnO2 nanozyme with a flower-like layered morphology has pores with a diameter of 10~80nm between its layers and a lattice fringe spacing of 3.5Å.

[0008] Preferably, the preparation method of the α-MnO2 nanozyme with nanorod morphology includes: mixing potassium permanganate solution and hydrochloric acid solution, and then carrying out a hydrothermal reaction to obtain the α-MnO2 nanozyme with nanorod morphology; the hydrothermal reaction temperature is 140℃ and the time is 12h; The preparation method of the δ-MnO2 nanozyme with a mixed morphology of flower-like and nanotube features includes: mixing potassium permanganate solution and hydrochloric acid solution, and then carrying out a hydrothermal reaction to obtain the δ-MnO2 nanozyme with a mixed morphology of flower-like and nanotube features; the hydrothermal reaction is carried out at a temperature of 110°C for a time of 6 hours. The preparation method of the δ-MnO2 nanozyme with flower-like lamellar morphology includes: mixing potassium permanganate solution and manganese sulfate, and then carrying out a hydrothermal reaction to obtain the δ-MnO2 nanozyme with flower-like lamellar morphology; the hydrothermal reaction temperature is 160℃ and the time is 12h.

[0009] Preferably, in the first channel, the concentration of MnO2 nanozyme is 2 mM and the concentration of TMB is 0.5 mM; In the second channel, the concentration of MnO2 nanozyme is 1 mM and the concentration of TMB is 2 mM; In the third channel, the concentration of MnO2 nanozyme is 2 mM and the concentration of TMB is 1 mM. Each channel contains 0.1M HAc-NaAc buffer at pH 3.0.

[0010] The present invention also provides a paper-based colorimetric sensor, comprising: A solid support, wherein the solid support is a filter paper sheet; The three-channel MnO2 nanoenzyme colorimetric sensor array described above, loaded on the solid support.

[0011] This invention also provides the application of the three-channel MnO2 nanoenzyme colorimetric sensor array or the paper-based colorimetric sensor described in the above technical solutions in the instant identification of flavonoid isomers or the identification of citrus varieties.

[0012] The present invention also provides a sample identification method, which uses the three-channel MnO2 nanoenzyme colorimetric sensor array or the paper-based colorimetric sensor described in the above technical solution; The test samples were added to the three channels respectively to carry out the reaction; the test samples included flavonoid isomers or citrus extracts. Detect the absorbance or RGB value signals of each channel at 370nm, 450nm and 652nm; The obtained absorbance or RGB values ​​are combined to form a fingerprint spectrum, and the fingerprint spectrum is analyzed using machine algorithms to achieve sample identification.

[0013] Preferably, the reaction temperature is 37°C and the reaction time is 15 min.

[0014] Preferably, the RGB value signal is: the first channel uses the R / (G+B) value, the second channel uses the B / (R+B) value, and the third channel uses the G / (R+B) value; The machine algorithms include hierarchical clustering analysis, principal component analysis, and three-dimensional linear discriminant analysis.

[0015] Preferably, the flavonoid isomers include at least one of senna-2, neo-senna-2, rutin, naringin, hesperidin, and neo-hesperidin; The detection limit of the sample identification method is 1 μg / mL.

[0016] This invention constructs a colorimetric sensor array composed of three different forms of MnO2 nanozymes for the real-time identification of representative citrus flavonoids and functional citrus products. Under dynamic electron transfer catalysis, the TMB / oxTMB system exhibits specific color changes (corresponding to green, yellow, and blue) at three wavelengths (370 nm, 450 nm, and 652 nm), a process potentially synergistically regulated by flavonoid structure, redox, and coordination interactions. By integrating the three wavelength / RGB signals with a three-channel sensor, a highly cross-reactive fingerprint database is generated. Combining hierarchical clustering analysis, principal component analysis, and linear discriminant analysis, 100% accuracy, high sensitivity, and interference resistance in identifying flavonoid isomers (1–500 μg / mL) are achieved. A further constructed paper-based platform also achieves 100% accuracy in real-time detection of related citrus samples. This invention provides theoretical guidance for the design of novel sensor arrays targeting highly similar analytes and offers a reliable real-time detection tool for food adulteration control. Furthermore, this invention systematically elucidates the potential interaction mechanism between MnO2 surface and flavonoids, aiming to provide a new theoretical basis for identifying flavonoid isomers based on synergistic mechanism-regulated multichannel sensor arrays, and to provide a rapid and accurate solution for distinguishing closely related citrus plants in the field. Attached Figure Description

[0017] Figure 1 SEM images of MnO2-I (A), MnO2-II (B), and MnO2-III (C); Figure 2 HRTEM images of MnO2-I (A,B), MnO2-II (C,D), and MnO2-III (E,F); Figure 3 EDS spectra of MnO2-I (A), MnO2-II (C), and MnO2-III (E), and SAED spectra of MnO2-I (B), MnO2-II (D), and MnO2-III (F); Figure 4 FTIR (A), XRD (B), and XPS (C) scan spectra of MnO2-I, MnO2-II, and MnO2-III; Figure 5 UV-vis spectra (A) of reaction systems with different oxidase-like behaviors of MnO2-I, MnO2-II and MnO2-III, and relative activities (BD) of MnO2-I, MnO2-II and MnO2-III nanozymes at different pH, time and temperature. Figure 6Michaelis-Menten and Lineweaver-Burk plots for MnO2-I (AB), MnO2-II (CD), and MnO2-III (EF); Figure 7 The reproducibility (AC), storage stability (DF), and relative activity of MnO2-I, MnO2-II, and MnO2-III nanozymes were evaluated. Figure 8 The UV-Vis spectra of six flavonoid compounds (at a concentration of 0.5 mg / mL) in a simulated MnO2-I / II / III oxidase system at different wavelengths of 450 nm (A), 652 nm (B), and 370 nm (C) are shown. Figure 9 The structural formulas for three pairs of flavonoid isomers; Figure 10 A schematic diagram (A) of a 6-target × 3-channel colorimetric sensor array with 3 signal channels (450nm (channel I), 652nm (channel II), and 370nm (channel III)), a PCA score diagram of the analysis of 6 flavonoids (500 μg / mL) with three signal channels (370nm, 450nm, and 652nm) (B), a 652nm single signal channel (C), and a 450 / 652nm dual signal channel (D); Figure 11 A dendrogram of HCA for six flavonoid compounds based on a three-channel colorimetric sensor array; Figure 12 PCA scores for Eri, Neo, Nar, Nari, Hes, and NH at the same concentrations: 200 μg / mL (A), 100 μg / mL (B), 75 μg / mL (C), 50 μg / mL (D), 30 μg / mL (E), 20 μg / mL (F), 10 μg / mL (G), 2 μg / mL (H), and 1 μg / mL (I). Figure 13 PCA score charts for Eri(A), Neo(B), Nar(C), Nari(D), Hes(E), and NH(F) at different concentrations; Figure 14 HPLC chromatograms of six citrus products; Figure 15 3D scoring LDA plots of binary mixtures of Eri and Hes (A), quaternary mixtures of Nar / Nari / Hes / NH (B), and hexaternary mixtures of six flavonoids (C); evaluation of the anti-interference properties of 3D scoring LDA plots for biomolecules (D), metal ions (E), and other flavonoids (F) at 500 μg / mL. Figure 16For colorimetric identification of six flavonoids using a paper-based three-channel sensor array, the paper sensor array visual image (A); fingerprints based on RGB values ​​(B), HCA tree diagram (C), PCA scoring map (D), and 3D LDA scoring based on R / (G+B) (channel I), B / (R+B) (channel II), and G / (R+B) (channel III) (E); Figure 17 Powder and plant morphology of six Citrus samples (illustration) (A), indoor and outdoor images of paper-based colorimetric sensor array (B), RGB fingerprint spectrum (C), PCA scoring map (D) and 3D LDA scoring map (E) for real-time identification of the six Citrus samples. Figure 18 The RGB fingerprint (A), HCA tree diagram (B), and PCA scoring diagram (C) of six citrus products were used for real-time detection based on solution-RGB extraction. Figure 19 To enable real-time adulteration detection of mixed citrus samples based on a paper-based sensor array, HCA dendritic diagrams (A), PCA scoring diagrams (B), and 3D LDA scoring diagrams (C) of HYP powders with different CP mass fractions were obtained. Figure 20 MnO2-III in DMPO-•OH (1:2:2:1) (A), DMPO-O2 - (1:1:1:1)(B) and TEMP- 1 EPR spectra of O2 (1:1:1) (C) under different trapping agents; UV-vis spectra of MnO2-I (D), MnO2-II (E), and MnO2-III (F) catalyzed TMB, TMB+NH (co-incubation), and TMB+NH (added later); Figure 21 UV-vis (A) of MnO2-I / II / III, NH and MnO2-I / II / III-NH; FTIR (B), XRD patterns (C), SEM images (DF) and high-resolution O1s (G) and Mn2p (H) XPS spectra of MnO2-III and MnO2-III-NH; Schematic diagram of the structure-activity relationship of flavonoids on the oxidase-mimicking activity of MnO2 nanozymes (I); Figure 22 HRTEM images of MnO2-I-NH (AB), MnO2-II-NH (CD), and MnO2-III-NH (EF); Figure 23 XPS full spectrum scans of MnO2-I-NH, MnO2-II-NH and MnO2-III-NH; Figure 24The UV-vis spectra of the MnO2-I oxidation system mediated by Eri (A), Neo (B), Nar (C), Nari (G), Hes (H), and NH (I) were analyzed; the concentrations of 0~300 μg / mL (D), 0~200 μg / mL (E), 0~3000 μg / mL (F), 0~3000 μg / mL (J), 0~75 μg / mL (K), and 0~50 μg / mL (L) showed a linear relationship with the absorbance intensity at 652 nm. Figure 25 The diagram shows the structural formulas of flavonoids, the structure-activity relationship of flavonoids on the oxidase-mimicking activity of MnO2 nanozymes, and a schematic diagram of the three-channel sensing array. Detailed Implementation

[0018] The present invention provides a three-channel MnO2 nanozyme colorimetric sensing array, comprising three detection channels, each channel comprising an MnO2 nanozyme with oxidase-like activity; The first channel includes an α-MnO2 nanozyme with a nanorod morphology; The second channel includes δ-MnO2 nanozymes with a mixed morphology of flower-like and nanotube structures; The third channel includes δ-MnO2 nanozymes with a flower-like lamellar morphology; The MnO2 nanozyme exhibits specific color responses at 370nm, 450nm, and 652nm in the TMB chromogenic system, forming a cross-reactive fingerprint spectrum.

[0019] In this invention, the diameter of the α-MnO2 nanozyme with the nanorod morphology is preferably 25~45nm, the length is preferably 350~820nm, and the lattice fringe spacing is preferably 2.4Å.

[0020] In this invention, the preferred method for preparing the α-MnO2 nanozyme (MnO2-I) with a nanorod morphology includes: mixing a potassium permanganate solution and a hydrochloric acid solution, followed by a hydrothermal reaction to obtain the α-MnO2 nanozyme with a nanorod morphology. In this invention, the preferred ratio of potassium permanganate to deionized water in the potassium permanganate solution is 0.3214 g:30 mL; the preferred concentration of the hydrochloric acid is 37 wt%; the preferred volume ratio of the potassium permanganate solution to the hydrochloric acid is 30:0.7; the mixing is preferably carried out under stirring; the preferred stirring time is 30 min; the preferred temperature of the hydrothermal reaction is 140 °C; the preferred time is 12 h; and the preferred hydrothermal reaction is carried out in a high-pressure reactor lined with Teflon. In this invention, after the hydrothermal reaction, it is preferred to perform a post-treatment, which is preferably: filtering the obtained system through a 0.22 μm filter membrane, washing the product with deionized water and anhydrous ethanol three times in sequence; drying the final product at 60°C for 24 h, and storing it in a sealed container at room temperature.

[0021] In this invention, the lattice fringe spacing of the δ-MnO2 nanozyme (MnO2-II) with a mixed flower-like and nanotube morphology is preferably 2.4 Å. The preferred preparation method of the δ-MnO2 nanozyme with a mixed flower-like and nanotube morphology includes: mixing a potassium permanganate solution and a hydrochloric acid solution, followed by a hydrothermal reaction to obtain the δ-MnO2 nanozyme with a mixed flower-like and nanotube morphology; the preferred ratio of potassium permanganate to deionized water in the potassium permanganate solution is 0.51 g:35 mL; the preferred temperature of the hydrothermal reaction is 110 °C, and the preferred time is 6 h; the specific process and post-treatment of the preparation method are preferably the same as those for the preparation and post-treatment of the α-MnO2 nanozyme with a nanorod-like morphology described above, except that the temperature and time of the hydrothermal reaction are changed, and the stirring time is adjusted to 15 min, which will not be repeated here.

[0022] In this invention, the δ-MnO2 nanozyme (MnO2-III) with a flower-like layered morphology preferably has pores with a diameter of 10-80 nm between its layers, and the lattice fringe spacing is preferably 3.5 Å. In this invention, the preparation method of the δ-MnO2 nanozyme with a flower-like layered morphology preferably includes the following steps: mixing potassium permanganate solution and manganese sulfate, and then performing a hydrothermal reaction to obtain the δ-MnO2 nanozyme with the flower-like layered morphology; the preferred ratio of potassium permanganate to deionized water in the potassium permanganate solution is 0.948 g:35 mL; the preferred manganese sulfate is MnSO4·H2O; the mass ratio of potassium permanganate to manganese sulfate is 0.948:0.169; the mixing is preferably carried out under stirring conditions, and the stirring time is preferably 30 min; the preferred temperature of the hydrothermal reaction is 160 °C, and the preferred time is 12 h; the hydrothermal reaction is preferably carried out in a high-pressure reactor with a Teflon lining. In this invention, after the hydrothermal reaction, it is preferable to perform a post-treatment, wherein the post-treatment preferably involves filtering the obtained system through a 0.22 μm filter membrane, washing the product three times sequentially with deionized water and anhydrous ethanol, and drying the final product at 60°C for 24 h and storing it in a sealed container at room temperature.

[0023] In this invention, in the first channel, the concentration of MnO2 nanozyme is preferably 2 mM and the concentration of TMB is preferably 0.5 mM; in the second channel, the concentration of MnO2 nanozyme is preferably 1 mM and the concentration of TMB is preferably 2 mM; in the third channel, the concentration of MnO2 nanozyme is preferably 2 mM and the concentration of TMB is preferably 1 mM; each channel preferably includes HAc-NaAc buffer solution with a concentration of 0.1 M and a pH of 3.0.

[0024] The present invention does not impose any special limitations on the construction method of the three-channel MnO2 nanoenzyme colorimetric sensing array; the required amount of raw materials can be added to the multi-well plate.

[0025] The present invention also provides a paper-based colorimetric sensor, comprising: A solid support, wherein the solid support is a filter paper sheet; The three-channel MnO2 nanoenzyme colorimetric sensor array described above, loaded on the solid support.

[0026] In this invention, the size of the filter paper sheet is preferably 6. 6mm; the filter paper is preferably Whatman filter paper.

[0027] In this invention, the preferred method for preparing the paper-based colorimetric sensor includes the following steps: immersing filter paper in MnO2 nanozyme solutions of different concentrations and then drying it; adding a mixed solution containing TMB solution and HAc-NaAc buffer solution dropwise onto the dried filter paper; preferably, the amount of TMB solution on a single filter paper is 10 μL, and the amount of HAc-NaAc buffer solution is 70 μL. In this invention, the immersion time is preferably 30 min, the drying temperature is preferably 45°C, and the drying time is preferably 15 min. In this invention, when using the paper-based colorimetric sensor for detection, the sample to be tested is preferably added to the mixed solution containing TMB solution and HAc-NaAc buffer solution before being added dropwise.

[0028] This invention also provides the application of the three-channel MnO2 nanoenzyme colorimetric sensor array or the paper-based colorimetric sensor described in the above technical solutions in the instant identification of flavonoid isomers or the identification of citrus varieties.

[0029] The present invention also provides a sample identification method, which uses the three-channel MnO2 nanoenzyme colorimetric sensor array or the paper-based colorimetric sensor described in the above technical solution; The test samples were added to the three channels respectively to carry out the reaction; the test samples included flavonoid isomers or citrus extracts. Detect the absorbance or RGB value signals of each channel at 370nm, 450nm and 652nm; The obtained absorbance or RGB values ​​are combined to form a fingerprint spectrum, and the fingerprint spectrum is analyzed using machine algorithms to achieve sample identification.

[0030] In this invention, the flavonoid isomers preferably include at least one of Eri, Neo, Nar, Nari, Hes, and Neohesperidin.

[0031] In this invention, the preferred method for preparing the citrus extract includes: drying the citrus sample, pulverizing it using a grinder, passing it through a No. 4 sieve, mixing it with an ethanol solution, and then performing ultrasonic extraction to obtain the citrus extract. In this invention, the citrus sample preferably includes at least one of the following: *Citrus aurantium* (ZQ), *Citrus aurantium* (ZS), *Citrus reticulata* (CP), *Citrus medica* flower (HYF), *Citrus medica* and *Citrus aurantium* peel (QZQ), and *Citrus medica* peel (HYP). In this invention, the volume concentration of the ethanol solution is preferably 75%, and the volume ratio of the citrus sample to the ethanol solution is preferably 1 mg: 2 mL; the ultrasonic extraction power is preferably 100 W, the frequency is preferably 40 kHz, and the time is preferably 45 min. After ultrasonic extraction, this invention also preferably includes dilution, and the concentration of the citrus extract is preferably 500 μg / mL; the amount of citrus extract on a single filter paper during detection is preferably 10 μL.

[0032] In this invention, the reaction temperature is preferably 37°C and the reaction time is preferably 15 min; the reaction is preferably carried out under sealed conditions.

[0033] This invention does not impose any particular limitation on the method for detecting the absorbance value; any method well-known to those skilled in the art can be used. In this invention, the RGB value signal is preferably: the first channel preferably uses an R / (G+B) value, the second channel preferably uses a B / (R+B) value, and the third channel preferably uses a G / (R+B) value. In this invention, the machine algorithm preferably includes hierarchical clustering analysis (HCA), principal component analysis (PCA), and three-dimensional linear discriminant analysis (LDA).

[0034] In this invention, the detection limit of the sample identification method is preferably 1 μg / mL.

[0035] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0036] Figure 25 The diagram shows the structural formulas of flavonoids, the structure-activity relationship of flavonoids on the oxidase-mimicking activity of MnO2 nanozymes, and a schematic diagram of the three-channel sensing array.

[0037] The following are examples and samples used in the tests: Preparation of MnO2-I: 0.3214 g of potassium permanganate was dissolved in 30 mL of deionized water. 0.7 mL of hydrochloric acid (37 wt%) was added to the mixture and stirred for 0.5 h. The mixture was then transferred to a Teflon-lined autoclave (100 mL) and reacted at 140 °C for 12 h. After the reaction was completed, the mixture was filtered through a 0.22 μm filter membrane. The product was washed three times with deionized water and anhydrous ethanol, respectively. The final product was dried at 60 °C for 24 h and then sealed and stored at room temperature.

[0038] Preparation of MnO2-II: Dissolve 0.51 g of potassium permanganate in 35 mL of deionized water, slowly add hydrochloric acid (0.7 mL, 37 wt%), stir for 15 min, transfer to a Teflon-lined autoclave, and react at 110 °C for 6 h. The post-treatment process is the same as that for MnO2-I.

[0039] Preparation of MnO2-III: Dissolve 0.948 g of potassium permanganate in 35 mL of deionized water, add 0.169 g of MnSO4·H2O and stir for 0.5 h. Then transfer the mixture to a Teflon-lined autoclave and react at 160 °C for 12 h. The post-treatment process is the same as that for MnO2-I.

[0040] 3,3',5,5'-Tetramethylbenzidine stock solution (TMB, 2mM): Accurately weigh 6.2714 mg of TMB and dissolve it in 10 mL of methanol solution. Mix well in the dark and sonicate until dissolved to prepare a 2 mM TMB stock solution; prepare and use immediately.

[0041] MnO2 nanozyme solution (10mM): Accurately weigh 3.4776 mg of MnO2 nanozyme and disperse it in 4 mL of ultrapure water. Mix well and dissolve by sonication to obtain a 10 mM stock solution for later use.

[0042] HAc-NaAc buffer (0.1M, pH 3.0): Accurately weigh 0.14g of anhydrous sodium acetate using an electronic balance, place it in a 1L beaker, add approximately 800mL of ultrapure water, and stir on a magnetic stirrer until completely dissolved; accurately pipette 5.63mL of glacial acetic acid and slowly add it to the sodium acetate solution, continuing to stir for 5-10 minutes to ensure thorough mixing; while maintaining stirring, add 1mol / L HCl or 1mol / L NaOH solution dropwise, monitoring the pH meter reading in real time, until the solution pH stabilizes at 3.00; transfer the prepared buffer to a sterile reagent bottle and store at 4°C protected from light.

[0043] The preparation method of the nanozyme-flavonoid complex is as follows: MnO2-I / II / III powder (34.8 mg, 10 mM) was dispersed in 40 mL of ultrapure water; the MnO2-I / II / III solution was mixed with a 5 mg / mL methanol solution of neohesperidin (NH) and stirred at 37 °C for 15 min; after the reaction was completed, the mixture was filtered (0.22 μm membrane), and the obtained product was washed with deionized water and anhydrous ethanol (each washed 3 times); the final product was dried at 60 °C for 24 h and named MnO2-I / II / III-NH, and stored in a sealed container at room temperature for later use.

[0044] Data processing: RGB values ​​were extracted using the "Fcolor picker" mobile application, and visualization processing such as RGB fingerprinting, HCA, and PCA was performed using Origin 2022 software. LDA data was processed using SPSS 27.0 software and plotted in Origin 2022. Lattice and SAED images were processed using Digital Micrograph; XPS spectra were processed using Avantage software.

[0045] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0046] Example 1 A three-channel MnO2 nanozyme colorimetric sensor array was constructed, and six flavonoid isomers, namely Eri, Neo, Nar, Nari, Hes, and Neohesperidin (NH), were detected. The sensor array was constructed using a 96-well plate; the total volume of the reaction system was 200 μL, containing HAc-NaAc buffer (0.1 M, pH 3.0); the array design included 6 flavonoid targets × 3 detection channels × 3 parallel replicates. The reaction conditions for the three detection channels were as follows: Channel I: MnO2-I concentration 2 mM, TMB concentration 0.5 mM; Channel II: MnO2-II concentration 1 mM, TMB concentration 2 mM; Channel III: MnO2-III concentration 2 mM, TMB concentration 1 mM. Different target flavonoids (fixed concentrations) were added to their respective wells, and after reacting at 37℃ for 15 min, the absorbance values ​​at wavelengths of 450 nm, 652 nm, and 370 nm were measured using a microplate reader.

[0047] Example 2 A paper-based colorimetric sensor was constructed, and extracts of Citrus aurantium (ZQ), Citrus aurantium (ZS), Citrus reticulata (CP), Citrus aurantium flower (HYF), Citrus aurantium and Citrus aurantium (QZQ) and Citrus aurantium peel (HYP) were identified. Preparation of extract: 1 mg of dried citrus sample that has been pulverized by a grinder and passed through a No. 4 sieve was added to 2 mL of 75% (v / v) ethanol solution and ultrasonically extracted for 45 min in an ultrasonic cleaner (40 kHz, 100 W); the extract was then appropriately diluted to prepare a test solution with a concentration of 500 μg / mL. Paper-based colorimetric sensor: Whatman filter paper was cut into 6×6mm pieces and immersed in MnO2 nanozyme solutions of different concentrations (Channel I: MnO2-I, 2mM; Channel II: MnO2-II, 1mM; Channel III: MnO2-III, 2mM) for 30 min, and then dried in a 45℃ oven for 15 min. A reaction mixture containing TMB solution (Channel I: 0.5mM, 10μL; Channel II: 2mM, 10μL; Channel III: 1mM, 10μL), HAc-NaAc buffer (0.1M, pH 3.0, 70μL), and analyte solution (500μg / mL, 10μL) was added to each dried filter paper piece. After adding the solution, place the filter paper in a petri dish and seal it. Incubate at 37°C for 15 minutes to allow it to fully react and develop color. Take an image of the paper chip under constant light source conditions. Use the "Fcolor picker" mobile application to extract the RGB (red, green, blue) color values ​​of each channel from the image. For further data analysis, calculate the following color ratios: R / (G+B) (channel I), B / (R+B) (channel II) and G / (R+B) (channel III).

[0048] Images were acquired and RGB values ​​were extracted. Different citrus varieties were distinguished and identified through HCA, PCA, and LDA analysis.

[0049] Performance testing and results analysis I. Characterization of MnO2-I / II / III nanomaterials Three MnO2 nanostructures with different morphologies (labeled MnO2-I, MnO2-II, and MnO2-III, respectively) were successfully synthesized via a hydrothermal method. Scanning electron microscope images (…) Figure 1 The images showed that MnO2-I exhibited a nanorod-like morphology (25–45 nm in diameter and 350–820 nm in length); MnO2-II displayed a mixed morphology of flower-like and nanotube-like structures; and MnO2-III formed assemblies with a clear flower-like structure, with pores of 10–80 nm in diameter between the layers. High-resolution transmission electron microscopy images further confirmed the structures of the three MnO2 variants.

[0050] like Figure 2 As shown in Figures AB, the nanorod-shaped MnO2-I exhibits a lattice spacing of 2.4 Å, corresponding to the (211) crystal plane of α-MnO2. The mixed flower-like / nanotube-like MnO2-II exhibits a lattice spacing of 2.4 Å, corresponding to the (101) crystal plane. Figure 2 The CD); while the flower-like MnO2-III exhibits a larger 3.5 Å lattice spacing, attributed to the (006) crystal plane ( Figure 2The lattice spacings measured for MnO2-II and MnO2-III are consistent with those for δ-MnO2.

[0051] In addition, energy dispersive X-ray spectroscopy elemental mapping (EDS-mapping) Figure 3 The A, C, and E values ​​indicate that Mn and O elements are uniformly distributed in all three MnO2 materials. Selected area electron diffraction (SAED) patterns (…) Figure 3 The B, D, and F samples showed clear diffraction spots, confirming their crystal structure.

[0052] Fourier transform infrared spectroscopy ( Figure 4 A) shows the characteristic Mn-O vibrational peaks of MnO2 (505, 503, 499 cm⁻¹). -1 ) and Mn-O-Mn vibration peaks (705, 913, 921 cm⁻¹) -1 ).

[0053] X-ray diffraction pattern ( Figure 4 B) also verified the crystal structures of MnO2-I (α type, JCPDS:44-0141) and MnO2-II / III (δ type, JCPDS:52-0556); The specific XRD data are as follows: MnO2-I, JCPDS:44-0141: 12.6°, 17.8°, 25.6°, 28.6°, 36.4°, 37.4°, 41.0°, 41.8°, 49.7°, 55.9°, 60.0°, 65.1°, 69.7°, corresponding to the (110), (200), (220), (310), (400), (211), (330), (301), (411), (600), (521), (002), (541) crystal planes, respectively. MnO2-II, JCPDS:52-0556: 12.2°, 24.7°, 36.5°, 37.3°, 42.1°, 49.7°, 65.8°, corresponding to the (003), (006), (101), (012), (015), (018), and (110) crystal planes, respectively; MnO2-III, JCPDS:52-0556: 12.2°, 24.7°, 36.5°, 37.3°, 42.1°, 49.7°, 65.8°, corresponding to the (003), (006), (101), (012), (015), (018), and (110) crystal planes, respectively.

[0054] In addition, X-ray photoelectron spectroscopy full spectrum scanning ( Figure 4 C) confirmed that the material is composed of Mn and O elements. All the above results collectively confirm that this invention successfully prepared three MnO2 nanomaterials with different morphologies, good crystal structures, and uniform elemental compositions.

[0055] II. MnO2-I / II / III oxidase-like activity (1) To evaluate the oxidase-like activity of MnO2-I / II / III nanozymes, the following reaction system was used: 100 μL of MnO2-I / II / III aqueous solution (1 mM), 100 μL of TMB methanol solution (3,3',5,5'-tetramethylbenzidine, 2 mM), 700 μL of HAc-NaAc buffer (0.1 M, pH 3.0) and 100 μL of deionized water were added to a 1.5 mL centrifuge tube in sequence; after the mixed solution was incubated at 37 °C for 15 min, its absorption spectrum was scanned and recorded in the wavelength range of 200~800 nm using a UV-Vis spectrophotometer.

[0056] Different morphologies and crystal phases may be associated with differentiated catalytic performance. This invention employs a TMB-based colorimetric method to evaluate the oxidase-mimicking activities of MnO2-I / II / III.

[0057] like Figure 5 As shown in Figure A, when MnO2 and TMB coexist, obvious absorption peaks were observed at 370 nm, 450 nm, and 652 nm. These correspond to the two-electron oxidation product of TMB (diamine, 450 nm) and the charge-transfer complex formed with the original TMB molecule (i.e., the one-electron oxidation product, 370 nm and 652 nm), respectively, indicating that all three MnO2 variants have significant oxidase-like activity.

[0058] (2) Steady-state kinetic analysis: By varying the concentration of the substrate TMB (0.1~2.0 mM) and keeping the concentration of MnO2-I / II / III nanozymes constant (1 mM), the initial reaction rate was measured to evaluate its steady-state kinetics. The Lineweaver-Burk double reciprocal plotting method (formula: 1 / V=K) was used. m / V max (1 / [S]+1 / K m ) Calculate the Michaelis constant K m Value and maximum reaction rate V max Value. Where [S] is the concentration of the substrate TMB, and V is the reaction rate at the corresponding substrate concentration.

[0059] Under optimized conditions (reaction pH 3.0, reaction time 15 min, temperature 37℃), Figure 5 Steady-state dynamics analysis was performed on the BD model, and the results showed a good fit with the Mie model. Figure 6 The calculated kinetic parameters, including the Michaelis constant and the maximum initial reaction rate, are as follows for MnO2-I / II / III (with TMB as the substrate): K m =2.42μM, 1.63μM, 0.66μM; V max =5.64 μM / min, 2.84 μM / min, 1.40 μM / min. Compared with reported MnO2-based nanozyme oxidase mimics (Table 1), the MnO2-I / II / III oxidase mimics of the present invention exhibit higher substrate affinity. Furthermore, the MnO2-I / II / III nanozymes retained approximately 80% of their initial activity after storage at 4 °C for 7 weeks, demonstrating good reproducibility and storage stability. Figure 7 ).

[0060] Table 1 Comparison of kinetic parameters of manganese dioxide oxidase mimics

[0061] III. Design of Colorimetric Arrays for Flavonoid-Mediated Catalysis (1) The significant catalytic differences between MnO2-I / II / III provide a basis for constructing a multi-channel colorimetric sensor array. To explore the feasibility of using MnO2-I / II / III as an integrated three-channel sensor array for visual differentiation of closely related citrus plants, this invention selected six representative bioactive flavonoids (Eri, Neo, Nar, Nari, Hes, NH) screened previously as model compounds. By adjusting the amounts of MnO2 and TMB, the reaction products exhibited different colors from blue (MnO2-II channel), green (MnO2-III channel) to yellow (MnO2-I channel), indicating that the two oxidation products of TMB coexist, which may be related to the different reaction kinetics of MnO2-I / II / III. Notably, after co-incubation of MnO2-I / II / III with flavonoids, characteristic color spectra were produced, corresponding to significant absorbance changes at 450 nm, 652 nm, and 370 nm. Figure 8 ).

[0062] Therefore, this invention establishes a system composed of three pairs of flavonoid isomers ( Figure 9 A sensor array consisting of 6 targets × 3 channels and three MnO2-I / II / III nanozyme sensors (concentrations of 2, 1, and 2 mM, respectively) was observed, exhibiting a variety of color development patterns. Figure 10 A).

[0063] HCA (based on absorbance values ​​at 450 nm (channel I), 652 nm (channel II), and 370 nm (channel III) Figure 11 ) and PCA ( Figure 10 (B) verified that all six flavonoid targets could be completely distinguished, with a classification accuracy of 100%. In contrast, using only one or two signal channels would result in 42-83% data overlap. Figure 10 (CD).

[0064] (2) Concentration-dependent identification analysis: Each flavonoid was prepared into a series of concentration gradients (1, 2, 5, 10, 20, 50, 100, 200 μg / mL) and added to the sensor array above for reaction. The obtained data matrix has the following dimensions: n concentrations × 3 channels × 3 parallels. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were used to process the data to evaluate the array's ability to distinguish between different concentrations of flavonoids.

[0065] It is worth noting that the constructed three-channel colorimetric array can reliably distinguish six different flavonoids at concentrations as low as 1 μg / mL. Figure 12 It can also distinguish the same target flavonoid within a concentration gradient range of 1~200μg / mL. Figure 13 The six citrus products contain a range of highly similar components, with the main difference being the relative amounts of each component. Figure 14 ) (3) Identification and analysis of multiple groups of flavonoid mixtures: To evaluate the sensor array's ability to recognize complex mixtures, three types of mixture systems were prepared: binary mixtures: Eri and Hes mixed in different ratios (from 10:0 to 0:10, v / v); quaternary mixtures: Nar, Nari, Hes, and NH mixed in different ratios (ratios of 1:1:1:1, 1:1:1:2, 1:1:2:1, 1:2:1:1, and 2:1:1:1); and hexaternary mixtures: six flavonoids mixed in different ratios (ratios of 1:1:1:1:1:1:1, 1:1:1:1:1:5, 1:1:1:1:5:1, 1:1:1:5:1:1, 1:1:5:1:1:1, 1:5:1:1:1:1, and 5:1:1:1:1:1:1). In all mixtures, the concentration of each flavonoid was 500 μg / mL. After the mixture was added to the sensor array reaction, the resulting data matrix (dimensions of 11 / 5 / 7 mixtures × 3 channels × 3 parallels) was processed using linear discriminant analysis (LDA).

[0066] Based on this, the present invention tested the sensor array using models of increasing complexity (binary, quaternary, and hexaternary mixtures) to simulate its performance in real-world multi-component systems with similar compositions. Figure 15The AC results clearly show that all mixed combinations can be successfully separated by LDA.

[0067] (4) Anti-interference capability assessment To investigate the specificity of the sensor array, an anti-interference experiment was conducted. Six types of flavonoids (all at a concentration of 500 μg / mL) were mixed with potential interfering substances of equal concentration (500 μg / mL). These interfering substances included biomolecules: glucose, lysine, arginine, glycine, histidine, and serine; and metal ions: K+. + Na + Ca 2+ Mg 2+ Ce 3+ Ni 2+ Structurally similar flavonoids: luteolin, nobiletin, and naringenin. The above mixed solution was added to a standard reaction system and reacted at 37°C for 15 min. The absorbance values ​​at 450 nm, 652 nm, and 370 nm were measured and compared with the signals of pure flavonoid samples without interfering substances.

[0068] Its robustness in complex matrices was assessed through interference experiments. Common interfering agents (biomolecules) were also considered. Figure 15 D), metal ions ( Figure 15 E) and other flavonoids Figure 15 Even under conditions of F), the target flavonoids can still be clearly distinguished, confirming the excellent selectivity and practical sample application potential of the constructed three-channel sensor array. In summary, all the above results verify the distinguishing performance of the constructed three-channel colorimetric array on structurally similar flavonoids (even isomers), supporting its potential for accurate and sensitive analysis of complex citrus samples.

[0069] IV. Construction of Paper-Based Sensor Arrays for Real-Time Detection In addition to the changes in absorbance signals at three characteristic wavelengths, the yellow, blue, and green color features produced by the reaction can also be converted into three-channel RGB values ​​via a smartphone for chemometric analysis. Therefore, this invention proposes a paper-based visualization sensor array pre-loaded with MnO2-I / II / III nanozymes (…). Figure 16 (A). Notably, the post-reaction visual presentation is consistent with the solution-based visual detection results ( Figure 10 (A), and the RGB values ​​show a clear fingerprint pattern ( Figure 16 Based on the ratios of R / (G+B) (channel I), B / (R+B) (channel II), and G / (R+B) (channel III), hierarchical clustering analysis, principal component analysis, and three-dimensional linear discriminant analysis showed that all three pairs of flavonoid isomers could be clearly separated with a discrimination accuracy of 100%. Figure 16The presence of CE (Citrus Fiber Optic) indicates that the platform has real-time detection and analysis capabilities, which lays the research foundation for on-site identification of closely related citrus plants.

[0070] V. Immediate Identification of Citrus Products This study prepared citrus products from different origins (ZQ, ZS, CP) and different medicinal parts (HYF, QZQ, HYP) to verify the practical utility of the developed paper-based sensor array. Figure 17 (AB). This paper-based sensor array generates unique RGB fingerprint spectra for the tested citrus samples. Figure 17 The PCA results from three parallel measurements showed that the samples could be accurately clustered, with a classification accuracy of 100%. Figure 17 (D). Three-dimensional LDA further confirmed that all six samples were clearly separated without any overlap. Figure 17 The results effectively demonstrated the array's ability to distinguish different parts of closely related citrus fruits. Consistent results were obtained from the absorbance values ​​at 450 nm, 652 nm, and 370 nm extracted based on the colorimetric response in the solution sensing system. Figure 18 This indicates that the proposed three-channel sensor array has excellent discrimination performance on both paper-based and solution-based platforms.

[0071] In real-world scenarios, the visual differences in appearance between citrus medicinal materials and their powders are extremely small, making adulteration and unintentional mixing difficult to avoid. This invention further evaluates the ability of the developed paper-based colorimetric array to identify adulterated samples. HYP powder and CP were mixed at different mass fractions (0%~100%). HCA, PCA, and LDA (such as...) Figure 19 As shown in the figure, parallel samples with the same adulteration ratio clustered together, while groups with different mixing ratios were clearly separated from each other. In summary, these results confirm that the proposed three-channel colorimetric sensor array provides a fast, reliable, and readily implementable practical solution for market adulteration screening.

[0072] VI. Flavonoid-mediated MnO2 catalytic mechanism Flavonoids are generally considered to act primarily as reducing agents in catalytic colorimetric reaction systems. Therefore, this invention evaluated the reducing effect of flavonoids by co-incubating them with MnO2 and TMB and conducting free radical scavenging experiments. Figure 8 AC and Figure 20 As shown in AC, absorbance signals (450, 652, and 370 nm) and free radical intermediates (•OH, O2) are observed. - and 1 The decrease in O2 confirmed the reducing effect of flavonoids. However, in the MnO2-I / III system, Nar and Nari were observed to induce signal enhancement (…). Figure 8 (A, C). More interestingly, compared to co-incubation, the sequential addition of flavonoids after oxTMB formation resulted in a less severe signal decrease (A, C). Figure 20 (DF). All the above evidence strongly suggests that, in addition to reduction, there are other interaction mechanisms between flavonoids and MnO2-I / II / III nanozymes that affect the results of their catalytic colorimetric reactions.

[0073] First, this invention uses NH as a model flavonoid and focuses on the flavonoid-MnO2 interaction. The 5-hydroxyl and 4-carbonyl groups on the flavonoid core often serve as key coordination sites with metal oxides such as MnO2. Therefore, preliminary investigations were conducted using UV-Vis spectroscopy and Fourier transform infrared spectroscopy. Figure 21 The strong absorption in the 240–280 nm region of the UV-Vis spectrum of A corresponds to the π–π transition of the flavonoid skeleton benzoyl system, while the absorption in the 300–400 nm region originates from the π–π transition of the cinnamic yl group.

[0074] In all three MnO2-I / II / III-NH systems, a key blue shift was observed in the 300–400 nm region, indicating that the electronic transition occurred in the cinnamoyl moiety (B ring). FTIR analysis ( Figure 21 As can be seen from B), Mn-O-Mn (e.g., 705cm) -1 Move to 700cm -1 ) and Mn-O (such as 505cm) -1 Move to 500cm -1 The significant redshift of the vibration further confirms the coordination interaction between NH (through C=O and OH groups) and Mn sites on the MnO2 surface, indicating the formation of the MnO2-I / II / III-NH complex.

[0075] Secondly, the changes in microstructure and electronic structure after the interaction of MnO2-I / II / III with NH were investigated. Slightly shifted matching peaks in the diffraction angles were observed in the XRD pattern. Figure 21 The changes (C) confirmed that no new crystalline phase was formed. These changes indicate that the redox reaction altered the exposure of the active crystal facets or the surface band structure, thereby affecting electron transfer. Subsequently, the morphology of MnO2-I / II / III-NH was investigated in more detail using SEM and HRTEM. Figure 21 DF and Figure 22 The results showed that the introduction of NH etched the surface of the MnO2 nanostructure to varying degrees. This is attributed to the active phenolic hydroxyl groups on the flavonoid core, which may introduce new defect structures and active catalytic sites through surface reduction and coordination processes. The electronic structures of MnO2 (I, II, and III) before and after interaction with NH were characterized by XPS. Figure 4 C (front) Figure 23 (After) and Figure 21 Specifically, a decrease in the atomic percentages of Mn and O was observed, while the C content increased significantly (compared to GH). Figure 4 The presence of background carbon signals in the C indicates that carbon-containing functional groups or a carbonaceous layer were successfully introduced onto the MnO2 surface during NH modification, potentially partially masking the Mn and O signals of the substrate MnO2. O1s spectrum ( Figure 21 In the MnO2-III-NH complex, the peaks at 533.0 eV, 531.5 eV, and 529.7 eV correspond to OC=O, Mn-OH, and Mn-O-Mn, respectively. In the MnO2-III-NH complex, OC=O, Mn-OH, and Mn-O-Mn all shift towards higher binding energies, confirming the transfer of electrons from NH to MnO2. The lattice oxygen (Mn-O-Mn) content decreased significantly from 88.82% to 47.66%, indicating the introduction of structural defects. Furthermore, the Mn2p spectrum revealed changes in the main valence state of manganese (…). Figure 21 H), characterized by Mn 4+ Reduce, while Mn 3+ / Mn 2+ The oxidation state change is consistent with the electron transfer observed in the O1s spectrum. This electron transfer is mediated by flavonoid-induced surface modification, which also plays a key role in catalytic redox processes.

[0076] From the perspective of the common structural characteristics of flavonoids, the interaction mode between citrus flavonoids and MnO2 is closely related to the number and position of hydroxyl (-OH), methoxy (-OCH3), and glycosidic groups (-Glc). Figure 21 Interestingly, the catechol structure (4,5-dihydroxyl group on ring B) is a key part occupying the electron transfer site of MnO2 and may inhibit catalytic activity. For example, Eri and Neo (with 4,5-dihydroxyl group on ring B) Figure 24 A, D, B, E) showed better performance than Nar and Nari (which have only one 4-hydroxyl group). Figure 24 The C, F, G, and J groups exhibit significantly stronger inhibitory effects. Furthermore, the methoxy group, acting as an electron donor, may enhance reducing power when accessible, while its steric position ultimately determines the net outcome of the interaction due to steric hindrance. Additionally, the hydrophilicity and large steric volume of the glycosidic group introduce steric hindrance, hindering close contact between the flavonoid core and the catalyst Mn site, potentially masking the crucial coordinating hydroxyl groups. This established structure-activity relationship explains the unique dose-response curves and color fading kinetics of the six structurally similar flavonoids. Figure 24 This aligns with their unique ability to inhibit or enhance the catalytic performance of MnO2.

[0077] Overall, the sensing mechanism of MnO2-I / II / III for flavonoids / citrus plants is not a simple redox or coordination process. The synergistic interaction between the catalytic center of the MnO2 nanozyme and the structure of the flavonoid is crucial, mediating dynamic electron transfer and catalytic sensing mechanisms. These diverse mechanisms actively contribute to the multi-response properties of the constructed colorimetric array, thereby aiding in the generation of a cross-reaction database for accurate identification. Importantly, the proposed structure-dependent activity regulation may provide key guidance for designing novel sensing arrays to effectively identify highly similar target substances.

[0078] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A three-channel MnO2 nanozyme colorimetric sensing array, characterized in that, It includes three detection channels, each containing a MnO2 nanozyme with oxidase-like activity; The first channel includes an α-MnO2 nanozyme with a nanorod morphology; The second channel includes δ-MnO2 nanozymes with a mixed morphology of flower-like and nanotube structures; The third channel includes δ-MnO2 nanozymes with a flower-like lamellar morphology; The MnO2 nanozyme exhibits specific color responses at 370nm, 450nm, and 652nm in the TMB chromogenic system, forming a cross-reactive fingerprint spectrum.

2. The three-channel MnO2 nanozyme colorimetric sensing array according to claim 1, characterized in that, The α-MnO2 nanozyme with a nanorod morphology has a diameter of 25~45nm, a length of 350~820nm, and a lattice fringe spacing of 2.4Å. The lattice fringe spacing of the δ-MnO2 nanozyme with a mixed morphology of flower-like and nanotube is 2.4 Å. The δ-MnO2 nanozyme with a flower-like layered morphology has pores with a diameter of 10~80nm between its layers and a lattice fringe spacing of 3.5Å.

3. The three-channel MnO2 nanozyme colorimetric sensing array according to claim 1 or 2, characterized in that, The preparation method of the α-MnO2 nanozyme with nanorod morphology includes: mixing potassium permanganate solution and hydrochloric acid solution, and then carrying out a hydrothermal reaction to obtain the α-MnO2 nanozyme with nanorod morphology; the hydrothermal reaction temperature is 140℃ and the time is 12h. The preparation method of the δ-MnO2 nanozyme with a mixed morphology of flower-like and nanotube features includes: mixing potassium permanganate solution and hydrochloric acid solution, and then carrying out a hydrothermal reaction to obtain the δ-MnO2 nanozyme with a mixed morphology of flower-like and nanotube features; the hydrothermal reaction is carried out at a temperature of 110°C for a time of 6 hours. The preparation method of the δ-MnO2 nanozyme with flower-like lamellar morphology includes: mixing potassium permanganate solution and manganese sulfate, and then carrying out a hydrothermal reaction to obtain the δ-MnO2 nanozyme with flower-like lamellar morphology; the hydrothermal reaction temperature is 160℃ and the time is 12h.

4. The three-channel MnO2 nanozyme colorimetric sensing array according to claim 1, characterized in that, In the first channel, the concentration of MnO2 nanozyme is 2 mM and the concentration of TMB is 0.5 mM; In the second channel, the concentration of MnO2 nanozyme is 1 mM and the concentration of TMB is 2 mM; In the third channel, the concentration of MnO2 nanozyme is 2 mM and the concentration of TMB is 1 mM. Each channel contains 0.1M HAc-NaAc buffer at pH 3.

0.

5. A paper-based colorimetric sensor, characterized in that, include: A solid support, wherein the solid support is a filter paper sheet; The three-channel MnO2 nanoenzyme colorimetric sensor array according to any one of claims 1 to 4, loaded on the solid support.

6. The application of the three-channel MnO2 nanoenzyme colorimetric sensor array according to any one of claims 1 to 4 or the paper-based colorimetric sensor according to claim 5 in the instant identification of flavonoid isomers or the identification of citrus varieties.

7. A method for sample identification, characterized in that, The three-channel MnO2 nanoenzyme colorimetric sensor array according to any one of claims 1 to 4 or the paper-based colorimetric sensor according to claim 5 is used; The test samples were added to the three channels respectively to carry out the reaction; the test samples included flavonoid isomers or citrus extracts. Detect the absorbance or RGB value signals of each channel at 370nm, 450nm and 652nm; The obtained absorbance or RGB values ​​are combined to form a fingerprint spectrum, and the fingerprint spectrum is analyzed using machine algorithms to achieve sample identification.

8. The sample identification method according to claim 7, characterized in that, The reaction was carried out at a temperature of 37°C for 15 minutes.

9. The sample identification method according to claim 7, characterized in that, The RGB value signal is as follows: the first channel uses the R / (G+B) value, the second channel uses the B / (R+B) value, and the third channel uses the G / (R+B) value. The machine algorithms include hierarchical clustering analysis, principal component analysis, and three-dimensional linear discriminant analysis.

10. The sample identification method according to claim 7, characterized in that, The flavonoid isomers include at least one of the following: senna-2, neo-senna-2, rutin, naringin, hesperidin, and neo-hesperidin; The detection limit of the sample identification method is 1 μg / mL.

Images