Spinel-structured nano enzyme as well as application and detection method thereof

Abstract

The invention belongs to the field of nano-materials, and particularly relates to a nano-enzyme with a spinel structure as well as application and a detection method of the nano-enzyme. The spinel-structured nano enzyme has oxidase-like activity or peroxidase-like activity, and is used for preparing oxidase-like, peroxidase-like, reagents, kits, materials, sensing platforms or sensors for detecting organic matters or inorganic matters. The method for detecting the organic matter or the inorganic matter based on the spinel structure nano enzyme has the advantages of high sensitivity, rapidness, low cost and strong specificity, and is particularly suitable for constructing a detection platform by taking alkaline phosphatase as a target detection object.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterials, and specifically relates to a spinel-structured nanozyme and an application and detection method thereof. Background Art

[0002] Alkaline phosphatase (ALP) is a phosphate monoester hydrolase widely distributed in the human body, which is mainly responsible for the hydrolysis of bioactive molecules containing phosphate groups. In biological systems, the activity state of ALP is closely related to human health. The determination of ALP activity level has been used clinically for the diagnosis of many diseases, such as obstructive jaundice, cholestatic hepatitis, osteoporosis, prostate cancer, etc. To date, a variety of methods for analyzing ALP activity have been established, such as electrochemistry, chromatography, and fluorescence. However, most of these analytical strategies require sophisticated instruments, complex operations, and high costs. These necessary requirements limit the application of these methods to conveniently detect alkaline phosphatase. At present, colorimetric methods based on enzymatic reactions and specific chromogenic substrates have attracted much attention due to their simple operation and low cost, providing a fast and convenient strategy for determining biological enzyme activity.

[0003] Compared with conventional artificial enzymes, nanozymes have higher catalytic activity and controllability. A large number of experiments have shown that regulating the size and morphology of nanozymes can significantly improve the catalytic performance of nanozymes. In addition, nanozymes are widely used because of their excellent physical and chemical properties (such as magnetic, photothermal and other properties) and catalytic properties. The medical concept of nanozymes is extending from "in vitro diagnosis" to "the prevention and treatment of major diseases such as tumors, infectious diseases, degenerative lesions, and strokes," which has laid the foundation for the clinical application of nanozymes. The new concepts, new materials and new technologies related to nanozymes have not only brought new vitality to traditional artificial enzymes and broadened the scope of innovation and application of enzyme engineering technology, but also effectively integrated the theories and technologies of multiple disciplines such as nanotechnology, chemistry, and biomedicine, making it a new battlefield for cross-integration.

[0004] Nanozyme-based colorimetric assays for alkaline phosphatase activity have been extensively studied. In practical applications, to achieve convenient detection of ALP activity, the operation needs to be simplified. Now smartphones can obtain the RGB (red, green, blue) values ​​of color images through image processing. Therefore, the combination of smartphones and color reactions can be used for quantitative detection of certain analytes. This research has far-reaching significance for improving sensitivity and accuracy and developing rapid and obvious colorimetric reactions. Summary of the invention

[0005] The present invention provides a highly sensitive, rapid, low-cost, highly specific, and easy-to-operate detection method and detection platform, which is based on the spinel-structured nanozyme Li x Mn2O4.

[0006] The present invention provides a spinel-structured nanozyme having oxidase-like activity, oxidase-mimicking enzyme activity, peroxidase-like activity, or peroxidase-mimicking enzyme activity, and containing at least one of the following spinel-structured lithium manganate Li x Mn2O4, x=any number in the range of 0.1-2.

[0007] Further, x=any number in the range of 0.5-1.5; further, x=any number in the range of 0.7-1.3; further, x=any number in the range of 0.9-1.2, and further, x=any number in the range of 0.9-1; such as x=0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3.

[0008] The method for preparing the spinel structure nanozyme of the present invention comprises the following steps: mixing lithium nitrate and manganese (II) nitrate hydrate in ethylene glycol, adding sodium acetate and polyethylene glycol, and performing a hydrothermal reaction after mixing.

[0009] The molar ratio of sodium acetate, lithium nitrate and manganese (II) nitrate hydrate is 8-10:0.35-0.65:1, preferably 8.5-9.5:0.5-0.6:1; the concentration of sodium acetate is 0.8-1.2 mol / L, preferably 0.9-1.1 mol / L; the mass volume ratio of polyethylene glycol to ethylene glycol is 0.8-1.2 g:40 mL, preferably 0.9-1 g:40 mL.

[0010] The hydrothermal reaction is carried out in a hydrothermal reactor, the temperature of the hydrothermal reaction is 190-200° C., and the reaction time is 18-20 hours.

[0011] The product obtained by the hydrothermal reaction is washed and dried. A preferred embodiment of the present invention is as follows: washing with water and ethanol three times in sequence, and drying under vacuum at 80°C.

[0012] The spinel-structured nanozyme provided by the present invention has oxidase-like activity, or oxidase-mimicking enzyme activity, or peroxidase-like activity, or peroxidase-mimicking enzyme activity, catalyzes the redox reaction of a substrate (color indicator, such as TMB), or catalyzes a substance with oxidative activity to participate in the redox reaction of a substrate (color indicator, such as TMB), the substrate (color indicator, such as TMB) is oxidized and changes color, and qualitative and / or quantitative determination is performed by measuring the absorbance value of the ultraviolet-visible absorption spectrum, or extracting the RGB value of the image, and the quantitative determination includes an external standard method or an internal standard method, and the external standard method also includes a standard curve method or a correction factor method. The above-mentioned substance with oxidative activity is H2O2.

[0013] Further, the substance with reduction activity can reduce the oxidized substrate (color indicator, such as TMB), and the oxidized substrate (color indicator, such as TMB) changes color due to reduction. The detection of the substance with reduction activity is converted into the detection of the substrate (color indicator, such as TMB) through the redox reaction of the substrate (color indicator, such as TMB), and the absorbance value of the ultraviolet-visible absorption spectrum is measured, or the RGB value of the image is extracted to perform qualitative or quantitative determination. The quantitative determination includes an external standard method or an internal standard method, and the external standard method includes a standard curve method or a correction factor method. The above-mentioned substance with reduction activity includes ascorbic acid (AA), trivalent cobalt salts, persulfates, peroxides, potassium dichromate, potassium permanganate, oxoacid salts, concentrated sulfuric acid, etc.

[0014] As demonstrated in the present invention, ascorbic acid AA is reducing and can reduce oxidized substrates (chromogenic indicators, TMB). Ascorbic acid AA is obtained by hydrolyzing L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate AA2P by alkaline phosphatase ALP. There is a correlation between the AA2P hydrolysis reaction catalyzed by alkaline phosphatase and the redox reaction of the substrate (chromogenic indicator, TMB) catalyzed by the spinel-structured nanozyme provided by the present invention. The two reactions are combined to convert the detection of ascorbic acid AA into qualitative and / or quantitative detection of oxidized and / or reduced substrates (chromogenic indicators, such as TMB), especially to convert the detection of alkaline phosphatase into qualitative and / or quantitative detection of oxidized and / or reduced substrates (chromogenic indicators, such as TMB).

[0015] Furthermore, the substance with associated activity with the spinel structure nanozyme provided by the present invention, the above-mentioned substance with oxidation activity, and the above-mentioned substance with reduction activity, through the redox reaction of the substrate (color indicator, such as TMB) as described above, converts its qualitative and or quantitative detection into qualitative and / or quantitative detection of the substrate (color indicator, such as TMB). The above-mentioned substance with associated activity can be bound to the spinel structure nanozyme provided by the present invention, to the above-mentioned substance with oxidation activity, and to the above-mentioned substance with reduction activity through chemical bonds, intermolecular forces, etc., and can also establish an associated connection between the above-mentioned substance with associated activity and the spinel structure nanozyme provided by the present invention, the above-mentioned substance with oxidation activity, and the above-mentioned substance with reduction activity through an associated chemical reaction, thereby realizing the conversion of the above-mentioned substance with associated activity through the redox reaction of the substrate (color indicator, such as TMB) into qualitative and / or quantitative detection of the substrate (color indicator, such as TMB). For example, in the detection of alkaline phosphatase ALP, the qualitative and / or quantitative detection of alkaline phosphatase ALP is converted into the qualitative and / or quantitative detection of substrate (color indicator, TMB) by virtue of the association between the reaction of alkaline phosphatase ALP catalyzing the hydrolysis of AA2P to generate ascorbic acid AA and the redox reaction of the substrate (color indicator, TMB) catalyzed by the nanozyme of the spinel structure provided by the present invention in which ascorbic acid AA participates. For example, in the detection of target nucleic acid or protein, the target nucleic acid or protein can be bound to the nanozyme of the spinel structure provided by the present invention, to the above-mentioned substance with oxidation activity, and to the above-mentioned substance with reduction activity by virtue of base pairing between nucleic acid sequences, protein affinity reagents, and chemical organic bonding, thereby converting the detection of target nucleic acid or protein from qualitative and / or quantitative detection to qualitative and / or quantitative detection of substrate (color indicator, TMB). The above-mentioned substances with associated activity include organic matter and inorganic matter. Organic matter includes fat, amino acid, protein, sugar, hemoglobin, chlorophyll, enzyme, hormone, nucleic acid, etc., and inorganic matter includes oxides, acids, alkalis and salts, etc.

[0016] The substrate is a color indicator, and the color indicator is a redox color indicator that changes color due to redox, including TMB.

[0017] The spinel-structured nanozyme provided by the present invention can be used to prepare:

[0018] 1) Oxidase-like enzymes or oxidase-mimicking enzymes;

[0019] 2) Peroxidase-like or peroxidase-mimicking enzymes;

[0020] 3) Reagents, detection systems, kits, materials, detection platforms, or detectors for detecting oxidatively active substances;

[0021] 4) Reagents, detection systems, kits, materials, detection platforms, or detectors for detecting reducing active substances;

[0022] 5) The nanozyme, oxidative active substance, or reductive active substance having a spinel structure has a reagent for detecting a related active substance, or a detection system, or a kit, or a material, or a detection platform, or a detector.

[0023] The following products provided by the present invention contain the spinel-structured nanozyme provided by the present invention:

[0024] 1) Oxidase-like enzymes or oxidase-mimicking enzymes;

[0025] 2) Peroxidase-like or peroxidase-mimicking enzymes;

[0026] 3) Reagents, detection systems, kits, materials, detection platforms, or detectors for detecting oxidatively active substances;

[0027] 4) Reagents, detection systems, kits, materials, detection platforms, or detectors for detecting reducing active substances;

[0028] 5) The nanozyme, oxidative active substance, or reductive active substance having a spinel structure has a reagent for detecting a related active substance, or a detection system, or a kit, or a material, or a detection platform, or a detector.

[0029] The spinel structure nanozyme provided by the present invention can be used to detect oxidatively active substances, reductively active substances, and to detect substances having associated activities with the spinel structure nanozyme, oxidatively active substances, or reductively active substances.

[0030] The above-mentioned oxidizing active substance is H2O2.

[0031] The above-mentioned reducing active substances include ascorbic acid (AA), trivalent cobalt salts, persulfates, peroxides, potassium dichromate, potassium permanganate, oxoacid salts, concentrated sulfuric acid and the like.

[0032] The above-mentioned substances with associated activity establish associations with spinel-structured nanozymes, or oxidative active substances, or reductive active substances through chemical bonds, intermolecular forces, and associated chemical reactions. They include organic and inorganic substances. Organic substances include fats, amino acids, proteins, sugars, heme, chlorophyll, enzymes, hormones, nucleic acids, etc. Enzymes include alkaline phosphatase. Inorganic substances include oxides, acids, bases, and salts, etc.

[0033] The present invention provides a colorimetric sensing detection method for oxidatively active substances based on spinel structure nanozymes, the steps comprising:

[0034] The analyte containing oxidative active substances, the spinel structured nanozyme provided by the present invention, and the color indicator are placed in a reaction system for reaction, and qualitative and / or quantitative determination is performed by measuring the absorbance value of the ultraviolet-visible absorption spectrum or extracting the RGB value of the image.

[0035] The present invention provides a colorimetric sensing detection method for reducing active substances based on spinel structure nanozymes, the steps comprising:

[0036] The spinel-structured nanozyme and color indicator provided by the present invention are placed in a reaction system for reaction, and then reacted with an analyte containing a reducing active substance, and qualitative and / or quantitative determination is performed by measuring the absorbance value of the ultraviolet-visible absorption spectrum or extracting the RGB value of the image.

[0037] Oxidative active substances may also be added to the reaction system containing the spinel structure nanozyme and the color indicator.

[0038] The present invention provides an instant colorimetric sensing detection method for spinel structure nanozymes, the steps comprising:

[0039] The spinel structured nanozyme and color indicator provided by the present invention are placed in a reaction system for reaction, and then reacted with the reducing active substance. Qualitative and / or quantitative determination is performed by measuring the absorbance value of the ultraviolet-visible absorption spectrum or extracting the RGB value of the image.

[0040] The nanozyme or reduction-active substance with a spinel structure has an associated active substance that reacts with the nanozyme or reduction-active substance with a spinel structure associated therewith.

[0041] An oxidatively active substance may also be added to the reaction system containing the spinel-structured nanozyme and the color indicator, and the active substance associated with the oxidatively active substance reacts with the associated oxidatively active substance.

[0042] The above-mentioned color indicator is a redox color indicator, which changes color due to redox, including TMB.

[0043] As an embodiment of the present invention, when performing alkaline phosphatase detection, the present invention provides an instant colorimetric sensing detection method of spinel structure nanozyme, the steps comprising:

[0044] Prepare a mixed solution B, add the spinel-structured nanozyme provided by the present invention and TMB into a fixed buffer for reaction to obtain a mixed solution B;

[0045] Prepare a mixed solution A, add a test substance containing alkaline phosphatase ALP and L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate AA2P into an incubation buffer for incubation to obtain a mixed solution A;

[0046] After the mixed solution B is mixed with the mixed solution A, qualitative and / or quantitative determination is performed by measuring the absorbance value of the UV-visible absorption spectrum or extracting the RGB value of the image.

[0047] Furthermore, after the mixed solution B is mixed with the mixed solution A, it is dropped into the paper base, and the RGB value of the paper base image is extracted for qualitative and / or quantitative determination.

[0048] Furthermore, the quantitative determination includes an external standard method or an internal standard method, and the external standard method includes a standard curve method or a correction factor method. The standard curve method is a linear curve constructed by fitting the RGB value and the corresponding concentration of alkaline phosphatase ALP.

[0049] Furthermore, the fixing buffer is 10 mM Tris-HCl, pH 4.0; the incubation buffer is 50 mM Tris-HCl, pH 8.0, containing 0.1 mM MgCl2.

[0050] When alkaline phosphatase is used as the target for analysis and detection, the spinel nanozyme Li x The redox reaction of Mn2O4 and TMB was detected by extracting the RGB values ​​of the image through a smartphone, and a colorimetric detection platform based on spinel structure nanozymes was established.

[0051] Compared with the prior art, the advantages of the present invention are:

[0052] The present invention provides a highly sensitive, rapid, low-cost, and highly specific detection platform. As shown in the embodiments of the present invention, it is particularly suitable for detecting ALP concentration. The present invention utilizes the spinel structure nanozyme Li x Mn2O4 (especially Li 0.9 Mn2O4) has the characteristics of simple synthesis and high catalytic activity. At the same time, combined with the advantages of convenient and fast extraction of RGB by smartphone, rapid detection and low detection cost, the colorimetric detection of ALP concentration is realized, and the minimum detection limit of colorimetric detection is 2.01mU / mL. In addition, the combination of smartphone and color reaction can be used for quantitative detection of certain analytes. This research has far-reaching significance for improving sensitivity and accuracy and developing fast and obvious colorimetric reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of the principle of colorimetric detection of ALP concentration based on instant detection of spinel nanozymes.

[0054] Figure 2 The spinel nanozyme Li 0.9 SEM image of Mn2O4.

[0055] Figure 3The law of ALP concentration and absorbance (a) and the linear relationship between ALP concentration and absorbance (b).

[0056] Figure 4 It is a linear standard curve of different ALP concentration values ​​and RGB values.

[0057] Figure 5 To verify the specificity of the detection platform. DETAILED DESCRIPTION

[0058] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] The mechanism of colorimetric sensing of ALP concentration based on spinel nanozymes is as follows Figure 1 As shown. On the one hand, Li 0.9 Mn2O4 spinel structure nanozyme has oxidase-like activity (OXD), which can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to turn blue and produce a colorimetric signal. On the other hand, under alkaline conditions, ALP can hydrolyze AA2P to decompose ascorbic acid (AA). The more AA is produced, the stronger the reducing property is, which can weaken the colorimetric signal. By extracting the RGB value of the colorimetric signal through a smart phone, a linear relationship between the ALP concentration and the RGB value can be established. Therefore, the present invention provides a highly sensitive, rapid, low-cost, and highly specific ALP detection platform.

[0060] Example 1 Spinel Nanozyme Li 0.9 Synthesis of Mn2O4

[0061] Weigh 0.189g lithium nitrate (2.75mmol) and 0.895g manganese (II) nitrate hydrate (5mmol) into 40mL ethylene glycol and stir magnetically for 10 minutes to make them evenly dispersed. Add 3.6g sodium acetate and 1.0g polyethylene glycol 400 and continue stirring for 20 minutes. Then transfer the mixed solution to a 100mL polytetrafluoroethylene reactor and heat treat at 200℃ for 20 hours. After the reaction is cooled, transfer it to a beaker and wash it with deionized water and anhydrous ethanol three times in sequence. Use a centrifuge to separate the nanopowder during each washing process, and finally place it in a vacuum drying oven at 80℃ overnight to obtain Li 0.9 Mn2O4 spinel nanopowder. Figure 2 shown.

[0062] Example 2 ALP concentration and absorbance law and linear relationship

[0063] 50 μL of ALP of different concentrations and 50 μL of 15 mM AA2P substrate were mixed in 100 μL Tris-HCl (50 mM, pH 8.0). Then, the above mixture was reacted at 37°C for 1 hour to obtain mixture A. 10 μL of 1 mg / mL Li 0.9 Mn2O4 spinel nanozyme solution and 3μL 10mM 3,3',5,5'-tetramethylbenzidine solution were added to 287μL Tris-HCl (10mM, pH 4.0) in turn, incubated at room temperature for 10 minutes, and centrifuged to obtain supernatant B. 10μL of mixture A was added to 290μL supernatant B, incubated at room temperature for 1 minute, and the absorbance of the mixed solution at 652nm was measured. The regular relationship between ALP concentration and absorbance was obtained as follows: Figure 3 (a) As shown. The linear relationship between absorbance value and ALP concentration was obtained by fitting a linear equation as shown in Figure 3 As shown in (b), the detectable ALP concentration range is 0-7.5 mU / mL, and its minimum detection limit is 0.68 mU / mL.

[0064] Example 3 Application of spinel structure nanozyme to detect alkaline phosphatase

[0065] Repeat the steps of Example 2 to obtain mixture A and supernatant B. Take a circular colorimetric test paper with a diameter of 1 cm, drop 48 μL of supernatant B and 2 μL of mixture A on the test paper, respectively, and ultrasonically vibrate for 10 minutes to obtain a colorimetric test paper with uniform color. The color on the test paper is photographed by a smartphone to obtain the RGB value of the color. Fitting finds that the ALP concentration is linearly correlated with the value of (G+B) / R, such as Figure 4 The standard curve equation shown is y = 3.9320-0.2554x, and the correlation coefficient is R 2 =0.9667, and the detection limit is 2.01 mU / mL, indicating that the present invention can detect ALP of unknown concentration.

[0066] Example 4 Specificity of the detection platform

[0067] Papain (Pap), sucrase (Inv), trypsin (Try), pepsin (Rep), glucose oxidase (Gox), lysozyme (Lys) and proteinase K (RapK) were added at 50 times the concentration of ALP, and the absorbance values ​​with and without ALP were compared. Figure 5 As shown, the absorbance value containing ALP is much smaller than the absorbance value without ALP, indicating that the detection platform based on instant detection of spinel structure nanozyme colorimetric detection of ALP has excellent specificity.

[0068] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A spinel-structured nanozyme, characterized in that: Lithium manganate Li having oxidase-like activity, oxidase-mimicking enzyme activity, peroxidase-like activity, or peroxidase-mimicking enzyme activity, containing at least one of the following spinel structures x Mn2O4, x=any number in the range of 0.1-2.

2. The nanozyme according to claim 1, characterized in that x = any number in the range of 0.5-1.

5.

3. The nanozyme according to claim 1 or 2 comprises at least one of the following applications: 1) preparing oxidase-like enzymes or oxidase mimetic enzymes; 2) preparing peroxidase-like enzymes or peroxidase mimetic enzymes; 3) preparing a reagent, a detection system, a kit, a material, a detection platform, or a detector for detecting oxidative active substances; 4) preparing a reagent, a detection system, a kit, a material, a detection platform, or a detector for detecting a reducing active substance; 5) Prepare a nanozyme with a spinel structure, or an oxidative active substance, or a reductive active substance having a reagent for detecting a related active substance, or a detection system, or a kit, or a material, or a detection platform, or a detector.

4. The product containing the nanozyme according to claim 1 or 2 comprises at least one of the following: 1) Oxidase-like enzymes or oxidase-mimicking enzymes; 2) Peroxidase-like or peroxidase-mimicking enzymes; 3) Reagents, detection systems, kits, materials, detection platforms, or detectors for detecting oxidatively active substances; 4) Reagents, detection systems, kits, materials, detection platforms, or detectors for detecting reducing active substances; 5) The nanozyme, oxidative active substance, or reductive active substance having a spinel structure has a reagent for detecting a related active substance, or a detection system, or a kit, or a material, or a detection platform, or a detector.

5. The nanozyme described in claim 1 or 2 can be used to detect oxidatively active substances, or reductively active substances, or substances with associated activities with the nanozymes, oxidatively active substances, or reductively active substances having the same spinel structure.

6. A colorimetric sensing method for detecting oxidative active substances based on spinel structure nanozymes, characterized in that the steps include: The analyte containing oxidative active substances, the nanozyme described in claim 1 or 2, and the color indicator are placed in a reaction system for reaction, and qualitative and / or quantitative determination is performed by measuring the absorbance value of the ultraviolet-visible absorption spectrum or extracting the RGB value of the image.

7. A colorimetric sensing method for detecting reduction-active substances based on spinel-structured nanozymes, characterized in that the steps include: The nanozyme and color indicator described in claim 1 or 2 are placed in a reaction system for reaction, and then reacted with the analyte containing the reducing active substance, and qualitative and / or quantitative determination is performed by measuring the absorbance value of the UV-visible absorption spectrum or extracting the RGB value of the image.

8. A method for instant colorimetric sensing of spinel nanozymes, characterized in that the steps include: The nanozyme and the color indicator according to claim 1 or 2 are placed in a reaction system for reaction, and then reacted with the reducing active substance, and qualitative and / or quantitative determination is performed by measuring the absorbance value of the ultraviolet-visible absorption spectrum or extracting the RGB value of the image; The substance having associated activity with the nanozyme or reduction-active substance as described in claim 1 or 2 reacts with its associated nanozyme or reduction-active substance.

9. The colorimetric sensing detection method according to claim 8, characterized in that: For the alkaline phosphatase test, the steps include: preparing a mixed solution B, adding the nanozyme according to claim 1 or 2 and TMB into a fixed buffer for reaction to obtain a mixed solution B; Prepare a mixed solution A, add a test substance containing alkaline phosphatase ALP and L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate AA2P into an incubation buffer for incubation to obtain a mixed solution A; After the mixed solution B is mixed with the mixed solution A, qualitative and / or quantitative determination is performed by measuring the absorbance value of the UV-visible absorption spectrum or extracting the RGB value of the image.

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