A MOFs nanoscale enzyme and application thereof in detection of new coronavirus

Abstract

A kind of MOFs nano-enzyme and application in new coronavirus detection, it relates to biological detection method field, MOFs-based nano-enzyme has peroxidase-like effect, can catalyze color developing agent discoloration;After the specific protein CD147 is modified on the surface of nano-enzyme, using the specific binding of CD147 and the S protein overexpressed on the surface of new coronavirus, new coronavirus can be efficiently combined on the surface of nano-enzyme to inhibit the catalytic color development of nano-enzyme;Based on the principle, the quantitative and qualitative detection of new coronavirus can be realized by colorimetric method;The present application realizes the rapid, high-sensitivity and visual detection of new coronavirus by the inherent catalytic advantage of nano-enzyme and the specific recognition and binding capacity of CD147 to new coronavirus without additional labeling process, and can effectively identify new coronavirus infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and clinical diagnostic technology, and specifically relates to a MOF nanozyme and its application in the detection of the novel coronavirus. Specifically, it is a method for visualizing, rapidly and highly sensitively detecting SARS-CoV-2 using MOF nanozymes. Background Technology

[0002] The novel coronavirus 2019-nCoV / SARS-CoV-2 is spreading globally, yet humanity lacks effective antiviral vaccines and specific drugs. Accurate detection and diagnosis of infected individuals or carriers are crucial for epidemic control. Currently, COVID-19 diagnostic strategies primarily rely on three methods: nucleic acid testing, antibody testing, and antigen testing. Nucleic acid testing is the "gold standard" for clinical diagnosis of COVID-19, detecting low levels of the virus based on nucleic acid amplification. Reverse transcription polymerase chain reaction (RT-PCR) combined with automated sample preparation systems can detect SARS-CoV-2 nucleic acid in nasopharynx or saliva in batches. However, nucleic acid test results are affected by factors such as patient disease progression, specimen collection, and the quality of testing reagents, with a positive detection rate of only 30-50%. Therefore, improving detection sensitivity and specificity remains an urgent problem to be solved. Furthermore, expensive and inflexible testing equipment limits its application in large-scale, multi-sample scenarios. Antibody testing primarily detects SARS-CoV-2-specific IgM and IgG antibodies in serum. It is a supplementary and rapid method for virus detection, but it is generally only applicable to those infected at least 10-14 days prior, which can lead to false negatives. To address these issues, various detection methods based on specific viral proteins (including antigens) have been proposed in recent years for identifying the pathogen due to their high sensitivity, selectivity, and rapid measurement. These methods can reduce the chance of cross-reactivity and effectively improve specificity. Among these methods, colorimetric assays are an attractive option due to their simplicity, cost-effectiveness, and ease of visual reading. This test has great potential for operation in remote locations without requiring complex equipment.

[0003] Enzyme catalysis, which catalyzes the color change of chromogenic molecules, is one of the most commonly used colorimetric methods. However, enzymes are easily inactivated under harsh conditions, limiting their application. In recent years, metal-organic frameworks (MOFs) have been widely used in the construction of colorimetric biosensors due to their peroxidase-like functions, such as decomposing H2O2 to produce ·OH, which oxidizes chromogenic reagents to produce color. Summary of the Invention

[0004] To overcome the shortcomings of existing COVID-19 detection methods, the present invention aims to provide a MOF nanozyme and its application in COVID-19 detection. This invention is a novel method for the simple, rapid, highly sensitive, label-free, and visual detection of SARS-CoV-2 based on MOF nanozymes. This method utilizes the unique peroxidase properties of MOF nanozymes to eliminate the need for additional labeling processes and achieves label-free, rapid, highly sensitive, and visual detection of SARS-CoV-2 through a simple colorimetric method.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] A MOF nanozyme uses terephthalic acid as a ligand to form a metallic MOF material through chelation with metal ions. The surface of the metallic MOF material has carboxyl groups, which condense with CD147 molecules containing amino groups, thereby fixing the CD147 molecules on the MOF surface. The metal ions are selected from one or more of Fe, Cu, Ni, and Pt.

[0007] Based on the above-mentioned application of MOF nanozymes in the detection of SARS-CoV-2, by utilizing the enzyme-like properties of MOFs and the specific binding ability of surface-modified CD147 protein to SARS-CoV-2, the molecular colorimetric ability of chromogenic molecules TMB, ABTS, OPD and PPD can be catalyzed to achieve colorimetric quantitative and qualitative detection of SARS-CoV-2.

[0008] The MOF nanozymes described above have peroxidase-like properties, which can catalyze the generation of hydroxyl radicals from hydrogen peroxide, oxidize chromogenic molecules TMB, ABTS, OPD, and PPD, and achieve the detection of the novel coronavirus by colorimetric method.

[0009] TMB is 3,3′,5,5′-tetramethylbenzidine,

[0010] ABT is 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate),

[0011] OPD stands for o-phenylenediamine.

[0012] PPD stands for p-phenylenediamine.

[0013] The MOF nanozyme has a surface modified with the specific protein CD147. By utilizing the specific binding of CD147 to the S protein overexpressed on the surface of SARS-CoV-2, the SARS-CoV-2 virus is bound to the surface of the nanozyme, thereby inhibiting the catalytic color development of the nanozyme and realizing the quantitative and qualitative analysis of SARS-CoV-2 virus.

[0014] After the MOF nanozymes were modified with CD147, the catalytic activity of the MOFs decreased, and their ability to oxidize TMB, ABTS, OPD and PPD molecules and change color was reduced.

[0015] The modified CD147 is formed by the condensation linking of the carboxyl group on the surface of the MOF nanozyme with the amino group of CD147.

[0016] The CD147 specifically binds to SARS-CoV-2, reducing the catalytic activity of MOF nanozymes and further weakening their ability to oxidize TMB, ABTS, OPD, and PPD molecules and cause discoloration.

[0017] Compared with existing biomolecular recognition and detection technologies, the advantages of this invention are:

[0018] 1. The preparation process of MOF nanozymes is simple and low in cost.

[0019] 2. MOFs have a large number of carboxyl functional groups on their surface, which enables the chemical fixation of CD147 molecules.

[0020] 3. MOF nanozymes possess high peroxidase-like properties, catalyzing the generation of ·OH from H₂O₂, which oxidizes the chromogenic molecules TMB, ABTS, OPD, and PPD, causing color changes. Upon recognition of SARS-CoV-2, the catalytic ability of MOFs decreases, and the ability to oxidize TMB, ABTS, OPD, and PPD weakens. The entire color development process is completed within 2 minutes. Utilizing this unique color development characteristic, rapid, visual, and highly sensitive detection of SARS-CoV-2 can be achieved.

[0021] 4. This invention is a novel label-free and visual detection method for SARS-CoV-2 that combines enzyme-catalyzed color development. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the principle of the detection method of the present invention for detecting SARS-CoV-2.

[0023] Figure 2 The images shown are electron microscope images of the MOFs prepared in Example 1 and schematic diagrams of the connection between metal ions and terephthalic acid, where (a) is an electron microscope image and (b) is a schematic diagram of the connection between metal ions and terephthalic acid.

[0024] Figure 3 The images show the infrared and Raman spectra of CD147 molecules immobilized in MOFs in Example 1, where (a) is the infrared spectrum after CD147 modification and (b) is the Raman spectrum after CD147 modification.

[0025] Figure 4This is the UV spectrum of the oxidation of TMB catalyzed by MOF nanozymes in Example 1 at pH 3.5, where curve ac represents TMB; H2O2 / TMB; and curve c represents MOFs / H2O2 / TMB.

[0026] Figure 5 The images show the UV spectra of TMB oxidation catalyzed by the surface modification of CD147 and SARS-CoV-2S-RBD proteins on the MOF nanozymes in Example 1. (a) shows the UV-Vis absorption spectra of TMB oxidation before and after modification with different substances, and (b) shows the absorbance of TMB oxidation at 652 nm after modification with different substances.

[0027] Figure 6 The image shows the UV spectra of TMB oxidation catalyzed by MOF nanozymes after modification with different concentrations of SARS-CoV-2S-RBD protein in Example 1.

[0028] Figure 7 The graphs show the relationship between the logarithm (ln C) and absorbance of different SARS-CoV-2S-RBD concentrations at 652 nm and the color change of the TMB solution in Example 1.

[0029] Figure 8 This is a graph showing the relationship between the logarithm of different SARS-CoV-2 pseudovirus concentrations (ln C) and absorbance at 652 nm in Example 1.

[0030] Figure 9 Ultraviolet spectra of TMB oxidation of different viral proteins.

[0031] Figure 10 This is a graph showing the relationship between the logarithm of different SARS-CoV-2 virus concentrations (ln C) and absorbance at 652 nm in Example 1. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0033] like Figure 1As shown, this invention discloses a MOF nanozyme and its application in the detection of SARS-CoV-2. It is a novel method for achieving highly sensitive and visual detection of SARS-CoV-2. Based on the unique enzyme-like properties of MOF nanozymes, under suitable conditions (pH 3-7), it can catalyze the color development of TMB, ABTS, OPD, and PPD molecules. The surface of the MOF nanozyme is rich in carboxyl functional groups, making it easy to modify. After modifying the nanozyme surface with the specific protein CD147, the specific binding of CD147 to the S protein overexpressed on the surface of SARS-CoV-2 can efficiently bind the SARS-CoV-2 to the nanozyme surface, thereby inhibiting the catalytic color development of the nanozyme. Using this color development principle, quantitative and qualitative analysis of SARS-CoV-2 can be achieved.

[0034] The MOF nanozymes described are MOF materials of one or more metals, such as Fe, Cu, Ni, and Pt, and are prepared using a microwave-assisted solvothermal method. The MOFs have a large number of carboxyl functional groups on their surface, which can be modified with the specific protein CD147 to construct a functionalized nanozyme system. Utilizing the specific binding of CD147 to the S protein overexpressed on the surface of the SARS-CoV-2 virus, the SARS-CoV-2 virus can be efficiently bound to the nanozyme surface, thereby inhibiting the catalytic color development of the nanozyme. This enables highly sensitive, rapid, and visual detection of SARS-CoV-2.

[0035] The MOF nanozymes described herein use terephthalic acid as a ligand, which can chelate with metal ions such as Fe, Cu, Ni, and Pt. The proportion of metal ions is controlled by adjusting the ratio of metal salts in the feed, resulting in MOF nanozymes with different metal ratios. The size and morphology of the MOF nanozymes can be controlled by adjusting the feed ratio, reaction time, and reaction temperature. This invention employs FeCu MOF nanozymes, with the metal ions linked to terephthalic acid as shown in the diagram. Figure 2 (b) in the middle.

[0036] The CD147 molecules are chemically immobilized on the surface of MOFs. Specifically, for example, if there are carboxyl groups on the surface of MOFs, the amino-containing CD147 molecules condense with the carboxyl groups of MOFs under the action of the biocrosslinking agents 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS).

[0037] like Figure 1As shown, this invention utilizes TMB chromogenic molecules. MOF nanozymes possess unique enzyme-like properties, which, under suitable conditions, can catalyze the blue color change of TMB molecules. Oxidized TMB exhibits maximum UV-Vis absorption at 652 nm. The specific protein molecule CD147 is immobilized on the MOF surface through the binding of its carboxyl and amino groups. The S protein overexpressed on the surface of the SARS-CoV-2 virus specifically binds to CD147, efficiently binding the SARS-CoV-2 virus to the nanozyme surface and thus inhibiting the nanozyme's catalytic color development. Based on this principle, colorimetric quantitative and qualitative detection of SARS-CoV-2 molecules can be achieved.

[0038] Example 1

[0039] (1) Preparation of MOF nanozymes.

[0040] Multimetallic metallo-metallic fossil fuel cells (MOFs) were prepared using a microwave-assisted solvothermal method. The morphology of the MOFs was controlled by adjusting the reaction temperature, feed ratio, and reaction time. Finally, CD147 molecules were chemically immobilized onto the MOF surface. The specific method is as follows:

[0041] (1-1) Bimetallic FeCu MOFs were prepared by microwave-assisted solvothermal method to chelate terephthalic acid with metal ions Fe and Cu. The morphology, size and ratio of MOFs can be controlled by the ratio of ligand to metal ions (1-10):(1-10), the ratio of different metal ions (1-50):(1-50), the reaction temperature (120-160℃) and the reaction time (2-12h).

[0042] (1-2) The product obtained in step (1-1) is washed with ethanol and dried;

[0043] (1-3) The MOFs obtained in step (1-2) are added to a CD147 solution. The CD147 solution is in PBS solvent and the concentration of CD147 is 7-200 μg / mL. The CD147 solution needs to be reacted with EDC for 10-30 minutes at a volume ratio of 8:(1-2). Then, the same volume of NHS as EDC is added. The concentration of EDC is 0.8-1.0 mg / mL and the concentration of NHS is 0.7-0.9 mg / mL. Chemical condensation is carried out at 4°C for 8-24 hours.

[0044] Figure 2 Image (a) shows the scanning electron microscope (SEM) characterization of the prepared MOFs, revealing a size of approximately 200-300 nm. As discussed in (1-3), CD147 is linked to the MOFs via amidation with -NH2 and -COOH groups. Figure 3Further infrared and Raman spectroscopy characterization was performed on the amide bonds formed by the esterification of -NH2 and -COOH. These characterizations confirm that CD147 was successfully immobilized on MOFs.

[0045] (2) MOF nanozyme catalytic system. For example... Figure 4 As shown, the catalytic properties of MOF nanozymes can catalyze the color change of the chromogenic molecule TMB to blue, and the ultraviolet spectrum of oxidized TMB shows maximum absorption at 652 nm.

[0046] (3) Detection of SARS-CoV-2 S-RBD using MOF nanozyme system. For example... Figure 5 As shown, CD147 probe modification inhibited the catalytic properties of MOFs-like enzymes, reducing their ability to catalyze the blue color change of the chromogenic molecule TMB. When different concentrations of SARS-CoV-2S-RBD solution bound to CD147-modified MOFs, the catalytic oxidation ability of MOF nanozymes further decreased due to protein molecules covering the catalytic active sites on the MOF surface. The UV-Vis absorption spectrum relationship curves under different conditions are shown in the figure. Figure 6 As shown. Absorbance values ​​at 652 nm were extracted for different concentrations, and a standard curve was constructed to represent the concentration gradient of SARS-CoV-2S-RBD, as shown. Figure 7 As shown in the figure, the detection method of this invention can achieve visualized, rapid, and highly sensitive detection of SARS-CoV-2S-RBD, with a detection limit as low as 6.2 × 10⁻⁶. -13 g / mL.

[0047] MOF nanozyme system for detecting SARS-CoV-2 pseudovirus

[0048] The method is the same as step 3, except that the SARS-CoV-2 S-RBD solution in step 3 is replaced with SARS-CoV-2 pseudovirus. Under the same conditions, the ultraviolet spectral curves are obtained similarly, as shown below. Figure 8 As shown, absorbance values ​​at 652 nm corresponding to different concentrations were extracted to construct a standard curve of the SARS-CoV-2 pseudovirus and its corresponding concentration gradient.

[0049] Selective experiment

[0050] The method is the same as step 3, except that SARS-CoV-2S-RBD is replaced with a different viral protein, and colorimetric analysis is performed under the same conditions. Figure 9 The values ​​are the changes in absorbance at 652 nm for different viral proteins. It can be seen that the nanozyme system proposed in this invention has high selectivity for SARS-CoV-2S-RBD.

[0051] Actual sample SARS-CoV-2 detection

[0052] The method is the same as step 3, except that the SARS-CoV-2S-RBD solution in step 3 is replaced with an actual SARS-CoV-2 solution. Under the same conditions, the ultraviolet spectral curve is also obtained, as shown below. Figure 10 As shown, absorbance values ​​at 652 nm were extracted for different concentrations to construct a standard curve for the corresponding concentration gradient of SARS-CoV-2. The figure demonstrates that the detection method of this invention enables visualized, rapid, and highly sensitive detection of SARS-CoV-2, with a detection limit as low as 3 PFU / mL.

Claims

1. The application of a MOF nanozyme in the detection of SARS-CoV-2 for non-disease diagnostic purposes, characterized in that, By utilizing the enzyme-like properties of MOFs and the specific binding ability of surface-modified CD147 protein to the novel coronavirus, the molecular colorimetric ability of chromogenic molecules TMB, ABTS, OPD and PPD can be catalyzed to achieve colorimetric quantitative and qualitative detection of SARS-CoV-2. The MOF nanozyme is an Fe and Cu MOF material, prepared by a microwave-assisted solvothermal method. The MOF nanozyme uses terephthalic acid as a ligand, which chelates with metal ions to form a metallic MOF material. The surface of the metallic MOF material has carboxyl groups, which then condense with CD147 molecules containing amino groups, thereby fixing the CD147 molecules on the MOF surface. The ratio of ligand to metal ions is (1-10):(1-10), the ratio of Fe to Cu ions is (1-50):(1-50), the reaction temperature is 120-160℃, and the reaction time is 2-12h. The MOF nanozyme described above has peroxidase-like properties and can catalyze hydrogen peroxide to generate hydroxyl radicals, which oxidize chromogenic molecules TMB, ABTS, OPD and PPD, enabling the detection of the novel coronavirus by colorimetric method. TMB is 3,3′,5,5′-tetramethylbenzidine; ABT is 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate); OPD stands for o-phenylenediamine; PPD stands for p-phenylenediamine; The MOF nanozyme has a surface modified with the specific protein CD147. By utilizing the specific binding of CD147 to the S protein overexpressed on the surface of SARS-CoV-2, the SARS-CoV-2 virus is bound to the surface of the nanozyme, thereby inhibiting the catalytic color development of the nanozyme and realizing the quantitative and qualitative analysis of SARS-CoV-2 virus. After the MOF nanozyme is modified with CD147, the catalytic activity of MOFs decreases, and the ability of oxidizing TMB, ABTS, OPD and PPD molecules to change color decreases; the modification of CD147 is the condensation connection between the carboxyl group on the surface of the MOF nanozyme and the amino group of CD147.

2. The application of the MOF nanozyme according to claim 1 in the detection of the novel coronavirus, characterized in that, The CD147 specifically binds to SARS-CoV-2, reducing the catalytic activity of MOF nanozymes and further weakening their ability to oxidize TMB, ABTS, OPD, and PPD molecules and cause discoloration.

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