CN114436338A

CN114436338A - Iron-molybdenum bimetal nano enzyme and preparation method and application thereof

Info

Publication number: CN114436338A
Application number: CN202210196497.3A
Authority: CN
Inventors: 许元红; 付雅萱; 赵珍
Original assignee: Qingdao University
Current assignee: Qingdao University
Priority date: 2022-03-01
Filing date: 2022-03-01
Publication date: 2022-05-06
Anticipated expiration: 2042-03-01
Also published as: CN114436338B

Abstract

The invention belongs to the technical field of preparation and application of biosensing materials, and relates to iron-molybdenum bimetal nano enzyme as well as a preparation method and application thereofSynthesizing bimetal nano enzyme Fe with excellent peroxidase-like activity by a hydrothermal method and a calcination method₂MoO₄Using it as colorimetric probe, and combining with Hybrid Chain Reaction (HCR) to analyze gene mutation; the kit can sensitively detect DNA mutation within a concentration range of 25pM to 4nM, has a detection limit as low as 2pM, and is superior to most colorimetric sensors reported in the past; in addition, the application of the sensor in a serum sample verifies the practicability, and has good accuracy and repeatability; fe of the invention₂MoO₄The preparation method of the material is simple, and the material has wide prospects in biochemical analysis and clinical application as a colorimetric sensor.

Description

Iron-molybdenum bimetal nano enzyme and preparation method and application thereof

The technical field is as follows:

the technology belongs to the technical field of preparation and application of biosensing materials, and relates to an iron-molybdenum bimetallic nanoenzyme Fe₂MoO₄A material and a preparation method and application thereof. Synthesizing a bimetallic nano-enzyme material by a hydrothermal method and calcination, taking the bimetallic nano-enzyme material as a colorimetric probe, and amplifying a signal by combining a hybridization chain reaction; the nucleic acid is loaded in an electrostatic adsorption mode, and the detection purpose is achieved by the color signal change generated by the inhibition of the nucleic acid on the activity of the nano enzyme.

Background art:

bladder cancer is the most common malignant tumor of the urinary system, is one of ten common tumors of the whole body, the incidence rate of bladder cancer in men is the seventh position of the malignant tumor of the whole body, and women are ranked after the tenth position. The medicine takes the first place of the disease rate of urogenital system tumors in China, and the high disease rate and mortality seriously threaten the health of human beings.

To avoid unnecessary surgery, effective treatment regimens are selected earlier, and there is an increasing need for risk prediction or accurate, rapid preliminary diagnosis of bladder cancer. In the past decade, genetic mutations have been considered as the genetic basis for differences in individual disease susceptibility, which may be involved in tumor carcinogenicity by affecting the function or expression of specific genes. Thus, gene mutations can be used as biomarkers for the prediction and diagnosis of disease. There are studies that indicate that PSCA rs2294008(C > T) in HOTAIR is associated with the risk of bladder cancer in humans.

The traditional detection method is time-consuming, requires complex equipment and professionals, has low detection cost, is convenient to carry, can be seen by naked eyes, does not need complex equipment, and is a feasible detection method. However, most of the reported colorimetric sensors generally have low sensitivity and poor selectivity. The Hybrid Chain Reaction (HCR) has the advantages of good amplification efficiency, isothermal conditions, enzyme-free amplification and the like, and is a good signal amplification strategy. However, the current sensing platform combined with HCR has partial defects, such as low detection sensitivity by utilizing the aggregation and depolymerization mechanism of gold nanoparticles; although the natural enzyme is used as a probe, the sensitivity is high, but the practical application of the natural enzyme is limited by the defects of difficult storage and transportation and the like of the natural enzyme.

The nano enzyme is a nano material with enzyme-like activity, can simulate the generation of hydroxyl radicals by natural enzyme, and is considered as a promising colorimetric probe in chemical or biological sensing due to the color development change of a substrate caused by the catalytic action of the nano enzyme. In addition, the DNA can interact with Fe through Van der Waals force, and the oxidation of 3,3',5,5' -tetramethyl benzidine (TMB) by the nanoenzyme is blocked or closed. Meanwhile, the molybdenum (VI) complex has different valence states, so that different forms of molybdenum oxide nanoenzymes can be generated. Among them, catalytic activity should be one of the most important factors determining the sensitivity of nanoenzyme assay. In order to greatly improve the catalytic activity, the synthesis of the bimetallic nano-enzyme is an effective choice. However, no reports on bimetallic nanoenzymes have been found so far.

The invention content is as follows:

the invention aims to overcome the defects in the prior art and provide Fe with high-efficiency peroxidase-like activity₂MoO₄The bimetallic nanoenzyme is used as a colorimetric sensing probe, and HCR is used as an amplification strategy, so that a sensitive, simple and convenient gene PSCA rs2294008 (C)>T) colorimetric analysis method, can be used for predicting the occurrence risk of bladder cancer.

In order to realize the aim, the invention provides the iron-molybdenum bimetallic nano enzyme which is Fe with a spherical structure₂MoO₄The grain diameter is 20-80 nm.

The invention also provides a preparation method of the iron-molybdenum bimetal nanoenzyme, the iron-molybdenum bimetal nanoenzyme is prepared by a hydrothermal method and a calcination method, and the preparation method comprises the following specific steps:

adding 0.2-0.8g of iron salt, 11-13g of sodium salt and 0.2-0.4g of urea into 40mL of deionized water, and adding 0.2-0.35g of polyacrylamide and 0.2-0.3g of molybdenum precursor; transferring the mixture into a polytetrafluoroethylene-lined reaction kettle, and heating at 200 ℃ for 10-12 hours; then centrifuging the reaction solution to obtain a precipitate, washing the precipitate with deionized water and ethanol respectively, drying at 60 ℃, and finally calcining the dried product in argon at 500-700 ℃ for 1-3h to obtain the Fe-Mo bimetallic nanoenzyme Fe₂MoO₄。

The ferric salt is ferric chloride hexahydrate or ferrous chloride.

The sodium salt is sodium citrate.

The molybdenum precursor is molybdenum trioxide or molybdenum trichloride.

The invention also provides a sensor prepared by using the iron-molybdenum bimetallic nanoenzyme.

The preparation method of the sensor comprises the following specific steps:

(1) and (3) hybrid chain reaction: heating the mixed solution of the H1 chain and the H2 chain, and gradually cooling after annealing; adding the target chain into the H1 chain H2 chain mixed solution, and incubating for 2H at 37 ℃ to obtain a hybrid chain reaction product;

(2) constructing a sensor: and (3) mixing the hybrid chain reaction product with the iron-molybdenum bimetallic nanoenzyme, and culturing for 8-10 minutes to obtain the sensor.

The H1 chain sequence was 5'-GCAGCACAGCCTTCATGGTCCTGGCCCACCAGTGACCATGA-3' (SEQ ID NO: 1).

The H2 chain sequence was 5'-GACCATGAAGGCTGTGCTGCTTCATGGTCACTGGTGGGCCA-3' (SEQ ID NO: 2).

The target chain is a mutated fragment of a part of a normal chain of PSCA rs2294008, and the nucleotide sequence is 5'-GACCATGAAGGCTGTGCTGCTTGCCCTGTTGA-3' (SEQ ID NO:3)

The sensitivity and selectivity performance characterization of the sensor is carried out through peroxidase test analysis, and the absorbance at 650nm is recorded; the detection limit of the sensor to the target chain is as low as 2 pM.

The invention also provides application of the sensor in preparing a kit for analyzing or detecting bladder cancer related gene mutation. The invention also provides application of the iron-molybdenum bimetallic nanoenzyme in preparation of a kit for analyzing or detecting bladder cancer related gene mutation.

The iron-molybdenum bimetallic nanoenzyme can be used for analyzing bladder cancer related gene mutation, and analyzing bladder cancer related gene PSCA rs2294008(C > T) by combining a colorimetric method with hybrid chain reaction, and comprises the following specific steps: (1) and (3) hybrid chain reaction: heating the mixed solution of the H1 chain and the H2 chain, and gradually cooling after annealing; adding the target chain into the H1 chain H2 chain mixed solution, and incubating for 2H at 37 ℃ to obtain a hybrid chain reaction product;

(2) constructing a sensor: culturing the hybrid chain reaction product mixed with the iron-molybdenum bimetallic nanoenzyme for 8-10 minutes to obtain a sensor;

(3) the sensitivity and selectivity performance of the constructed sensor were characterized by peroxidase test analysis, and the absorbance at 650nm was recorded.

The iron-molybdenum bimetallic nano-enzyme disclosed by the invention can prevent the formation of iron particles with low catalytic activity due to the dilution of molybdenum on iron, so that the catalytic activity of the iron-based catalyst can be adjusted. Therefore, the Fe/Mo bimetallic nanoenzyme is expected to interact with genes, has better enzyme simulation activity, and becomes an effective probe for bladder cancer risk prediction based on HCR, which is stable, selective and sensitive.

The application principle of the bimetal nano enzyme is as follows: the mutation site (target) of the bladder cancer-associated gene is designed as a target chain; the H1 and H2 sequences are rationally designed into a stable hairpin configuration that is unable to interact spontaneously without target; the introduction of the target strand will trigger the HCR process, and the final product is a long double-stranded DNA nanowire with a gap, which is the site where DNA is attached to the nanoenzyme particle. The sticky end part passes through DNA base and Fe₂MoO₄The Van der Waals force between the nano-enzyme and the material is strongly combined, so that the nano-enzyme surface is covered by DNA and is prevented from reacting with 3,3',5,5' -tetramethyl benzidine (TMB). The negatively charged long double stranded portion may be linked to Fe₂MoO₄Compete for positively charged TMB while rejecting Fe₂MoO₄. Furthermore, the length of the HCR product produced can reach 40nm, which exceeds the OH diffusion distance (20 nm). Therefore, the nanoenzyme and TMB bound to the distal portion of the nucleic acid double strand are not easily oxidized by OH. Therefore, the activity of the HCR product-modified nanoenzyme is decreased, which can be seen from the decrease in uv absorbance.

Compared with the prior art, the invention has the following beneficial effects:

(1) firstly synthesizes spherical Fe-Mo bimetal by hydrothermal method and calcination methodNano enzyme Fe₂MoO₄Large specific surface area, many active sites and excellent peroxidase activity.

(2) Establishment of Fe-based₂MoO₄The bladder cancer related gene PSCA rs2294008 (C) is analyzed by the colorimetric method combined with the Hybrid Chain Reaction (HCR)>T)。

(3)Fe₂MoO₄The nano enzyme is used as a tool of the colorimetric sensor, on one hand, the preparation is simple, the stability is good, and the defects of natural enzyme are overcome; on the other hand, it utilizes intrinsic peroxidase-like activity, rather than aggregation or disaggregation of gold nanoparticles, thereby improving sensitivity. The detection limit of the sensor is as low as 2pM, and the sensor is superior to the colorimetric method in the prior art in a wide linear dynamic range of target DNA concentration from 25pM to 4 nM. The results show that the hybrid chain reaction Fe is combined₂MoO₄Nanolase as colorimetric sensor for analyzing bladder cancer related gene PSCA rs2294008 (C)>T) has good accuracy and reproducibility. In addition, the application of the sensor in a serum sample verifies the practicability of the sensor, and the strategy has wide prospects in the aspects of biochemical analysis and clinical application.

Description of the drawings:

FIG. 1 is a schematic diagram of the principle of analyzing bladder cancer related genes by using Fe-Mo bi-metal nanoenzyme.

FIG. 2 shows Fe prepared by the present invention₂MoO₄In which A is Fe₂MoO₄Scanning electron microscope images of; b is Fe₂MoO₄Transmission electron microscopy images of; c is Fe₂MoO₄High resolution transmission electron microscopy images; d is Fe₂MoO₄Element distribution map of (c).

FIG. 3 shows Fe according to the present invention₂MoO₄The peroxidase detection scheme of (1), wherein curve a is TMB + H₂O₂(ii) a Curve b is Fe₂MoO₄+H₂O₂(ii) a Curve c is Fe₂MoO₄+TMB+H₂O₂。

FIG. 4 shows Fe according to the present invention₂MoO₄ESR test results for hydroxyl radical generation are shown schematically.

FIG. 5 is a schematic diagram of nucleic acid result analysis in the HCR process according to the present invention. Lane 1 is 2 μ M target strand; lane 2 is strand 2 μ M H1; lane 3 is strand 2 μ M H2; lane 4 is 2. mu. M H1+ 2. mu. M H2; lane 5 is 1. mu.M target strand + 2. mu. M H1+ 2. mu. M H2; lane 6 is 2. mu.M target strand + 2. mu. M H1+ 2. mu. M H2; lane 7 is 4 μ M target strand +2 μ M H1+2 μ M H2; m is DNA Marker.

FIG. 6 shows Fe according to the present invention₂MoO₄And HCR product interaction, where a is a hydrated particle size diagram; b is a zeta potential diagram.

FIG. 7 shows Fe-based alloys according to the present invention₂MoO₄A condition optimization schematic diagram of a sensing platform, wherein A is pH value optimization of a reaction solution; b, optimizing the reaction temperature; c is optimized by H chain concentration (H1: H2 ═ 1: 1); d is Fe₂MoO₄And (4) optimizing the concentration.

FIG. 8 shows Fe-based alloys according to the present invention₂MoO₄The performance of the sensing platform is shown schematically, wherein A is a sensitivity diagram and B is a selectivity diagram.

The specific implementation mode is as follows:

the present invention will now be described in detail by way of specific examples with reference to the accompanying drawings.

Example 1:

this example relates to a method for preparing Fe-Mo bimetallic nanoenzyme, Fe₂MoO₄The bimetallic nanoenzyme is synthesized by a hydrothermal method and a calcination method, and comprises the following specific steps:

(1) adding 0.54g of ferric chloride hexahydrate, 12.941g of sodium citrate and 0.3g of urea into 40mL of deionized water, and stirring until the ferric chloride hexahydrate, the sodium citrate and the urea are completely dissolved; adding 0.3g of polyacrylamide and 0.229g of molybdenum trioxide while stirring until the polyacrylamide and the molybdenum trioxide are completely dissolved; transferring the mixture into a 100mL polytetrafluoroethylene-lined reaction kettle, and heating at 200 ℃ for 12 hours;

(2) taking out the reacted sample, centrifuging for 10min at 8000r/min, and removing the supernatant to obtain precipitate; adding deionized water into the precipitate, centrifuging at 8000r/min for 10min, washing, and adding ethanol for centrifuging; finally, drying the obtained precipitate sample at 60 ℃ for 10 h;

(3) calcining the obtained sample at 600 ℃ for 2h in argon gas, and raising the temperature at 3 ℃ for min^-1(ii) a Then naturally cooling to room temperature to obtain the product Fe-Mo bimetal nano enzyme₂MoO₄。

This example uses a scanning electron microscope and a high resolution transmission electron microscope to prepare Fe₂MoO₄The characterization was performed, and the results are shown in fig. 2. As shown in the 2A scanning electron micrograph, Fe₂MoO₄The prepared nano particles are uniform spherical, the particle size is about 50nm, the specific surface area is large, and the number of active sites is large; the morphology of FIG. 2A is consistent with that of FIG. 2B. Fe₂MoO₄The high resolution TEM image (FIG. 2C) shows that the lattice spacing of the nanoparticle crystal structure is about 0.28nm, Fe₂MoO₄(JCPDS No.25-1430) has the diffraction plane of (422) coincident. To provide further evidence, Fe was analyzed using elemental mapping₂MoO₄The composition and distribution of the elements of (a) are shown in fig. 2D, showing uniformity.

This example utilizes hydrogen peroxide (H)₂O₂) And 3,3',5,5' -tetramethyl benzidine (TMB) pair prepared Fe-Mo bimetallic nanoenzyme Fe₂MoO₄The enzyme activity of the material is characterized, the result is shown in figure 3, Fe₂MoO₄Has excellent peroxidase and enzyme properties. For Fe₂MoO₄Electron Spin Resonance (ESR) was performed to further verify the form of active oxygen, and the results are shown in FIG. 4, Fe₂MoO₄The characteristic peak 1:2:2:1 appears, which indicates Fe₂MoO₄Is capable of converting hydrogen peroxide into a hydroxyl radical which develops color of the TMB.

Example 2:

(1) adding 0.8g of ferric chloride hexahydrate, 11g of sodium citrate and 0.4g of urea into 40mL of deionized water, and stirring until the ferric chloride hexahydrate, the sodium citrate and the urea are completely dissolved; adding 0.2g of polyacrylamide and 0.3g of molybdenum trioxide while stirring until the materials are completely dissolved; transferring the mixture into a 100mL polytetrafluoroethylene-lined reaction kettle, and heating at 200 ℃ for 10 hours;

(2) taking out the reacted sample, centrifuging for 15min at 8000r/min, and removing the supernatant to obtain precipitate; adding deionized water into the precipitate, centrifuging at 8000r/min for 15min, washing, and adding ethanol for centrifuging; finally, drying the obtained precipitate sample at 60 ℃ for 8 h;

(3) calcining the obtained sample at 700 ℃ for 3h in argon gas, wherein the heating rate is 3 ℃ for min^-1(ii) a Then naturally cooling to room temperature to obtain the product Fe-Mo bimetal nano enzyme₂MoO₄。

Example 3:

this example relates to a hybrid chain reaction of a target chain and a hairpin chain, specifically performed according to the following steps, hairpin 1 chain (H1) and hairpin 2 chain (H2) in a volume ratio of 1:1, mixing, heating the mixed solution at 95 ℃ for 5min, and gradually cooling to room temperature; an equal volume of target strand was then added to the H1 chain H2 chain mixture and incubated at 37 ℃ for 2H, triggering the HCR reaction. The solutions used to dilute the DNA were all 1 XSPSC buffer (pH7.5,50mM Na)₂HPO₄,1M NaCl)。

The HCR products were analyzed by agarose gel electrophoresis (2% agarose gel, 150V,25 min) and nucleic acid imaging system, and the results are shown in FIG. 5. As can be seen from FIG. 5, after incubation of the target strands with H1, H2, the bands H1, H2 disappeared gradually and a new band became bright as the concentration of the target strands increased (lanes 5-7). Since the HCR process consumes two hairpins, the band intensities corresponding to H1 and H2 are significantly reduced. The appearance of new bands is the result of the formation of long DNA by HCR.

In summary, the H1 sequence and the H2 sequence designed in this example can trigger HCR reaction by the target strand, the product is a long double-stranded DNA nanowire with a gap, and the HCR product is mixed with Fe₂MoO₄Mixing with nanoenzymes, Fe₂MoO₄The nanoenzyme surface is covered with HCR product DNA to prevent TMB and Fe with peroxidase activity₂MoO₄Reacts with hydroxyl radicals generated by the reaction of hydrogen peroxide. The negatively charged long double stranded portion may be linked to Fe₂MoO₄Compete for positively charged TMBRepelling Fe₂MoO₄. The activity of the nanoenzyme modified by the HCR product is reduced. Whereas the wild type (non-mutated normal gene) did not trigger HCR and the absorbance change was insignificant. The DNA sequences used are shown in Table 1.

TABLE 1 sequence Listing of target strands, H1, H2 and wild type

Name (R)	Sequence of
		Target chain	5’-GACCATGAAGGCTGTGCTGCTTGCCCTGTTGA-3’
Chain H1	5’-GCAGCACAGCCTTCATGGTCCTGGCCCACCAGTGACCATGA-3’
		Chain H2	5’-GACCATGAAGGCTGTGCTGCTTCATGGTCACTGGTGGGCCA-3’
Wild type	5’-GACCACGAAGGCTGTGCTGCTTGCCCTGTTGA-3’

Wild type: normal genotype without mutation.

Example 4:

this example relates to Fe based on example 1₂MoO₄The preparation method of the nano enzyme sensor comprises the following specific processes of adding 1.5 mu mol/L (2 mu L) of target chain into H1 chain H2 chain mixed solution (4 mu L) obtained in the embodiment 3 after annealing and natural cooling to room temperature, and incubating for 2H at 37 ℃ to obtain HCR product; the HCR product was reacted with 25. mu.lLFe₂MoO₄Mixing the nano material acetic acid-sodium acetate buffer solution (the concentration is 1mg/mL) uniformly, and then incubating for 10 minutes at room temperature to obtain the Fe-based nano material₂MoO₄A sensor of nano-enzyme. The particle size and potential analysis of the sensor showed that the results are shown in FIG. 6, and the results are Fe in FIG. 6A₂MoO₄The particle size of the nanomaterial is increased after the interaction with the HCR product, and the HCR product with negative electricity shown in FIG. 6B and Fe₂MoO₄The electric potential changes after the nanometer material is combined. These results demonstrate successful sensor construction.

Example 5:

this example relates to the Fe-based alloy described in example 3₂MoO₄The optimization test of the preparation conditions of the nano-enzyme sensor respectively optimizes the pH value, the H chain concentration and the material concentration in the preparation process and optimizes the performance temperature under the condition of the same other conditions, and comprises the following specific steps:

(1) optimizing the pH value: preparing sensors by using acetic acid-sodium acetate buffers with different pH values (3, 3.5, 4, 4.5 and 5) and carrying out peroxidase activity test on the sensors; (2) temperature optimization: carrying out peroxidase activity tests on the prepared sensor under the conditions of different temperatures (20, 30, 40, 50 and 60 ℃); (3) h chain concentration (H1: H2 ═ 1:1) optimization: selecting 4 concentrations of H chains (0.5, 1, 2 and 3 mu M) to respectively perform HCR reaction with different concentrations of target chains (0.5, 1, 2, 3 and 4 mu M), preparing a sensor by using the obtained product, and performing peroxidase activity test on the sensor; (4) optimizing the material concentration: equal amounts of HCR product with varying concentrations of Fe₂MoO₄Incubating the nano material (20, 25, 30 mu M), and carrying out a peroxidase activity test on the obtained product; the results are shown in FIG. 7. As can be seen from FIG. 7, 2. mu. M H chain and 25. mu.g/mL Fe at pH3.5, 50 ℃₂MoO₄The best test result can be obtained under the condition, and the subsequent tests are all carried out under the condition.

Example 6:

this example relates to a process based on Fe₂MoO₄The sensor performance characterization of the nano enzyme comprises the following specific steps:

(1) and (3) testing the sensitivity: in thatpH3.5, 50 ℃, 2. mu. M H chain and 25. mu.g/mL Fe₂MoO₄Carrying out peroxidase activity tests with different concentrations of target chain HCR products under the condition; collecting absorbance at 650 nm; (2) and (3) selective testing: at pH3.5, 50 ℃, 2. mu. M H chain and 25. mu.g/mL Fe₂MoO₄Incubating the HCR product with the target strand and the wild-type strand, respectively, under the same conditions, the other group is not incubated under the same conditions; then carrying out peroxidase activity test; the results are shown in FIG. 8.

As can be seen from the graph in fig. 8A, the change in absorbance (Δ a ═ a0-a at 650nm) increased linearly with increasing concentration of target strands in the 25pM to 4nm range, where a0 and a are the absorbance of the system with and without HCR product; fitting a linear regression equation of y being 0.4353x +0.7841, and R2 being 0.994; the limit of detection (LOD) was evaluated to be 2pM (S/N.gtoreq.3); accuracy of Sensors (CVs)<3%, n ═ 3); the CV values were within acceptable ranges, indicating good reproducibility and accuracy of the sensor. As can be seen from fig. 8B, the presence of the target strand triggers the HCR reaction, the signal is amplified and the color is reduced, which is reflected by the difference in absorbance; whereas HCR reactions cannot be triggered without incubation or without target strands, which is manifested as a decrease in absorbance difference. These results show that Fe is based on₂MoO₄The colorimetric sensor of the nano enzyme has higher selectivity.

Example 7:

the present example relates to Fe-based₂MoO₄The sensor of the nano enzyme is applied to the test in the serum sample. To demonstrate its potential for practical applications, it will be based on Fe₂MoO₄The colorimetric sensor of (a) was used in diluted serum to demonstrate its feasibility. HCR products triggered by target chains with different concentrations (0.5, 1 and 2nM) are obtained firstly, then a serum sample diluted by 10 times is added into the obtained HCR products, and then a sensor is constructed by the method described in the embodiment 4 and the optimized parameters obtained in the embodiment 5 to carry out peroxidase activity test and detect the concentration of the target chains in the serum. The results calculated after collecting the ultraviolet absorbance at 650nm are summarized in Table 2, indicating that the experimental results obtained are consistent with the theoretical values. The recovery rate of the target chain is 92.2-102 percent9%, the detection accuracy is better. The Relative Standard Deviation (RSD) of the 3 detected concentrations is less than 6.5%, indicating that the method has good reproducibility. These results indicate that the biosensor performs well in the simulated analysis of serum samples.

TABLE 2 Fe basis₂MoO₄Test result of nano enzyme sensor applied to serum sample

Sequence listing

<110> Qingdao university

<120> iron-molybdenum bimetallic nano-enzyme and preparation method and application thereof

<130> 2022

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<170> SIPOSequenceListing 1.0

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Claims

1. The Fe-Mo bimetal nano enzyme is characterized in that Fe with a spherical structure₂MoO₄The grain diameter is 20-80 nm.

2. The method for preparing the Fe-Mo bimetallic nanoenzyme as in claim 1, wherein the ferric salt, the sodium salt and the urea are added into deionized water and stirred until completely dissolved; adding polyacrylamide and molybdenum precursor while stirring until the polyacrylamide and the molybdenum precursor are completely dissolved; transferring the mixture into a polytetrafluoroethylene-lined reaction kettle, and heating at 200 ℃ for 10-12 hours; taking out the reacted sample, centrifuging, removing the supernatant, and washing the precipitate; the sample obtained was dried at 60 ℃; then calcining the mixture for 1 to 3 hours at the temperature of 500 ℃ and 700 ℃ in argon, and naturally cooling the mixture to the room temperature to obtain the product, namely the Fe-Mo bimetallic nanoenzyme Fe₂MoO₄。

3. The method for preparing the Fe-Mo bi-metal nanoenzyme according to claim 2, wherein the iron salt is ferric trichloride hexahydrate or ferrous chloride.

4. The method for preparing the iron-molybdenum bi-metal nanoenzyme according to claim 2, wherein the sodium salt is sodium citrate.

5. The method for preparing the iron-molybdenum bi-metal nanoenzyme according to claim 2, wherein the molybdenum precursor is molybdenum trioxide or molybdenum trichloride.

6. The method for preparing the iron-molybdenum bi-metal nanoenzyme according to claim 2, wherein the temperature rise rate during the calcination is 3 ℃/min.

7. A sensor prepared by using the iron-molybdenum bi-metal nanoenzyme of claim 1.

8. The sensor of claim 7, wherein the method comprises the steps of:

the H1 chain sequence is 5'-GCAGCACAGCCTTCATGGTCCTGGCCCACCAGTGACCATGA-3'; the H2 chain sequence is 5'-GACCATGAAGGCTGTGCTGCTTCATGGTCACTGGTGGGCCA-3'; the target strand sequence is 5'-GACCATGAAGGCTGTGCTGCTTGCCCTGTTGA-3'.

9. Use of the iron-molybdenum bi-metal nanoenzyme of claim 1 in the preparation of a kit for detecting bladder cancer-associated gene mutations.

10. Use of the sensor of any one of claims 7 or 8 in the manufacture of a kit for detecting mutations in bladder cancer-associated genes.