CN114436338B - Fe-Mo bimetallic nano-enzyme and preparation method and application thereof - Google Patents
Fe-Mo bimetallic nano-enzyme and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 229910017116 Fe—Mo Inorganic materials 0.000 title claims description 22
- 238000000034 method Methods 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 238000001354 calcination Methods 0.000 claims abstract description 11
- DSMZRNNAYQIMOM-UHFFFAOYSA-N iron molybdenum Chemical compound [Fe].[Fe].[Mo] DSMZRNNAYQIMOM-UHFFFAOYSA-N 0.000 claims abstract 8
- 239000000047 product Substances 0.000 claims description 23
- 206010005003 Bladder cancer Diseases 0.000 claims description 15
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 15
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 15
- 238000009396 hybridization Methods 0.000 claims description 13
- 239000002244 precipitate Substances 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- JKQOBWVOAYFWKG-UHFFFAOYSA-N molybdenum trioxide Chemical group O=[Mo](=O)=O JKQOBWVOAYFWKG-UHFFFAOYSA-N 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 230000035772 mutation Effects 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000007795 chemical reaction product Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 claims description 5
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 5
- 239000004202 carbamide Substances 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 5
- 159000000000 sodium salts Chemical class 0.000 claims description 5
- 238000011282 treatment Methods 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 4
- 229910052786 argon Inorganic materials 0.000 claims description 4
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical group O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 4
- 229910052750 molybdenum Inorganic materials 0.000 claims description 4
- 239000011733 molybdenum Substances 0.000 claims description 4
- 229920002401 polyacrylamide Polymers 0.000 claims description 4
- 239000002243 precursor Substances 0.000 claims description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical group O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 230000000630 rising effect Effects 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 229960002089 ferrous chloride Drugs 0.000 claims description 2
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- ZSSVQAGPXAAOPV-UHFFFAOYSA-K molybdenum trichloride Chemical compound Cl[Mo](Cl)Cl ZSSVQAGPXAAOPV-UHFFFAOYSA-K 0.000 claims description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 17
- 239000000523 sample Substances 0.000 abstract description 17
- 238000001514 detection method Methods 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract description 11
- 210000002966 serum Anatomy 0.000 abstract description 8
- 238000001027 hydrothermal synthesis Methods 0.000 abstract description 6
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- 238000012742 biochemical analysis Methods 0.000 abstract description 2
- 239000011540 sensing material Substances 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 230000009946 DNA mutation Effects 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 21
- 108090000790 Enzymes Proteins 0.000 description 21
- 238000012360 testing method Methods 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 13
- 102000003992 Peroxidases Human genes 0.000 description 12
- 108040007629 peroxidase activity proteins Proteins 0.000 description 12
- 238000002835 absorbance Methods 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 8
- 238000005457 optimization Methods 0.000 description 8
- 230000035945 sensitivity Effects 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 5
- 239000002086 nanomaterial Substances 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 101001136592 Homo sapiens Prostate stem cell antigen Proteins 0.000 description 4
- 239000000956 alloy Substances 0.000 description 4
- 229910045601 alloy Inorganic materials 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000004737 colorimetric analysis Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000002105 nanoparticle Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
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- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
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- 102000053602 DNA Human genes 0.000 description 2
- 238000004435 EPR spectroscopy Methods 0.000 description 2
- 102000020897 Formins Human genes 0.000 description 2
- 108091022623 Formins Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000005411 Van der Waals force Methods 0.000 description 2
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
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- 239000007974 sodium acetate buffer Substances 0.000 description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 2
- 230000001960 triggered effect Effects 0.000 description 2
- LDXJRKWFNNFDSA-UHFFFAOYSA-N 2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound C1CN(CC2=NNN=C21)CC(=O)N3CCN(CC3)C4=CN=C(N=C4)NCC5=CC(=CC=C5)OC(F)(F)F LDXJRKWFNNFDSA-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 108091007417 HOX transcript antisense RNA Proteins 0.000 description 1
- 108091060210 Heavy strand Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- 238000002173 high-resolution transmission electron microscopy Methods 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
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- 229910000476 molybdenum oxide Inorganic materials 0.000 description 1
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- 125000003729 nucleotide group Chemical group 0.000 description 1
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- 238000007254 oxidation reaction Methods 0.000 description 1
- PQQKPALAQIIWST-UHFFFAOYSA-N oxomolybdenum Chemical compound [Mo]=O PQQKPALAQIIWST-UHFFFAOYSA-N 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- 230000002485 urinary effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G49/00—Compounds of iron
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
- B01J23/70—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper
- B01J23/76—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper combined with metals, oxides or hydroxides provided for in groups B01J23/02 - B01J23/36
- B01J23/84—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper combined with metals, oxides or hydroxides provided for in groups B01J23/02 - B01J23/36 with arsenic, antimony, bismuth, vanadium, niobium, tantalum, polonium, chromium, molybdenum, tungsten, manganese, technetium or rhenium
- B01J23/85—Chromium, molybdenum or tungsten
- B01J23/88—Molybdenum
- B01J23/881—Molybdenum and iron
-
- B01J35/40—
-
- B01J35/51—
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/60—Particles characterised by their size
- C01P2004/64—Nanometer sized, i.e. from 1-100 nanometer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention belongs to the technical field of preparation and application of biological sensing materials, and relates to an iron-molybdenum bimetallic nano-enzyme, a preparation method and application thereof, wherein a hydrothermal method and a calcination method are used for synthesizing a bimetallic nano-enzyme Fe with excellent peroxidase-like activity 2 MoO 4 Taking the sample as a colorimetric probe and analyzing gene mutation by combining with a Hybrid Chain Reaction (HCR); the DNA mutation in the concentration range of 25pM-4nM can be sensitively detected, the detection limit is as low as 2pM, and the detection limit is superior to most colorimetric sensors reported in the past; in addition, the application of the sensor in serum samples verifies the practicability, and has good accuracy and repeatability; fe of the present invention 2 MoO 4 The preparation method of the material is simple, and the material has wide prospect in the aspects of biochemical analysis and clinical application as a colorimetric sensor.
Description
Technical field:
the technology belongs to the technical field of preparation and application of biological sensing materials, and relates to Fe serving as Fe-Mo bimetallic nano enzyme 2 MoO 4 Materials, and methods of making and using the same. Synthesizing a bimetallic nano enzyme material by a hydrothermal method and calcining, 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 by using an electrostatic adsorption mode, and the detection purpose is achieved by inhibiting the activity of the nano enzyme by the nucleic acid to generate a color development signal change.
The background technology is as follows:
bladder cancer is the most common malignancy of the urinary system, is one of ten common tumors of the whole body, and the incidence rate of male bladder cancer is the seventh position of the whole body malignancy, and women are ranked after the tenth position. The disease rate of urogenital tumor is the first place in China, and the high disease rate and death rate seriously threaten the health of human beings.
In order to avoid unnecessary surgery, the need for early selection of effective treatment regimens, for risk prediction or accurate, rapid preliminary diagnosis of bladder cancer, is growing. In the last decade, gene mutations have been considered as the genetic basis for differences in susceptibility to disease in individuals, which may be involved in the oncogenicity of tumors by affecting the function or expression of specific genes. Thus, gene mutations can serve as biomarkers for predicting and diagnosing disease. There are studies showing that PSCA rs2294008 (C > T) in HOTAIR is associated with risk of bladder cancer in humans.
The traditional detection method is time-consuming, requires complex equipment and professionals, and the colorimetric method is low in detection cost, convenient to carry and visible to naked eyes, does not require complex equipment, and is a relatively feasible detection method. However, most reported colorimetric sensors are generally low in sensitivity and poor in selectivity. The Hybridization Chain Reaction (HCR) has the advantages of good amplification efficiency, isothermal condition, enzyme-free amplification and the like, and is a good signal amplification strategy. However, the current sensing platform combined with HCR has a partial disadvantage such as low detection sensitivity by using aggregation and deagglomeration mechanisms of gold nanoparticles; the use of natural enzymes as probes, although highly sensitive, has limited practical applications due to the inherent difficulty in storage and transportation of natural enzymes.
Nanoenzymes are nanomaterials with enzyme-like activity that mimic the production of hydroxyl radicals by natural enzymes, and are considered promising colorimetric probes in chemical or biological sensing because of the chromogenic change of the substrate caused by their catalytic action. In addition, DNA can interact with Fe by van der waals forces to block or shut down the oxidation of 3,3', 5' -Tetramethylbenzidine (TMB) by nanoenzymes. Meanwhile, the molybdenum (VI) complex has different valence states, so that different forms of molybdenum oxide nano-enzyme can be produced. Among them, catalytic activity should be one of the most important factors determining the assay sensitivity of nanoenzymes. In order to greatly improve the catalytic activity, the synthesis of the bimetallic nano-enzyme is an effective choice. However, no report has been found so far on the bimetallic nanoenzyme.
The invention comprises the following steps:
the invention aims to overcome the defects existing in the prior art and provide Fe with high-efficiency peroxidase-like activity 2 MoO 4 The bimetallic nano-enzyme is used as a colorimetric sensing probe, and HCR is used as an amplification strategy, so that a sensitive and simple gene PSCA rs2294008 (C)>T) colorimetric assay, useful for predicting risk of bladder carcinogenesis.
In order to achieve the above object, the present invention provides an Fe-Mo bimetallic nanoenzyme which is Fe having a spherical structure 2 MoO 4 The particle size is 20-80nm.
The invention also provides a preparation method of the Fe-Mo bimetallic nano-enzyme, which is prepared by a hydrothermal method and a calcining method, and comprises the following specific steps:
adding 0.2-0.8g of ferric 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; centrifuging the reaction solution to obtain a precipitate, washing the precipitate with deionized water and ethanol respectively, drying at 60 ℃, and calcining the dried product at 500-700 ℃ in argon for 1-3h to obtain the Fe-Mo bimetallic nano-enzyme Fe 2 MoO 4 。
The ferric salt is ferric trichloride 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 Fe-Mo bimetallic nano-enzyme.
The preparation method of the sensor specifically comprises the following steps:
(1) Hybridization chain reaction: heating the mixed solution of the H1 chain and the H2 chain, annealing and gradually cooling; adding a target chain into H1 chain and H2 chain mixed solution, and incubating for 2 hours at 37 ℃ to obtain a hybridization chain reaction product;
(2) Building a sensor: and (3) mixing the hybridization chain reaction products with the Fe-Mo bimetallic nano-enzyme, 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 is 5'-GACCATGAAGGCTGTGCTGCTTCATGGTCACTGGTGGGCCA-3' (SEQ ID NO: 2).
The target strand is a mutated fragment of a portion of the normal strand of PSCA rs2294008, having a nucleotide sequence of 5'-GACCATGAAGGCTGTGCTGCTTGCCCTGTTGA-3' (SEQ ID NO: 3)
The invention performs sensitivity and selectivity performance characterization on the sensor through peroxidase test analysis, and records absorbance at 650 nm; the detection limit of the sensor on the target chain is as low as 2pM.
The invention also provides application of the sensor in preparation of a kit for analyzing or detecting bladder cancer related gene mutation. The invention also provides application of the Fe-Mo bimetallic nano-enzyme in preparing a kit for analyzing or detecting the mutation of the bladder cancer related gene.
The Fe-Mo bimetallic nano-enzyme can be used for analyzing the mutation of a bladder cancer related gene, and analyzing the bladder cancer related gene PSCA rs2294008 (C > T) by combining a colorimetric method with a hybridization chain reaction, and comprises the following specific steps: (1) hybridization chain reaction: heating the mixed solution of the H1 chain and the H2 chain, annealing and gradually cooling; adding a target chain into H1 chain and H2 chain mixed solution, and incubating for 2 hours at 37 ℃ to obtain a hybridization chain reaction product;
(2) Building a sensor: mixing the hybridization chain reaction products with Fe-Mo bimetallic nano-enzyme, and culturing for 8-10 minutes to obtain a sensor;
(3) The constructed sensor was characterized for sensitivity and selectivity performance by peroxidase test analysis and absorbance at 650nm was recorded.
The Fe-Mo bimetallic nano-enzyme disclosed by the invention can prevent the formation of Fe particles with lower catalytic activity due to the dilution of Fe by Mo, so that the catalytic activity of the Fe-based catalyst can be adjusted. Therefore, fe/Mo bimetallic nano-enzyme is expected to interact with genes, has better enzyme simulation activity and becomes an effective probe for predicting the risk of the bladder cancer based on the HCR, wherein the effective probe is stable, selective and sensitive.
The application principle of the bimetal nano enzyme is as follows: the mutation site (target) of the bladder cancer-related gene is designed as a target strand; the H1 and H2 sequences are rationally designed to be stable hairpin configurations that do not interact spontaneously without the target; 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 the DNA is attached to the nano-enzyme particle. The cohesive end part is formed by DNA base and Fe 2 MoO 4 The Van der Waals force between the two is strongly combined with the material, so that the nano enzyme surface is covered by DNA and prevented from reacting with 3,3', 5' -tetramethyl benzidine (TMB). The negatively charged long double-stranded portion can be bonded to Fe 2 MoO 4 Competing for positively charged TMB while rejecting Fe 2 MoO 4 . Furthermore, the length of the HCR product produced can reach 40nm, which exceeds the diffusion distance of OH (20 nm). Thus, the nanoenzyme and TMB bound to the distal portion of the nucleic acid duplex are not easily oxidized by OH. Thus, the activity of the HCR product-modified nanoenzyme may be reduced, as can be seen from the reduction in uv absorbance.
Compared with the prior art, the invention has the following beneficial effects:
(1) Spherical Fe-Mo bimetallic nano-enzyme Fe is synthesized by a hydrothermal method and a calcining method for the first time 2 MoO 4 The specific surface area is large, the active sites are more, and the peroxidase activity is excellent.
(2) Build based on Fe 2 MoO 4 Colorimetric method of (C) and Hybrid Chain Reaction (HCR) analysis of bladder cancer related gene PSCA rs2294008 (C)>T)。
(3)Fe 2 MoO 4 On one hand, the nano enzyme is used as a tool of a colorimetric sensor, has simple preparation and good stability, and overcomes the defects of natural enzymes; on the other hand, it exploits the inherent peroxidase-like activity, rather than aggregation or deaggregation 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 the target DNA concentration from 25pM to 4 nM. The results show that the hybrid chain reaction Fe is combined 2 MoO 4 Analysis of bladder cancer related Gene PSCA rs2294008 (C) by Nano-enzyme as colorimetric sensor>The method of T) has good accuracy and repeatability. In addition, the application of the sensor in serum samples verifies the practicability, and the strategy has wide prospects in biochemical analysis and clinical application.
Description of the drawings:
FIG. 1 is a schematic diagram of the principle of analysis of bladder cancer related genes by using Fe-Mo bimetallic nano-enzyme according to the present invention.
FIG. 2 shows Fe prepared according to the present invention 2 MoO 4 Wherein A is Fe 2 MoO 4 Scanning electron microscope images of (2); b is Fe 2 MoO 4 Is a transmission electron microscope image; c is Fe 2 MoO 4 Is a high resolution transmission electron microscope image; d is Fe 2 MoO 4 Is a pattern of elements of the pattern.
FIG. 3 shows Fe according to the present invention 2 MoO 4 Wherein curve a is TMB+H 2 O 2 The method comprises the steps of carrying out a first treatment on the surface of the Curve b is Fe 2 MoO 4 +H 2 O 2 The method comprises the steps of carrying out a first treatment on the surface of the Curve c is Fe 2 MoO 4 +TMB+H 2 O 2 。
FIG. 4 shows Fe according to the present invention 2 MoO 4 Results of ESR test for hydroxyl radical generation are schematically shown.
FIG. 5 is a schematic diagram showing analysis of nucleic acid results of HCR process according to the present invention. Lane 1 is 2 μm target strand; lane 2 is 2 μ M H1 chain; lane 3 is 2 μ M H2 chain; lane 4 is 2μ M H1+2μ M H2; lane 5 is 1 μm target strand +2 μ M H1+2 μ M H2; lane 6 is 2 μm target strand +2 μ M H1+2 μ M H2; lane 7 is 4 μm target strand +2μ M H1+2μ M H2; m is DNA Marker.
FIG. 6 shows Fe according to the present invention 2 MoO 4 Schematic of interaction with HCR product, wherein a is a hydrated particle size schematic; b is a zeta potential schematic diagram.
FIG. 7 is a view of the Fe-based alloy according to the present invention 2 MoO 4 A condition optimization schematic diagram of a sensing platform, wherein A is pH value optimization of a reaction solution; b is reaction temperature optimization; c is optimized for H chain concentration (h1:h2=1:1); d is Fe 2 MoO 4 And (5) optimizing the concentration.
FIG. 8 shows the Fe-based alloy according to the present invention 2 MoO 4 The performance of the sensing platform is shown in schematic form, wherein A is the sensitivity and B is the selectivity.
The specific embodiment is as follows:
the invention will now be described in more 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, fe 2 MoO 4 The bimetallic nano-enzyme is synthesized by a hydrothermal method and a calcining method, and comprises the following specific steps:
(1) Adding 0.54g of ferric trichloride hexahydrate, 12.941g of sodium citrate and 0.3g of urea into 40mL of deionized water, and stirring until the mixture is completely dissolved; adding 0.3g polyacrylamide and 0.229g molybdenum trioxide while stirring until 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 supernatant to obtain precipitate; adding deionized water into the precipitate, centrifuging for 10min at 8000r/min, and then adding ethanol for centrifuging and washing; finally, drying the obtained precipitate sample at 60 ℃ for 10 hours;
(3)calcining the obtained sample in argon at 600 ℃ for 2h, wherein the temperature rising rate is 3 ℃ for min -1 The method comprises the steps of carrying out a first treatment on the surface of the Then naturally cooling to room temperature, and obtaining a product which is the Fe-Mo bimetallic nano-enzyme Fe 2 MoO 4 。
In this example, fe was prepared by using a scanning electron microscope and a high-resolution transmission electron microscope pair 2 MoO 4 Characterization was performed and the results are shown in fig. 2. As shown in a 2A scanning electron microscope, fe 2 MoO 4 The prepared nano particles are in uniform spherical shape, the particle size is about 50nm, the specific surface area is large, and the active sites are more; FIG. 2A is consistent with the morphology of the transmission electron microscope of FIG. 2B. Fe (Fe) 2 MoO 4 The high resolution transmission electron microscopy image (FIG. 2C) shows that the lattice spacing of the nanoparticle crystal structure is about 0.28nm, fe 2 MoO 4 (422) diffraction plane of (JCPDS No. 25-1430) is uniform. To provide further evidence, fe was analyzed by elemental mapping 2 MoO 4 The results are shown in fig. 2D, which shows uniformity.
The present example uses hydrogen peroxide (H) 2 O 2 ) And 3,3', 5' -tetramethyl benzidine (TMB) pair prepared Fe-Mo bimetallic nano enzyme 2 MoO 4 The material is subjected to enzyme active matter characterization, and the result is shown in figure 3, fe 2 MoO 4 Has excellent peroxidase and enzyme properties. For Fe 2 MoO 4 Electron Spin Resonance (ESR) was performed to further verify the form of active oxygen, and the result is shown in fig. 4, fe 2 MoO 4 Characteristic peaks 1:2:2:1 appear, indicating Fe 2 MoO 4 Can convert hydrogen peroxide into hydroxyl radicals which develop TMB.
Example 2:
this example relates to a method for preparing Fe-Mo bimetallic nanoenzyme, fe 2 MoO 4 The bimetallic nano-enzyme is synthesized by a hydrothermal method and a calcining method, and comprises the following specific steps:
(1) Adding 0.8g of ferric trichloride hexahydrate, 11g of sodium citrate and 0.4g of urea into 40mL of deionized water, and stirring until the mixture is 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 a precipitate; adding deionized water into the precipitate, centrifuging for 15min at 8000r/min, and then adding ethanol for centrifuging and washing; finally, drying the obtained precipitate sample at 60 ℃ for 8 hours;
(3) Calcining the obtained sample at 700 ℃ in argon for 3 hours at a temperature rising rate of 3 ℃ for min -1 The method comprises the steps of carrying out a first treatment on the surface of the Then naturally cooling to room temperature, and obtaining a product which is the Fe-Mo bimetallic nano-enzyme Fe 2 MoO 4 。
Example 3:
this example relates to the hybridization chain reaction of a target strand and a hairpin strand, specifically performed according to the following steps, hairpin 1 strand (H1) and hairpin 2 strand (H2) in a volume ratio of 1:1, mixing, heating the mixed solution at 95 ℃ for 5min, and gradually cooling to room temperature; then, an equal volume of target strand was added to the H1 strand H2 strand mixture and incubated at 37℃for 2H, triggering the HCR reaction. The solutions used for diluting the DNA were 1 XSSC buffer (pH 7.5,50mM Na) 2 HPO 4 ,1M NaCl)。
The HCR product was analyzed by agarose gel electrophoresis (2% agarose gel, 150V,25 minutes) and a nucleic acid imaging system, and the results are shown in FIG. 5. As can be seen from FIG. 5, after incubation of the target strand with H1, H2, the H1, H2 bands gradually disappeared with increasing concentration of the target strand, and a new band became bright (lanes 5-7). Since the HCR process consumes two hairpins, the brightness of the bands corresponding to H1 and H2 is significantly reduced. The appearance of new bands is a result of the formation of long DNA by HCR.
In summary, the H1 sequence and 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 reacted with Fe 2 MoO 4 Nano-enzyme mixing, fe 2 MoO 4 The nano enzyme surface is covered by HCR product DNA to prevent TMB and Fe with peroxidase activity 2 MoO 4 And hydroxyl radicals generated by the reaction with hydrogen peroxide. The negatively charged long double-stranded portion can be bonded to Fe 2 MoO 4 Competing for positively charged TMB while rejecting Fe 2 MoO 4 . The activity of the HCR product modified nanoenzyme may be reduced. Whereas the wild type (normal gene without mutation) does not trigger HCR, the absorbance change is not significant. The DNA sequences used are shown in Table 1.
TABLE 1 sequence listing of target chain, H1, H2 chain and wild type
| Name of the name | Sequence(s) |
| Target strand | 5’-GACCATGAAGGCTGTGCTGCTTGCCCTGTTGA-3’ |
| H1 chain | 5’-GCAGCACAGCCTTCATGGTCCTGGCCCACCAGTGACCATGA-3’ |
| H2 chain | 5’-GACCATGAAGGCTGTGCTGCTTCATGGTCACTGGTGGGCCA-3’ |
| Wild type | 5’-GACCACGAAGGCTGTGCTGCTTGCCCTGTTGA-3’ |
Wild type: normal genotype without mutation.
Example 4:
this example relates to Fe according to example 1 2 MoO 4 The 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) of the annealing of the embodiment 3 after naturally cooling to room temperature, and incubating for 2 hours at 37 ℃ to obtain an HCR product; the HCR product was combined with 25. Mu. LFe 2 MoO 4 Mixing nano material acetic acid-sodium acetate buffer solution (with concentration of 1 mg/mL), and incubating at room temperature for 10 minutes to obtain Fe-based material 2 MoO 4 A sensor of nano enzyme. The particle size and potential analysis of the sensor is shown in FIG. 6, and the result of FIG. 6A is Fe 2 MoO 4 The particle size becomes larger after the nanomaterial interacts with the HCR product, and the negatively charged HCR product is shown in fig. 6B with Fe 2 MoO 4 The potential changes after the nanomaterial is bound. These results demonstrate the success of the sensor construction.
Example 5:
this example relates to the Fe-based alloy described in example 3 2 MoO 4 Under the condition that other conditions are the same, the 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, and the specific steps are as follows:
(1) pH value optimization: the sensor was prepared using acetate-sodium acetate buffers of different pH values (3, 3.5, 4, 4.5, 5) and subjected to peroxidase activity test; (2) temperature optimization: performing peroxidase activity tests on the prepared sensor at different temperatures (20, 30, 40, 50, 60 ℃); (3) optimization of H chain concentration (h1:h2=1:1): 4H chains (0.5, 1, 2 and 3 mu M) with different concentrations are selected to respectively carry out HCR reaction with target chains (0.5, 1, 2, 3 and 4 mu M), and a sensor is prepared by using the obtained products and is subjected to peroxidase activity test; (4) optimization of material concentration: equivalent HCR product and different concentration of Fe 2 MoO 4 The nanomaterials (20, 25, 30 μm) were incubated and the resulting products were tested for peroxidase activity; the results are shown in FIG. 7. As can be seen from FIG. 7, at pH3.5, 50 ℃, 2. Mu. M H chain and 25. Mu.g/mL Fe 2 MoO 4 The best test result can be obtained under the condition, and the subsequent tests are all carried out under the condition.
Example 6:
the present embodiment relates to Fe-based 2 MoO 4 The sensor performance of the nano enzyme is characterized by comprising the following specific steps:
(1) Sensitivity test: at pH3.5, 50DEG C, 2 mu M H chain and 25 mu g/mL Fe 2 MoO 4 Performing peroxidase activity test on target chain HCR products with different concentrations under the condition; collecting absorbance at 650 nm; (2) selectivity test: at pH3.5, 50 ℃, 2. Mu. M H strand and 25. Mu.g/mL Fe 2 MoO 4 Incubating the HCR product with the target strand and the wild-type strand, respectively, under the same conditions, the other group not being incubated under the same conditions; then, performing a peroxidase activity test; the results are shown in FIG. 8.
As can be seen from the graph of fig. 8A, the change in absorbance (Δa=a0-a, at 650 nm) increases linearly with increasing concentration of target chains in the range of 25pM to 4nm, where A0 and a are the absorbance of the system with and without HCR product; linear regression equation fit to y=0.4353x+0.7841, r2=0.994; the limit of detection (LOD) was evaluated as 2pM (S/N.gtoreq.3); accuracy of Sensor (CVs)<3%, n=3); the CV values are all 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, the color is diluted, and this is reflected by the difference in absorbance; whereas HCR reactions cannot be triggered without incubation or without target strand, this is manifested by a smaller absorbance difference. These results indicate that based on Fe 2 MoO 4 The colorimetric sensor of the nano enzyme has higher selectivity.
Example 7:
the present embodiment relates to Fe-based 2 MoO 4 The nanoenzyme sensor is applied to a test in a serum sample. To prove the potential for practical use, fe-based alloy will be 2 MoO 4 Is used in diluted serum to demonstrate its feasibility. Target chain-triggered HCR products with different concentrations (0.5, 1 and 2 nM) are obtained, a serum sample diluted 10 times is added into the obtained HCR product, and then a sensor is constructed by using the method described in example 4 and the optimized parameters obtained in example 5 to perform a peroxidase activity test to detect the concentration of the target chain in the serum. The results calculated after the UV absorbance at 650nm are collected are summarized in Table 2, indicating that the test results obtained are consistent with the theoretical values. The recovery rate of the target chain is 92.2-102.9%, and the detection is carried outThe measurement accuracy is good. The Relative Standard Deviation (RSD) of the 3 detection concentrations was less than 6.5%, indicating good reproducibility of the method. These results indicate that the biosensor performs well in the simulated analysis of serum samples.
TABLE 2 Fe-based 2 MoO 4 Test results of nanoenzyme sensor applied to serum sample
Sequence listing
<110> university of Qingdao
<120> an Fe-Mo bimetallic nano-enzyme, its preparation method and application
<130> 2022
<160> 2
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gcagcacagc cttcatggtc ctggcccacc agtgaccatg a 41
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gaccatgaag gctgtgctgc ttcatggtca ctggtgggcc a 41
Claims (9)
1. The preparation method of the Fe-Mo bimetallic nano-enzyme is characterized in that ferric salt, sodium salt and urea are added into deionized water and stirred until the ferric salt, the sodium salt and the urea are completely dissolved; adding polyacrylamide and molybdenum precursor while stirringUntil 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 supernatant, and washing precipitate; the resulting sample was dried at 60 ℃; calcining at 500-700 deg.c in argon for 1-3 hr, and naturally cooling to room temperature to obtain Fe product 2 MoO 4 The method comprises the steps of carrying out a first treatment on the surface of the The sodium salt is sodium citrate.
2. The method for preparing the iron-molybdenum bimetallic nanoenzyme according to claim 1, wherein the ferric salt is ferric trichloride hexahydrate or ferrous chloride.
3. The method for preparing the iron-molybdenum bimetallic nanoenzyme according to claim 1, wherein the molybdenum precursor is molybdenum trioxide or molybdenum trichloride.
4. The method for preparing the iron-molybdenum bimetallic nanoenzyme according to claim 1, wherein the temperature rising rate during calcination is 3 ℃/min.
5. The iron-molybdenum bimetallic nanoenzyme according to claim 1, wherein the iron-molybdenum bimetallic nanoenzyme is Fe having a spherical structure 2 MoO 4 The particle size is 20-80nm.
6. A sensor prepared using the iron molybdenum bimetallic nanoenzyme of claim 5.
7. The sensor according to claim 6, wherein the method steps for preparing the sensor comprise:
(1) Hybridization chain reaction: heating the mixed solution of the H1 chain and the H2 chain, annealing and gradually cooling; adding a target chain into H1 chain and H2 chain mixed solution, and incubating for 2 hours at 37 ℃ to obtain a hybridization chain reaction product;
(2) Building a sensor: mixing the hybridization chain reaction products with Fe-Mo bimetallic nano-enzyme, and culturing for 8-10 minutes to obtain a sensor;
the H1 chain sequence is 5'-GCAGCACAGCCTTCATGGTCCTGGCCCACCAGTGACCATGA-3'; the H2 chain sequence is 5'-GACCATGAAGGCTGTGCTGCTTCATGGTCACTGGTGGGCCA-3'; the target strand sequence is 5'-GACCATGAAGGCTGTGCTGCTTGCCCTGTTGA-3'.
8. Use of the iron-molybdenum bimetallic nanoenzyme according to claim 5 for the preparation of a kit for detecting a mutation in a gene associated with bladder cancer.
9. Use of a sensor according to any one of claims 6 or 7 in the manufacture of a kit for detecting a mutation in a gene associated with bladder cancer.
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