CN110988359A - Preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application of Au @ HIF-1 aptamer @ Au nanoenzyme in detection of latent coronary heart disease - Google Patents
Preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application of Au @ HIF-1 aptamer @ Au nanoenzyme in detection of latent coronary heart disease Download PDFInfo
- Publication number
- CN110988359A CN110988359A CN201911238301.7A CN201911238301A CN110988359A CN 110988359 A CN110988359 A CN 110988359A CN 201911238301 A CN201911238301 A CN 201911238301A CN 110988359 A CN110988359 A CN 110988359A
- Authority
- CN
- China
- Prior art keywords
- hif
- solution
- aptamer
- nanoenzyme
- room temperature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102100032742 Histone-lysine N-methyltransferase SETD2 Human genes 0.000 title claims abstract description 90
- 238000001514 detection method Methods 0.000 title claims abstract description 33
- 208000029078 coronary artery disease Diseases 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 101000654725 Homo sapiens Histone-lysine N-methyltransferase SETD2 Proteins 0.000 claims abstract description 40
- 108091023037 Aptamer Proteins 0.000 claims abstract description 32
- 210000001808 exosome Anatomy 0.000 claims abstract description 29
- 210000002966 serum Anatomy 0.000 claims abstract description 26
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000010931 gold Substances 0.000 claims abstract description 19
- 229910052737 gold Inorganic materials 0.000 claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims abstract description 17
- 238000000151 deposition Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 105
- 238000005406 washing Methods 0.000 claims description 21
- 238000003756 stirring Methods 0.000 claims description 18
- 239000007853 buffer solution Substances 0.000 claims description 15
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000011258 core-shell material Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 238000009835 boiling Methods 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- 238000002791 soaking Methods 0.000 claims description 10
- 239000000758 substrate Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 7
- 239000008280 blood Substances 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 6
- 239000001509 sodium citrate Substances 0.000 claims description 6
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 6
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 claims description 5
- 229910017912 NH2OH Inorganic materials 0.000 claims description 5
- QZPSXPBJTPJTSZ-UHFFFAOYSA-N aqua regia Chemical compound Cl.O[N+]([O-])=O QZPSXPBJTPJTSZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 238000011161 development Methods 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 5
- LIKFHECYJZWXFJ-UHFFFAOYSA-N dimethyldichlorosilane Chemical compound C[Si](C)(Cl)Cl LIKFHECYJZWXFJ-UHFFFAOYSA-N 0.000 claims description 5
- 238000010812 external standard method Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 239000012982 microporous membrane Substances 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 239000011734 sodium Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 230000010100 anticoagulation Effects 0.000 claims description 4
- 238000005728 strengthening Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 230000003014 reinforcing effect Effects 0.000 abstract description 3
- 102000002177 Hypoxia-inducible factor-1 alpha Human genes 0.000 abstract description 2
- 108050009527 Hypoxia-inducible factor-1 alpha Proteins 0.000 abstract description 2
- 238000008157 ELISA kit Methods 0.000 abstract 1
- 102000016878 Hypoxia-Inducible Factor 1 Human genes 0.000 abstract 1
- 108010028501 Hypoxia-Inducible Factor 1 Proteins 0.000 abstract 1
- 230000014541 detection of hypoxia Effects 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 4
- 229910003771 Gold(I) chloride Inorganic materials 0.000 description 3
- 206010000891 acute myocardial infarction Diseases 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 3
- 230000002093 peripheral effect Effects 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 206010003211 Arteriosclerosis coronary artery Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010069729 Collateral circulation Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 208000000114 Pain Threshold Diseases 0.000 description 1
- 206010042434 Sudden death Diseases 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 208000026758 coronary atherosclerosis Diseases 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000001146 hypoxic effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 230000037040 pain threshold Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
- B22F1/05—Metallic powder characterised by the size or surface area of the particles
- B22F1/054—Nanosized particles
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
- B22F1/07—Metallic powder characterised by particles having a nanoscale microstructure
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F9/00—Making metallic powder or suspensions thereof
- B22F9/16—Making metallic powder or suspensions thereof using chemical processes
- B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
- B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Abstract
The invention reports preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application of Au @ HIF-1 α aptamer @ Au nanoenzyme in detection of hypoxia inducible factor-1 α (hypoxia inducible factor-1, HIF-1 α) in concealed coronary heart disease serum exosomes, firstly, nanogold is taken as a core, and sulfhydrylation HIF-1 α aptamer (HIF-1 α aptamer, the base sequence of which is 5' -HS- (CH)2)6-CCCACCCACCCATGTTGTTGTCTACGTGCT-3') orderly self-assembling on nanogold to form Au @ HIF-1 α aptamer complex, then recognizing and combining with HIF-1 α in serum exosome through the complex, and then depositing a circle of nanogold shell on the periphery of the material by using gold reinforcing liquid to form Au @ HIF-1 α aptamer @ Au shell core junctionFinally, the nano enzyme property of the nano gold shell is utilized to catalyze TMB/H2O2The result shows that compared with the prior ELISA kit, the kit has the advantages of low detection limit (up to 0.19ng/L), low detection cost, good stability of detection reagent and the like, and can be used for early warning detection of patients with the latent coronary heart disease.
Description
Technical Field
The invention relates to the field of immunoassay, in particular to preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application of the Au @ HIF-1 α aptamer @ Au nanoenzyme in detection of a hidden coronary heart disease serum exosome HIF-1 α.
Background
Latent coronary heart disease is asymptomatic, but the objective examination of coronary heart disease with myocardial ischemia is also called asymptomatic coronary heart disease. The patient had coronary atherosclerosis but had less disease or better collateral circulation, or the patient had a higher pain threshold and therefore no pain symptoms. It may suddenly turn into angina pectoris or myocardial infarction, or even sudden death. Therefore, the hidden coronary heart disease patient can be early warned as early as possible, and early treatment can be carried out in time, and the early warning device has important clinical significance and social value.
The hypoxia inducible factor-1 α (HIF-1- α) is a transcription factor promoting cell survival in a hypoxic environment, and becomes an important index for clinical diagnosis of coronary heart disease, HIF-1 α is unstable in serum and can be degraded to different degrees, HIF-1 α keeps stable expression in serum of a normal human population, and the newly increased expression of HIF-1 α in serum of a patient with occult coronary heart disease is not enough to be distinguished from a normal group after degradation, which is one of the main reasons for clinically improbable coronary heart disease non-imaging detection at present, the increased expression of HIF-1 α in exosome is relatively stable due to the absence of HIF-1 α degrading enzyme, so the increased expression of HIF-1- α in exosome is expected to become an important method for early warning of occult coronary heart disease by monitoring the content of the HIF-1 α in exosome, although the commercial HIF-1-672 kit can realize determination, the detection is higher than that the detection of the peripheral venous blood is limited by the peripheral venous blood, and the actual detection of the peripheral blood venous coronary heart disease can not be developed by a clinical detection method for realizing a lower sensitivity of the detection of the peripheral blood induction factor 734 and the peripheral venous coronary heart disease detection of the clinical occult detection.
Composite materials of nanoenzymes and aptamers (aptamers) are a hot spot of research in the field of materials in recent years. As a nano material with unique enzyme-like property, the nano enzyme can avoid the trouble of unstable structure of biological natural enzyme, save the trouble of complexity of the traditional enzyme simulation process, increase the convenience of high-efficiency catalysis under physiological mild conditions, can be produced in large scale at lower cost and has infinite potential of industrial application. The aptamer is a structured oligonucleotide sequence obtained by an in vitro screening technology-index enrichment ligand phylogenetic technology, has strict recognition capability and high affinity with corresponding target molecules (proteins, viruses, bacteria, cells and the like), and has the advantages of low price, easy synthesis, normal-temperature storage and the like.
Disclosure of Invention
The invention aims to provide a preparation method of Au @ HIF-1 α aptamer @ Au core-shell structure nanoenzyme, which has a core-shell structure and a large catalytic area, so that the lowest detection limit of HIF-1 α is greatly reduced, and meanwhile, the structural instability of biological natural enzyme can be avoided based on the catalytic performance of a nanogold shell, and the long-term storage and use of materials are facilitated.
The second purpose of the invention is to provide application of Au @ HIF-1 α aptamer @ Au core-shell structure nanoenzyme, namely, the nanoenzyme is used for catalyzing TMB/H2O2The system detects HIF-1 α in serum exosome and early warns patients with occult coronary heart disease.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a preparation method of Au @ HIF-1 α aptamer @ Au nanoenzyme and an application thereof in latent coronary heart disease detection are disclosed, wherein the technical route schematic diagram is shown in figure 1, and the preparation method specifically comprises the following steps:
(1) preparing nano gold: soaking all glass containers in aqua regia overnight, then soaking in a chloroform solution containing 5% dichlorodimethylsilane for 5-30min, airing at room temperature, finally washing with deionized water for three times, and drying for later use. 50ml of 0.05 to 1 percent of HAuCl is taken4The solution was placed in an erlenmeyer flask and heated to boiling with stirring. Then 5mL of 0.5% -5% sodium citrate solution is rapidly added into the solution, and the solution is kept boiling for 10-30min after being wine red; finally stopping heating, cooling to room temperature under stirring, using deionized water to fix the volume to 50mL to obtain a nano gold solution, and placing at 4 ℃ for later use;
(2) preparation of Au @ HIF-1 α aptamer by mixing 0.1mM HIF-1 α aptamer (base sequence 5' -HS- (CH)2) n-CCCACCCACCCATGTTGTTGTCTACGTGCT-3') solution and the nano gold obtained in the step (1)Mixing the solutions according to a volume ratio of 1: 10-1: 500, stirring for 4-16 h in a dark and mild way at room temperature, then dropwise adding 50 mu L of 0.5M Tris-acetate solution (pH 8.2) and 500 mu L of 1M NaCl solution under mild stirring, standing for 24h in a dark way at room temperature, finally, completely depositing the product at the bottom of an EP tube by centrifugation (16000g for 20min), washing twice with the Tris-acetate solution to obtain Au @ HIF-1 α aptamer solution, and storing for later use at 4 ℃;
(3) extracting exosomes in serum: the whole blood was allowed to stand at room temperature for 1 hour without anticoagulation, centrifuged (3000g, 10min), and the resulting supernatant was centrifuged again (3000g, 10min) to obtain serum. Diluting serum with PBS buffer solution 1:1, centrifuging (10000g, 30min), filtering the obtained supernatant with 0.2 μm microporous membrane, ultracentrifuging (100000g, 70min), washing the obtained precipitate with PBS buffer solution once, dispersing in PBS buffer solution to obtain exosome solution, and storing at 4 deg.C;
(4) preparing Au @ HIF-1 α aptamer @ Au nanoenzyme, adding 50 mu L of exosome solution sample to an enzyme-labeled plate coated with HIF-1 α antibody, incubating at 37 ℃ for 30min, then adding 50 mu L of Au @ HIF-1 α aptamer solution obtained in step (2), further incubating at room temperature for 30min, after washing three times with PBS, adding 100 mu L of gold-strengthening solution (5mM HAuCl)4·4H2O solution with 10mM NH2OH & HCl solution with equal volume mixing), incubating for 20min at 28 ℃, washing for 3 times by PBS and drying to obtain Au @ HIF-1 α aptamer @ Au core-shell structure nanoenzyme;
(5) and (3) quantitative detection: to each well of the microplate of step (4), 100. mu.L of a substrate solution was added for color development (4.5mL of a substrate buffer (2.84g of Na)2HPO4·12H2O, 1.92g citric acid, 100mL water) +0.5mL 2mg/mL TMB solution +0.32mL 30% H2O2Mixed well) at 28 ℃ for 15min and read the OD at 652nm652nmValues, HIF-1 α levels in exosomes were calculated by external standard method.
Compared with the prior art, the invention has the beneficial effects that:
(1) the detection limit is lowered. The prior art uses horseradish peroxidase (HRP) as a catalytic enzyme, in H2O2Exist ofThe invention adopts nano gold to replace HRP, not only realizing the purpose of oxidizing TMB, but also the constructed core-shell structure Au @ HIF-1 α aptamer @ Au material has a gold shell with larger surface area and obtains better catalytic effect than HRP, thereby greatly reducing the detection limit (up to 0.19ng/L), further not concentrating the sample and directly determining the content of HIF-1 α in the serum exosome.
(2) The invention uses HIF-1 α aptamer to replace HIF-1 α used in the prior art, thus greatly reducing the detection cost.
(3) The invention uses nano enzyme and HIF-1 α aptamer to replace HRP and HIF-1 α antibodies respectively, thereby greatly improving the stability of the detection reagent and being beneficial to long-term storage and use.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram of a technical scheme of the present invention;
FIG. 2 shows the dynamic light scattering pattern (A) of nanogold, the transmission electron microscopy pattern (B) of nanogold, and the UV contrast pattern (C) of nanogold and Au @ HIF-1 α aptamer.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
The embodiment provides a preparation method of Au @ HIF-1 α aptamer @ Au nanoenzyme and application thereof in detection of latent coronary heart disease, which comprises the following steps:
(1) preparing nano gold: soaking all glass containers in aqua regia overnight, then soaking for 5min by using a chloroform solution containing 5% dichlorodimethylsilane, airing at room temperature, finally washing for three times by using deionized water, and drying for later use. 50ml of 0.01 percent HAuCl is taken4The solution was placed in an erlenmeyer flask and heated to boiling with stirring. Then 5mL of 0.5% sodium citrate solution is rapidly added into the solution, and the solution is kept boiling for 10min after being wine red; finally stopping heating, cooling to room temperature under stirring, using deionized water to fix the volume to 50mL to obtain a nano gold solution, and placing at 4 ℃ for later use;
(2) preparation of Au @ HIF-1 α aptamer by mixing 0.1mM HIF-1 α aptamer (base sequence 5' -HS- (CH)2)2-CCCACCCACCCATGTTGTTGTCTACGTGCT-3') solution and the nano-gold solution obtained in the step (1) are mixed according to the volume ratio of 1:10, and are stirred for 4 hours in a dark and gentle way at room temperature, then under the gentle stirring, 50 mu L of 0.5M Tris-acetate solution (pH 8.2) and 500 mu L of 1M NaCl solution are dripped into the solution, and are kept for 24 hours in a dark way at room temperature, finally, the product is completely deposited at the bottom of an EP tube through centrifugation (16000g, 20min), and is washed twice by Tris-acetate solution, and then Au @ HIF-1 α aptamer solution is obtained through redispersion, and is kept for standby at 4 ℃;
(3) extracting exosomes in serum: taking whole blood of an acute myocardial infarction mouse for 1h, standing at room temperature without anticoagulation, standing for 1h, centrifuging (3000g, 10min), and centrifuging the obtained supernatant again (3000g, 10min) to obtain serum. Diluting serum with PBS buffer solution 1:1, centrifuging (10000g, 30min), filtering the obtained supernatant with 0.2 μm microporous membrane, ultracentrifuging (100000g, 70min), washing the obtained precipitate with PBS buffer solution once, dispersing in PBS buffer solution to obtain exosome solution, and storing at 4 deg.C;
(4) preparing Au @ HIF-1 α aptamer @ Au nanoenzyme, adding 50 mu L of exosome solution sample into an enzyme label plate coated with HIF-1 α monoclonal antibody, incubating for 30min at 37 ℃, and adding 50 mu L of exosome solution sample into the enzyme label plateThe Au @ HIF-1 α aptamer solution obtained in step (2) was incubated at room temperature for 30min, washed three times with PBS, and 100. mu.L of gold-enriched solution (5mM AuCl)4·4H2O solution with 10mM NH2OH & HCl solution with equal volume mixing), incubating for 20min at 28 ℃, washing for 3 times by PBS and drying to obtain Au @ HIF-1 α aptamer @ Au core-shell structure nanoenzyme;
(5) and (3) quantitative detection: to each well of the microplate of step (4), 100. mu.L of a substrate solution was added for color development (4.5mL of a substrate buffer (2.84g of Na)2HPO4·12H2O, 1.92g citric acid, 100mL water) +0.5mL 2mg/mL TMB solution +0.32mL 30% H2O2Mixed well) at 28 ℃ for 15min and read the OD at 652nm652nmThe minimum detection limit of the method is 0.19ng/L, and the content of HIF-1 α in the serum exosome of the sample is calculated to be 2.74 +/-0.12 ng/L by an external standard method.
Example 2
The embodiment provides a preparation method of Au @ HIF-1 α aptamer @ Au nanoenzyme and application thereof in detection of latent coronary heart disease, which comprises the following steps:
(1) preparing nano gold: soaking all glass containers in aqua regia overnight, then soaking in a chloroform solution containing 5% dichlorodimethylsilane for 5-30min, airing at room temperature, finally washing with deionized water for three times, and drying for later use. 50ml of 0.2% HAuCl was taken4The solution was placed in an erlenmeyer flask and heated to boiling with stirring. Then 5mL of 2% sodium citrate solution is rapidly added into the solution, and the solution is kept boiling for 20min after being wine red; finally stopping heating, cooling to room temperature under stirring, using deionized water to fix the volume to 50mL to obtain a nano gold solution, and placing at 4 ℃ for later use; the morphology was observed using a dynamic light scattering and transmission electron microscope, as shown in fig. 2A and 2B;
(2) preparation of Au @ HIF-1 α aptamer by mixing 0.1mM HIF-1 α aptamer (base sequence 5' -HS- (CH)2)6-CCCACCCACCCATGTTGTTGTCTACGTGCT-3') mixing the solution with the nano gold solution obtained in the step (1) according to the volume ratio of 1:100, and stirring for 10 hours at room temperature in dark and mild conditions; then 50. mu.L of 0.5M tris-acetate solution (pH 8.2) and 500. mu.L of 1M NaCl solution were added dropwise thereto under mild stirring, and the chamber was cooledStanding in a warm and dark place for 24h, finally, completely depositing the product at the bottom of an EP tube by centrifugation (16000g, 20min), washing twice with Tris-acetate solution, and then re-dispersing to obtain Au @ HIF-1 α aptamer solution, and storing at 4 ℃ for later use, wherein an ultraviolet spectrophotometer is used for comparing ultraviolet absorption spectra of the nanogold and the Au @ HIF-1 α aptamer, as shown in figure 2C;
(3) extracting exosomes in serum: taking whole blood of the acute myocardial infarction mouse for 2h, standing at room temperature without anticoagulation for 1h, centrifuging (3000g, 10min), and centrifuging the obtained supernatant again (3000g, 10min) to obtain serum. Diluting serum with PBS buffer solution 1:1, centrifuging (10000g, 30min), filtering the obtained supernatant with 0.2 μm microporous membrane, ultracentrifuging (100000g, 70min), washing the obtained precipitate with PBS buffer solution once, dispersing in PBS buffer solution to obtain exosome solution, and storing at 4 deg.C;
(4) preparing Au @ HIF-1 α aptamer @ Au nanoenzyme, adding 50 mu L of exosome solution sample into an enzyme label plate coated with an HIF-1 α polyclonal antibody, incubating at 37 ℃ for 30min, then adding 50 mu L of Au @ HIF-1 α aptamer solution obtained in the step (2), further incubating at room temperature for 30min, washing three times with PBS, and adding 100 mu L of gold reinforcing solution (5mM AuCl)4·4H2O solution with 10mM NH2OH & HCl solution with equal volume mixing), incubating for 20min at 28 ℃, washing for 3 times by PBS and drying to obtain Au @ HIF-1 α aptamer @ Au core-shell structure nanoenzyme;
(5) and (3) quantitative detection: to each well of the microplate of step (4), 100. mu.L of a substrate solution was added for color development (4.5mL of a substrate buffer (2.84g of Na)2HPO4·12H2O, 1.92g citric acid, 100mL water) +0.5mL 2mg/mL TMB solution +0.32mL 30% H2O2Mixed well) at 28 ℃ for 15min and read the OD at 652nm652nmThe minimum detection limit of the method is 0.19ng/L, and the content of HIF-1 α in the serum exosome of the sample is calculated to be 6.85 +/-0.62 ng/L by an external standard method.
Example 3
The embodiment provides a preparation method of Au @ HIF-1 α aptamer @ Au nanoenzyme and application thereof in detection of latent coronary heart disease, which comprises the following steps:
(1) preparing nano gold: soaking all glass containers in aqua regia overnight, then soaking for 30min by using a chloroform solution containing 5% dichlorodimethylsilane, airing at room temperature, finally washing for three times by using deionized water, and drying for later use. 50mL of 1% HAuCl was taken4The solution was placed in an erlenmeyer flask and heated to boiling with stirring. Then 5mL of 5% sodium citrate solution is rapidly added into the solution, and the solution is kept boiling for 30min after being wine red; finally stopping heating, cooling to room temperature under stirring, using deionized water to fix the volume to 50mL to obtain a nano gold solution, and placing at 4 ℃ for later use;
(2) preparation of Au @ HIF-1 α aptamer by mixing 0.1mM HIF-1 α aptamer (base sequence 5' -HS- (CH)2)10-CCCACCCACCCATGTTGTTGTCTACGTGCT-3') solution and the nano-gold solution obtained in the step (1) are mixed according to the volume ratio of 1: 500, and are stirred for 16 hours in a dark and gentle way at room temperature, then 50 mu L of 0.5M Tris-acetate solution (pH 8.2) and 500 mu L of 1M NaCl solution are dripped into the solution under the gentle way of stirring, and are kept for 24 hours in a dark way at room temperature, finally, the product is completely deposited at the bottom of an EP tube by centrifugation (16000g, 20min), and is washed twice by Tris-acetate solution, and then Au @ HIF-1 α aptamer solution is obtained by redispersion, and is kept for standby at 4 ℃;
(3) extracting exosomes in serum: whole blood of the acute myocardial infarction mouse is taken, is not anticoagulated, stands at room temperature for 1h, is centrifuged (3000g, 10min), and the obtained supernatant is centrifuged again (3000g, 10min) to obtain serum. Diluting serum with PBS buffer solution 1:1, centrifuging (10000g, 30min), filtering the obtained supernatant with 0.2 μm microporous membrane, ultracentrifuging (100000g, 70min), washing the obtained precipitate with PBS buffer solution once, dispersing in PBS buffer solution to obtain exosome solution, and storing at 4 deg.C;
(4) preparing Au @ HIF-1 α aptamer @ Au nanoenzyme, adding 50 mu L of exosome solution sample into an enzyme label plate coated with an HIF-1 α polyclonal antibody, incubating at 37 ℃ for 30min, then adding 50 mu L of Au @ HIF-1 α aptamer solution obtained in the step (2), further incubating at room temperature for 30min, washing three times with PBS, and adding 100 mu L of gold reinforcing solution (5mM AuCl)4·4H2O solution with 10mM NH2OH & HCl solution mixed in equal volume), and incubating for 20min at 28 ℃; followed byWashing the core-shell structure nano enzyme with PBS for 3 times and patting the core-shell structure nano enzyme to obtain Au @ HIF-1 α aptamer @ Au core-shell structure nano enzyme;
(5) and (3) quantitative detection: to each well of the microplate of step (4), 100. mu.L of a substrate solution was added for color development (4.5mL of a substrate buffer (2.84g of Na)2HPO4·12H2O, 1.92g citric acid, 100mL water) +0.5mL 2mg/mL TMB solution +0.32mL 30% H2O2Mixed well) at 28 ℃ for 15min and read the OD at 652nm652nmThe minimum detection limit of the method is 0.19ng/L, and the content of HIF-1 α in the serum exosome of the sample is calculated to be 8.70 +/-0.53 ng/L by an external standard method.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (6)
1. Preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application thereof in detection of latent coronary heart disease, which is characterized by comprising the following steps:
(1) preparing nano gold: soaking all glass containers in aqua regia overnight, then soaking in a chloroform solution containing 5% dichlorodimethylsilane for 5-30min, airing at room temperature, finally washing with deionized water for three times, and drying for later use. 50mL of 0.05% -1% HAuCl is taken4The solution was placed in an erlenmeyer flask and heated to boiling with stirring. Then 5mL of 0.5% -5% sodium citrate solution is rapidly added into the solution, and the solution is kept boiling for 10-30min after being wine red; finally stopping heating, cooling to room temperature under stirring, using deionized water to fix the volume to 50mL to obtain a nano gold solution, and placing at 4 ℃ for later use;
(2) preparing Au @ HIF-1 α aptamer, namely mixing 0.1mM HIF-1 α aptamer solution with the nanogold solution obtained in the step (1) according to the volume ratio of 1: 10-1: 500, stirring for 4-16 h at room temperature in a dark and mild manner, then dropwise adding 50 mu L of 0.5M Tris-acetate solution (pH 8.2) and 500 mu L of 1M NaCl solution under mild stirring, standing for 24h at room temperature in a dark manner, finally, completely depositing the product at the bottom of an EP tube by centrifugation (16000g, 20min), washing twice by using Tris-acetate solution, and then redispersing to obtain Au @ HIF-1 α aptamer solution, and storing at 4 ℃ for later use;
(3) extracting exosomes in serum: the whole blood was allowed to stand at room temperature for 1 hour without anticoagulation, centrifuged (3000g, 10min), and the resulting supernatant was centrifuged again (3000g, 10min) to obtain serum. Diluting serum with PBS buffer solution 1:1, centrifuging (10000g, 30min), filtering the obtained supernatant with 0.2 μm microporous membrane, ultracentrifuging (100000g, 70min), washing the obtained precipitate with PBS buffer solution once, dispersing in PBS buffer solution to obtain exosome solution, and storing at 4 deg.C;
(4) preparing Au @ HIF-1 α aptamer @ Au nanoenzyme, adding 50 mu L of exosome solution sample to an enzyme-labeled plate coated with HIF-1 α antibody, incubating at 37 ℃ for 30min, then adding 50 mu L of Au @ HIF-1 α aptamer solution obtained in step (2), further incubating at room temperature for 30min, after washing three times with PBS, adding 100 mu L of gold-strengthening solution (5mM HAuCl)4·4H2O solution and 10mM NH2OH & HCl solution with equal volume mixing), incubating for 20min at 28 ℃, washing for 3 times by PBS and drying to obtain Au @ HIF-1 α aptamer @ Au core-shell structure nanoenzyme;
(5) and (3) quantitative detection: to each well of the microplate of step (4), 100. mu.L of a substrate solution was added for color development (4.5mL of a substrate buffer (2.84g of Na)2HPO4·12H2O, 1.92g citric acid, 100mL water) +0.5mL 2mg/mL TMB solution +0.32mL 30% H2O2Mixed well) at 28 ℃ for 15min and read the OD at 652nm652nmValues, HIF-1 α levels in exosomes were calculated by external standard method.
2. The preparation method of Au @ HIF-1 α aptamer @ Au nanoenzyme according to claim 1, wherein the particle size of the nanogold prepared by the sodium citrate reduction method in the step (1) is 5-30 nm.
3. The method for preparing Au @ HIF-1 α aptamer @ Au nanoenzyme according to claim 1, wherein the HIF-1 α aptamer used in the step (2) has a base sequence of 5' -HS- (CH)2)n-CCCACCCACCCATGTTGTTGTCTACGTGCT-3', wherein n has a value in the range of 2-10.
4. The method for preparing Au @ HIF-1 α aptamer @ Au nanoenzyme according to claim 3, wherein the base sequence of the HIF-1 α aptamer used in the step (2) is 5' -HS- (CH)2)6-CCCACCCACCCATGTTGTTGTCTACGTGCT-3’。
5. The preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme according to claim 1, wherein the HIF-1 α antibody used in step (4) is HIF-1 α monoclonal antibody or HIF-1 α polyclonal antibody.
6. The preparation of the Au @ HIF-1 α aptamer @ Au nanoenzyme of claim 5, wherein the HIF-1 α antibody is a HIF-1 α monoclonal antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911238301.7A CN110988359A (en) | 2020-02-11 | 2020-02-11 | Preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application of Au @ HIF-1 aptamer @ Au nanoenzyme in detection of latent coronary heart disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911238301.7A CN110988359A (en) | 2020-02-11 | 2020-02-11 | Preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application of Au @ HIF-1 aptamer @ Au nanoenzyme in detection of latent coronary heart disease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110988359A true CN110988359A (en) | 2020-04-10 |
Family
ID=70090661
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911238301.7A Pending CN110988359A (en) | 2020-02-11 | 2020-02-11 | Preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application of Au @ HIF-1 aptamer @ Au nanoenzyme in detection of latent coronary heart disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110988359A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111773440A (en) * | 2020-05-22 | 2020-10-16 | 南京大学 | Anticoagulation material based on enzyme-like catalytic reaction |
| CN113930335A (en) * | 2021-12-17 | 2022-01-14 | 深圳市第二人民医院(深圳市转化医学研究院) | Nano enzyme cascade bioreactor and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011133504A2 (en) * | 2010-04-20 | 2011-10-27 | University Of Florida Research Foundation, Inc. | Nanozymes, methods of making nanozymes, and methods of using nanozymes |
| CN109061187A (en) * | 2018-08-16 | 2018-12-21 | 中国科学院成都生物研究所 | A kind of clinical medicine analysis method based on functionalized nano silica |
-
2020
- 2020-02-11 CN CN201911238301.7A patent/CN110988359A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011133504A2 (en) * | 2010-04-20 | 2011-10-27 | University Of Florida Research Foundation, Inc. | Nanozymes, methods of making nanozymes, and methods of using nanozymes |
| CN109061187A (en) * | 2018-08-16 | 2018-12-21 | 中国科学院成都生物研究所 | A kind of clinical medicine analysis method based on functionalized nano silica |
Non-Patent Citations (1)
| Title |
|---|
| WANG QL 等: "Colorimetric determination of the early biomarker hypoxia-inducible factor-1 alpha (HIF-1α) in circulating exosomes by using a gold seed-coated with aptamer-functionalized Au@Au core-shell peroxidase mimic" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111773440A (en) * | 2020-05-22 | 2020-10-16 | 南京大学 | Anticoagulation material based on enzyme-like catalytic reaction |
| CN113930335A (en) * | 2021-12-17 | 2022-01-14 | 深圳市第二人民医院(深圳市转化医学研究院) | Nano enzyme cascade bioreactor and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110988359A (en) | Preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application of Au @ HIF-1 aptamer @ Au nanoenzyme in detection of latent coronary heart disease | |
| CN106904656B (en) | A kind of method and its application based on polypeptide templated synthesis manganese dioxide nano-plates | |
| Emami et al. | Comparison of gold nanoparticle conjugated secondary antibody with non-gold secondary antibody in an ELISA kit model | |
| KR101990301B1 (en) | Optical biosensor | |
| Xia et al. | Colorimetric immunoassays based on pyrroloquinoline quinone-catalyzed generation of Fe (II)-ferrozine with tris (2-carboxyethyl) phosphine as the reducing reagent | |
| Wang et al. | Poly (amino acid) multilayers modified dendritic mesoporous silica nanoparticles achieve effective enzyme stability for ultrasensitive immunoassay | |
| CN113203781A (en) | Method for detecting GPC3 based on RGO-CS-Hemin @ Pt NPs nano material and aptamer | |
| Liu et al. | Porous inorganic materials for bioanalysis and diagnostic applications | |
| Panferov et al. | Methods for increasing sensitivity of immunochromatographic test systems with colorimetric detection | |
| Coşkun et al. | Serum and ascitic fluid nitrate levels in patients with cirrhosis | |
| Lin et al. | A smartphone-assisted “all-in-one” paper chip for one-pot noninvasive detection of salivary glucose level | |
| CN111346676A (en) | Iron-substituted tungstophosphoric acid polydopamine nano mimic enzyme and preparation method and application thereof | |
| Zhang et al. | Janus nanozyme based satellite structure immunosandwich colorimetric strategy for glycoproteins visual detection | |
| Liu et al. | Construction of Magnetic Core–Large Mesoporous Satellite Immunosensor for Long‐Lasting Chemiluminescence and Highly Sensitive Tumor Marker Determination | |
| CN110702670A (en) | Sarcosine detection method based on metal organic framework material | |
| Omidfar et al. | Development of a new sensitive immunostrip assay based on mesoporous silica and colloidal Au nanoparticles | |
| CN109490532A (en) | A method of enzymic-labelled antibody is prepared using metal-organic framework material | |
| CN116003818B (en) | Method for preparing functionalized multi-metal organic framework nano enzyme and application of peroxidase activity thereof | |
| Sun et al. | Development of an approach of high sensitive chemiluminescent assay for cystatin C using a nanoparticle carrier | |
| CN113929082B (en) | Method for preparing rice straw carbon quantum dot nano enzyme and application of peroxidase activity thereof | |
| Han et al. | Design of two electrode system for detection of antioxidant capacity with photoelectrochemical platform | |
| CN110441372B (en) | Preparation method and application of hydroxyl iron oxide composite material photoelectrochemical sensor with polyoxometallate as electron donor | |
| Cheng et al. | A colorimetry-impedance dual-mode ceruloplasmin detection strategy based on dendritic mesoporous silicon nanoparticles with CeO2 encapsulation inside and horseradish peroxidase enrichment outside | |
| CN112098648A (en) | Method for detecting serum biomarker of liver cancer patient | |
| Xi et al. | Recent advances in construction and application of metal-nanozymes in pharmaceutical analysis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200410 |