CN114410751A - Marker nucleic acid probe, preparation method thereof, test strip and application of polypyrrole nano particles - Google Patents
Marker nucleic acid probe, preparation method thereof, test strip and application of polypyrrole nano particles Download PDFInfo
- Publication number
- CN114410751A CN114410751A CN202111653244.6A CN202111653244A CN114410751A CN 114410751 A CN114410751 A CN 114410751A CN 202111653244 A CN202111653244 A CN 202111653244A CN 114410751 A CN114410751 A CN 114410751A
- Authority
- CN
- China
- Prior art keywords
- probe
- detection
- nucleic acid
- quality control
- polypyrrole
- 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.)
- Granted
Links
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 65
- 229920000128 polypyrrole Polymers 0.000 title claims abstract description 63
- 238000012360 testing method Methods 0.000 title claims abstract description 61
- 239000003550 marker Substances 0.000 title claims abstract description 54
- 108020004711 Nucleic Acid Probes Proteins 0.000 title claims abstract description 46
- 239000002853 nucleic acid probe Substances 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title abstract description 15
- 239000000523 sample Substances 0.000 claims abstract description 145
- 238000001514 detection method Methods 0.000 claims abstract description 136
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 19
- 238000003908 quality control method Methods 0.000 claims description 60
- 239000002773 nucleotide Substances 0.000 claims description 42
- 125000003729 nucleotide group Chemical group 0.000 claims description 42
- 239000006185 dispersion Substances 0.000 claims description 25
- 108020004414 DNA Proteins 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 14
- 108020004707 nucleic acids Proteins 0.000 claims description 14
- 102000039446 nucleic acids Human genes 0.000 claims description 14
- 150000007523 nucleic acids Chemical class 0.000 claims description 14
- 239000003381 stabilizer Substances 0.000 claims description 14
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 12
- 238000005859 coupling reaction Methods 0.000 claims description 12
- 239000002245 particle Substances 0.000 claims description 12
- 108010090804 Streptavidin Proteins 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 8
- 229920001223 polyethylene glycol Polymers 0.000 claims description 8
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 8
- APSBXTVYXVQYAB-UHFFFAOYSA-M sodium docusate Chemical compound [Na+].CCCCC(CC)COC(=O)CC(S([O-])(=O)=O)C(=O)OCC(CC)CCCC APSBXTVYXVQYAB-UHFFFAOYSA-M 0.000 claims description 8
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims description 6
- 229920001214 Polysorbate 60 Polymers 0.000 claims description 6
- 229960002685 biotin Drugs 0.000 claims description 6
- 235000020958 biotin Nutrition 0.000 claims description 6
- 239000011616 biotin Substances 0.000 claims description 6
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 6
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 6
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 6
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 5
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 5
- 102000053602 DNA Human genes 0.000 claims description 4
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 4
- 230000000295 complement effect Effects 0.000 claims description 4
- 235000019329 dioctyl sodium sulphosuccinate Nutrition 0.000 claims description 4
- 229960000878 docusate sodium Drugs 0.000 claims description 4
- 239000002250 absorbent Substances 0.000 claims description 3
- 230000002745 absorbent Effects 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 claims description 3
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 claims description 3
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 2
- -1 tween40 Polymers 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims 2
- 150000003573 thiols Chemical class 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 230000000007 visual effect Effects 0.000 abstract description 4
- 238000003860 storage Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 59
- 239000012488 sample solution Substances 0.000 description 15
- 241000341655 Human papillomavirus type 16 Species 0.000 description 13
- 239000000020 Nitrocellulose Substances 0.000 description 13
- 229920001220 nitrocellulos Polymers 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000012528 membrane Substances 0.000 description 12
- 238000010521 absorption reaction Methods 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 8
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical group [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 230000001590 oxidative effect Effects 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- 238000005507 spraying Methods 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 3
- 239000012149 elution buffer Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000007800 oxidant agent Substances 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 3
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000000635 electron micrograph Methods 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 229910002567 K2S2O8 Inorganic materials 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 229910017472 S2O8 Inorganic materials 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010483 polyoxyethylene sorbitan monopalmitate Nutrition 0.000 description 1
- 239000000249 polyoxyethylene sorbitan monopalmitate Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000012465 retentate Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229960002385 streptomycin sulfate Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
Images
Classifications
-
- 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
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
-
- 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application discloses a marker nucleic acid probe, a preparation method thereof, a test strip and application of polypyrrole nano particles. The label nucleic acid probe comprises a label and a detection probe loaded on the label, and the label is polypyrrole nano particles. The polypyrrole nano particles are creatively adopted as the markers, the polypyrrole nano particles have high storage stability, high conductivity and high light stability, the lateral chromatography test paper based on the polypyrrole nano particles can carry out sensitive and quantitative visual detection on DNA or RNA in a short time, the minimum concentration of the DNA which can be detected by naked eyes is 1pM, and the detection sensitivity of the lateral chromatography test paper is greatly improved.
Description
Technical Field
The application relates to the field of nucleic acid chromatography test paper, in particular to a marker nucleic acid probe, a preparation method thereof, test paper and application of polypyrrole nano particles.
Background
The detection of genes (DNA or RNA) is of great importance in gene therapy, clinical diagnosis and various biomedical research. The currently accepted method of gene detection is the Polymerase Chain Reaction (PCR). PCR is highly sensitive and accurate, but it is based on laboratory procedures, requiring relatively trained personnel. Furthermore, false positive results often occur when small amounts of fragmented DNA are present as a contaminant. Therefore, there is an urgent need to develop a simple, economical, highly sensitive and specific rapid in-situ detection technique for DNA.
The lateral chromatography is a technique for detecting a target substance in a sample based on a hybridization reaction between a nucleic acid probe and a target nucleic acid under the action of capillary chromatography. The lateral chromatography test strip consists of 4 parts, namely a sample pad, a combination pad, a test pad (usually a nitrocellulose membrane) and a water absorption pad, wherein the sample pad is used for quickly absorbing a sample to be tested; the combination pad is loaded with a marker nucleic acid probe, and the marker nucleic acid probe is combined with a detection target in a sample to be detected to form a detectable complex during detection; a detection line and a quality control line are arranged on the test pad and used for developing a color-marked compound and observing a detection result; the absorbent pad allows the sample to flow laterally over the test pad.
Among them, the labeled nucleic acid probe is one of the key factors affecting the sensitivity of the chromatography test paper, and the most widely used at present is a colloidal gold labeled probe, which is a detection probe combined on the surface of a colloidal gold particle. However, the sensitivity of the use of colloidal gold as a labeling material is low, and it is described in the literature that the minimum detection concentration of a nucleic acid chromatography strip based on colloidal gold is 500 pM. How to improve the detection sensitivity of the lateral chromatography test strip is a research hotspot in the field at present.
Disclosure of Invention
An object of the present application is to provide a marker nucleic acid probe and a preparation method thereof, which are beneficial to improving the detection sensitivity of a nucleic acid chromatography test strip.
Another object of the present application is to provide a nucleic acid chromatography test strip having high detection sensitivity.
It is a further object of the present application to provide a use of polypyrrole nanoparticles.
In order to achieve the above object, the present application provides a marker nucleic acid probe, including a marker and a detection probe loaded on the marker, where the marker is a polypyrrole nanoparticle.
Further, the average particle size of the polypyrrole nanoparticles is 10nm to 200nm, further the average particle size of the polypyrrole nanoparticles is 30nm to 80nm, and further the average particle size of the polypyrrole nanoparticles is 50nm to 60 nm.
Further, the detection probe is complementary and paired with the nucleotide sequence at the first end of the detection target, the detection probe is single-stranded DNA, the 5 'end of the detection probe is modified by sulfydryl, carboxyl or amino, and the detection probe is complementary and paired with the nucleotide sequence at the 3' end of the detection target. .
The application also provides a preparation method of the marker nucleic acid probe, which comprises the following steps:
s1, providing a dispersion of polypyrrole nanoparticles;
s2, adding deoxynucleotides, a first stabilizer and the detection probe into the dispersion liquid in sequence to carry out coupling reaction, so that the detection probe is loaded on the polypyrrole nano particles.
Further, the deoxynucleotide is selected from a mixture of one or more of: dATP, dCTP, dGTP, dTTP, the first stabilizer being selected from a mixture of one or more of: polyvinyl alcohol, sodium dodecyl sulfate, docusate sodium, polyethylene glycol, tween40, tween60 and polyvinylpyrrolidone.
Further, in step S2, in step S2, the mass fraction of the polypyrrole nanoparticles in the dispersion is 1.5 to 2.5 wt%, a solution of the deoxynucleotide at a concentration of 0.1 to 10mmol/L, a solution of the first stabilizer at a concentration of 0.1 to 10 wt%, and a solution of the detection probe at a concentration of 1 to 10 μ g/mL are added to the dispersion, and the volume ratio of the dispersion, the solution of the deoxynucleotide, the solution of the first stabilizer, and the solution of the detection probe is (400 to 600): (1-10): (5-15): (50-100).
The application still provides a test paper strip, include the bottom plate and set gradually sample pad, combination pad, test pad and the pad that absorbs water on the bottom plate, the load is gone up to the combination pad and is had the aforementioned marker nucleic acid probe of this application.
Furthermore, a detection line and a quality control line are arranged on the test pad, the detection line is close to the combination pad, the quality control line is close to the water absorption pad, a conjugate of a capture probe is arranged on the detection line, the capture probe is complementarily paired with a nucleotide sequence at the second end of a detection target, a conjugate of a quality control probe is arranged on the quality control line, and the quality control probe is complementarily paired with the detection probe. .
Further, the conjugate of the capture probe is a conjugate of the capture probe and streptavidin, the 3 'end of the capture probe is labeled by biotin, and the capture probe is complementarily paired with the nucleotide sequence at the 5' end of the detection target; the conjugate of the quality control probe is a conjugate of the quality control probe and streptavidin, and the 3' end of the quality control probe is labeled by biotin.
The application also provides application of the polypyrrole nano particles as a marker in a nucleic acid chromatography test strip.
Compared with the prior art, the beneficial effect of this application lies in: the polypyrrole nano particles are creatively applied to the marker nucleic acid probe, the polypyrrole nano particles have high storage stability, high conductivity and high light stability, the marker nucleic acid probe based on the polypyrrole nano particles is applied to lateral chromatography test paper, sensitive and quantitative visual detection can be carried out on DNA or RNA in a short time, the minimum concentration of the DNA which can be detected by naked eyes is 1pM, the minimum detection concentration is 20 times smaller than that of the existing gold nano particles (AuNPs), and the detection sensitivity of the detection test paper is greatly improved. The marker nucleic acid probe provides a new idea for DNA detection, and has wide prospects in clinical application and biomedical diagnosis.
Drawings
FIG. 1 is a schematic representation of one embodiment of a test strip of the present application;
FIG. 2 shows that the test strip detects a target nucleic acid;
FIG. 3 shows the target nucleic acid not detected by the strip;
fig. 4 is an electron micrograph of polypyrrole nanoparticles of example 1 of the present application.
Fig. 5 is an electron micrograph of polypyrrole nanoparticles of example 2 of the present application.
Fig. 6 is a test photograph of each test strip in example 6 of the present application.
Fig. 7 is a test photograph of each test strip in example 7 of the present application.
Fig. 8 is a photograph of each test strip in example 11 of the present application.
Fig. 9 is a photograph of each test strip of example 12 of the present application.
Detailed Description
The present application is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
In the description of the present application, it should be noted that, for the terms of orientation, such as "central", "lateral", "longitudinal", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc., it indicates that the orientation and positional relationship shown in the drawings are based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, but does not indicate or imply that the device or element referred to must have a specific orientation, be constructed in a specific orientation, and be construed as limiting the specific scope of protection of the present application.
It is noted that the terms first, second and the like in the description and in the claims of the present application are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order.
The terms "comprises," "comprising," and "having," and any variations thereof, in the description and claims of this application, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The application provides a marker nucleic acid probe, which comprises a marker and a detection probe loaded on the marker, wherein the detection probe is complementarily paired with a nucleotide sequence at one end of a detection target, and the marker is a polypyrrole nano particle.
The existing marker nucleic acid probe usually adopts colloidal gold (or gold nanoparticles) as a marker, the inventor creatively adopts polypyrrole nanoparticles as the marker, the polypyrrole nanoparticles have higher storage stability, higher conductivity and high light stability, the lateral chromatography test paper based on the polypyrrole nanoparticles can perform sensitive and quantitative visual detection on DNA or RNA in a short time, the minimum concentration of the DNA which can be detected by naked eyes is 1pM, and the detection sensitivity of the lateral chromatography test paper is greatly improved. The marker nucleic acid probe based on the polypyrrole nanoparticles provides a new idea for DNA detection, and has wide prospects in clinical application and biomedical diagnosis.
The principle that the polypyrrole nanoparticles can be used as the markers is that the polypyrrole nanoparticles are dark in color, a black band visible to naked eyes can be formed when the polypyrrole nanoparticles are aggregated in a small amount in a detection line, and the detection line is darker in color when the polypyrrole nanoparticles are aggregated more, so that the polypyrrole nanoparticles can be used for qualitative or semi-quantitative detection. In addition, the connection between the detection probe and the polypyrrole nanoparticle is mainly adsorption, but it is not excluded that the detection probe and the polypyrrole nanoparticle are connected by a chemical bond.
In some embodiments, the polypyrrole nanoparticles have an average particle size of 10nm to 200 nm. Preferably, the polypyrrole nanoparticles have an average particle size of 30nm to 80 nm. Further preferably, the polypyrrole nanoparticles have an average particle diameter of 50nm to 60 nm.
In some embodiments, the detection target of the present application is a single-stranded DNA or RNA with 15-50 bases, the detection probe is a single-stranded DNA, the 5 'end of the detection probe is modified by a thiol group, a carboxyl group or an amino group, and the detection probe is complementary-paired with the 3' end nucleotide sequence of the detection target.
The marker nucleic acid probe of the present application can be used for various types of gene detection, and therefore the present application does not limit the type of detection probe. For example, the nucleotide sequence of the detection target may be 5'-atgacctatgaattgacagac-3', and the nucleotide sequence of the detection probe may be 5 '-Sulfhydroyl-gtctgtcaa-3'; for another example, the nucleotide sequence of the assay target may be 5'-ggcatttgttggggtaaccaactatttgtt-3', and the nucleotide sequence of the detection probe may be SH-C6-5'-aacaaatagttg-3'; for another example, the nucleotide sequence of the assay target may be 5'-UAGCUUAUCAGACUGAUGUUGA-3' (microRNA21), and the nucleotide sequence of the detection probe may be 5 '-Thiol-CCCCCTAGACACCGTGTTCAACATCAGT-3'.
The application also provides a preparation method of the marker nucleic acid probe, which comprises the following steps:
s1, providing a dispersion of polypyrrole nanoparticles;
s2, adding deoxynucleotides, a first stabilizer and a detection probe into the dispersion liquid in sequence to carry out coupling reaction, so that the detection probe is coupled on the polypyrrole nano particles.
In some embodiments, in step S1, the polypyrrole nanoparticles are dispersed in deionized water to obtain the dispersion, and the mass fraction of the polypyrrole nanoparticles in the dispersion is 1.5 wt% to 2.5 wt%.
In some embodiments, the polypyrrole nanoparticles are prepared by a method comprising: adding pyrrole monomerAdding the polypyrrole nano-particles into a first solution containing a second stabilizer, then adding an oxidant for oxidative polymerization, finally adding a terminator to terminate the reaction, and centrifugally purifying the reacted solution to obtain the polypyrrole nano-particles. Wherein, the solvent of the first solution can be but is not limited to water, methanol, diethyl ether; the second stabilizer is selected from a mixture of one or more of: polyvinyl alcohol (PVA), Sodium Dodecyl Sulfate (SDS), docusate sodium (Sodiumdocusate or AOT), polyethylene glycol (PEG), Tween40 (Polyoxyethylenesorbitan monopalmitate or TWEEN40), Tween60 [ Poly (ethyleneglycol) sorbitan or TWEEN60]Polyvinylpyrrolidone (PVP); the oxidant is selected from any one of the following: FeCl3，K2S2O8，H2O2，(NH4)S2O8，AgNO3，CuCl2(ii) a The terminating agent may be, but is not limited to, methanol. Preferably, the molar ratio of the oxidant to the pyrrole monomer is (1-3): 1.
in some embodiments, in step S2, the deoxynucleotides are selected from a mixture of one or more of: dATP, dCTP, dGTP, dTTP; the first stabilizer is selected from a mixture of one or more of: polyvinyl alcohol (PVA), Sodium Dodecyl Sulfate (SDS), docusate sodium (Sodiumdocusate or AOT), polyethylene glycol (PEG), Tween40 (Polyoxyethylenesorbationmonopalmitate or TWEEN40), Tween60 [ Poly (ethyleneglycol) sorbitan monostate or TWEEN60], polyvinylpyrrolidone (PVP).
In some embodiments, in step S2, a solution of the deoxynucleotide at a concentration of 0.1 to 10mmol/L is added to the dispersion, a solution of the first stabilizer at a concentration of 0.1 to 10 wt% is added to the dispersion, and a solution of the detection probe at a concentration of 1 to 10 μ g/mL is added to the dispersion.
In some embodiments, the volume ratio of the dispersion, the solution of deoxynucleotides, the solution of the first stabilizer, and the solution of the detection probe is (400-600): (1-10): (5-15): (50-100).
In some embodiments, after each addition of one solution to the dispersion, shaking mixing is performed for 10 to 30min at a temperature of 20 to 30 ℃, preferably for 20min at a temperature of 25 ℃.
In some embodiments, in step S2, the coupling reaction conditions are: and (4) performing shaking table incubation at room temperature for 2-8 h, preferably performing shaking table incubation at room temperature for 4 h.
In some embodiments, in step S2, after the coupling reaction, the solution of the marker nucleic acid probe is obtained through the steps of centrifugation, collection of the precipitate, washing and resuspension.
It is worth mentioning that the preservation temperature of the marker nucleic acid probe solution is 2-8 ℃, and the preferable preservation temperature is 4 ℃.
The application also provides a nucleic acid chromatography test strip, which comprises a bottom plate, and a sample pad, a combination pad, a test pad and a water absorption pad which are sequentially arranged on the bottom plate, wherein the combination pad is loaded with the marker nucleic acid probe.
Further, the test pad is provided with a detection line and a quality control line, the detection line is close to the binding pad, the quality control line is close to the water absorption pad, the detection line is provided with a conjugate of a capture probe, the capture probe is complementarily paired with a nucleotide sequence at the second end of the detection target (the nucleotide sequence at the first end of the detection target is complementarily paired with the detection probe, so that the detection target can be combined with the detection probe, and the quality control line is provided with a conjugate of a quality control probe, and the quality control probe is complementarily paired with the detection probe.
The application method of the test strip provided by the application is as follows: and (2) immersing the sample pad into a buffer solution containing target DNA or RNA, transferring the liquid to the water absorption pad, and carrying out visual evaluation on the detection area and the quality control area within 10min, wherein the detection area and the detection area are positive when strips appear, the quality control area is negative when strips appear and no strip exists in the test area, and the detection is invalid when no strip exists in the quality control area.
As shown in fig. 2, when the sample pad is immersed in a solution containing a detection target, the detection target first reaches the binding pad to bind to the marker nucleic acid probe, and then the marker nucleic acid probe bound to the detection target and the marker nucleic acid probe unbound to the detection target reach the test pad, the detection target binds to the capture probe at the detection line, a band appears at the detection line, and the marker nucleic acid probe unbound to the detection target reaches the quality control line to bind to the quality control probe, and a band appears at the quality control line. And the detection line and the quality control line are both colored, which indicates that the sample contains a detection target.
As shown in FIG. 3, when the sample pad is immersed in a solution containing no detection target or the concentration of the detection target in the solution is low, the labeled nucleic acid probe is bound to the quality control probe only when the solution reaches the quality control line based on the principle of lateral chromatography, and a band appears at the quality control line. Indicating that the sample does not contain the detection target or that the concentration of the detection target in the sample is below the minimum detection limit.
In some embodiments, the conjugate of the capture probe is a conjugate of the capture probe and streptavidin. Further, the 3 'end of the capture probe is labeled with biotin, and the capture probe is complementarily paired with the nucleotide sequence at the 5' end of the detection target.
In some embodiments, the conjugate of the quality control probe is a conjugate of the quality control probe and streptavidin. Further, the 3' end of the quality control probe is labeled by biotin.
In one embodiment, the capture probe or the quality control probe is prepared by the following steps: mixing 50nmol of capture probe or quality control probe with streptavidin aqueous solution (80 μ L, 2.5mg/mL) for coupling reaction (preferably incubating at 25 ℃ for 1h) to obtain a coupling reaction solution, mixing the coupling reaction solution with Phosphate Buffered Saline (PBS) with the concentration of 500mg/L, placing the mixture in a sample dialysis tube with the cut-off molecular weight of 30000, centrifuging at 6000rpm for 20min to remove the unreacted capture probe or quality control probe, and collecting the cut-off solution to obtain a capture probe conjugate solution or a quality control probe conjugate solution. Wherein, the mol ratio of the capture probe or the quality control probe to the streptavidin is preferably 100: 1; the temperature of centrifugation is preferably 4 ℃ and the centrifugation step is preferably repeated three times, each time the centrifuged product is dissolved in 500. mu.L of PBS and placed in a dialysis tube for centrifugation.
And selecting a proper capture probe and a proper quality control probe according to the difference between the detection target and the detection probe.
For example, the nucleotide sequence of the detection target is 5'-atgacctatgaattgacagac-3', the nucleotide sequence of the detection probe is 5 '-sulfo-hydroxy-gtctgtcaa-3', the nucleotide sequence of the capture probe is 5 '-ataggtcat-Biotin-3', and the nucleotide sequence of the quality control probe is 5 '-Biotin-MC 6-D-ttgacagac-3'.
For another example, the nucleotide sequence of the detection target is 5'-ggc att tgt tgg ggt aac caa cta ttt gtt-3', the nucleotide sequence of the detection probe is SH-C6-5'-aac aaa tag ttg-3', the nucleotide sequence of the capture probe is 5'-cca aca aat gcc-3' -Biotin, and the nucleotide sequence of the quality control probe is Biotin-5'-caa cta ttt gtt-3'.
For another example, the nucleotide sequence of the detection target is 5'-UAG CUUA UCA GAC UGA UGU UGA-3' (microRNA21), the nucleotide sequence of the detection probe is 5 '-Thiol-CCCCCT AGA CAC CGT GTT CAA CATC AGT-3', the nucleotide sequence of the capture probe is 5 '-CTG ATA AGC TAC CCCC-Biotin-3', and the nucleotide sequence of the quality control probe is 5 '-Biotin-ACACGG TGT CTA GGG GG-3'.
The application also provides a preparation method of the test strip, which comprises the following steps:
a1, sticking a nitrocellulose membrane on a bottom plate, and then fixing a combination pad, a sample pad and a water absorption pad in sequence;
a2, loading the marker nucleic acid probe on the bonding pad, spraying the capture probe conjugate solution on the detection line of the nitrocellulose membrane, and spraying the quality control probe conjugate solution on the quality control line of the nitrocellulose membrane.
In some embodiments, the label nucleic acid probe solution is adsorbed on the conjugate pad in an amount of 8. mu.L/cm2～12μL/cm2The concentration of the marker nucleic acid probe solution is 2 x 10-4mg/uL～8×10-4mg/uL, preferably 10. mu.L/cm2At a concentration of 6X 10-4mg/uL。
The spraying amount of the capture probe conjugate solution and the quality control probe conjugate solution is 0.5-1 mu L, and the solution concentration is 0.1 nmol/mu L-0.5 nmol/mu L; preferably, the amount sprayed is 1. mu.L, and the solution concentration is 0.2 nmol/. mu.L.
The application also provides application of the polypyrrole nano particles, and the polypyrrole nano particles are used as a marker to be applied to the nucleic acid chromatography test strip.
[ example 1 ]
Preparing a polypyrrole nanoparticle dispersion liquid: a500 mL Erlenmeyer flask was charged with 100mL of water, PVA (M)w31000)8g, stirring at 900rpm for 20min at room temperature; adding 500mg of pyrrole monomer, and stirring at 900rpm for 20min at room temperature; after 50mL (56mg/mL) of ferric chloride aqueous solution was rapidly added, stirring was carried out at 1500rpm for 6 hours at room temperature, 20mL of methanol was added to terminate the reaction, centrifugation was carried out at 15000rpm for 30 minutes to remove the supernatant, the obtained solid was washed with hot water three times, and then dispersed in water for use, the mass fraction of polypyrrole nanoparticles in the dispersion was 2.5 wt% o, and the average particle diameter of the polypyrrole nanoparticles was 46nm, as shown in FIG. 8.
[ example 2 ]
Preparing a polypyrrole nanoparticle dispersion liquid: a500 mL Erlenmeyer flask was charged with 100mL of water, PVA (M)w9000)8g, stirring at 900rpm for 20min at room temperature; adding 500mg of pyrrole monomer, and stirring at 900rpm for 20min at room temperature; after 50mL (56mg/mL) of ferric chloride aqueous solution was rapidly added, stirring was carried out at 1500rpm for 6 hours at room temperature, 20mL of methanol was added to terminate the reaction, centrifugation was carried out at 15000rpm for 30 minutes to remove the supernatant, the obtained solid was washed with hot water three times, and then dispersed in water for use, the mass fraction of polypyrrole nanoparticles in the dispersion was 2.5 wt% o, and the average particle size of the polypyrrole nanoparticles was 89nm, as shown in FIG. 9.
[ example 3 ]
Detecting the nucleotide sequence of target HPV 16: 5'-ggc att tgt tgg ggt aac caa cta ttt gtt-3' are provided.
HPV16 marker nucleic acid probe preparation: 500. mu.L of the polypyrrole nanoparticle dispersion of example 1 was added with 15. mu.L of dATP at a concentration of 1mmol/L and then shaken at 25 ℃ for 30min, 7.5. mu.L of a1 wt% aqueous solution of sodium dodecyl sulfate was added and then shaken at 25 ℃ for 10min, and then 10. mu.L of an HPV16 detection probe (nucleotide sequence) at a concentration of 1.0OD/mL was addedComprises the following steps: SH-C6-5'-aac aaa tag ttg-3'), and carrying out shaking table incubation for 4h at room temperature; then, the mixture was centrifuged at 8000rpm for 8min, the precipitate was collected, washed 3 times with phosphate buffered saline, and resuspended in an elution buffer to prepare an HPV16 marker nucleic acid probe solution (concentration: about 6X 10)-4mg/. mu.L) was stored at 4 ℃ for later use.
The preparation method of the elution buffer solution comprises the following steps: 304mg of Na was added to 40g of water in this order3PO4·12H2O, 2.0g of bovine serum albumin, 4.0g of sucrose and 0.1g of Tween-20, and mixing uniformly.
[ example 4 ]
Preparation of HPV16 capture probe conjugates:
(1)100uL of HPV16 capture probe (nucleotide sequence: 5'-cca aca aat gcc-3' -Biotin) with concentration of 0.5nmol/uL and streptavidin aqueous solution (80 uL, 2.5mg/mL) were mixed for coupling reaction, and the molar ratio of HPV16 capture probe to streptavidin was about 100: 1, incubating for 1h at 25 ℃ under the coupling reaction condition to obtain a coupling reaction solution;
(2) mixing the coupling reaction solution with Phosphate Buffered Saline (PBS) with the concentration of 500mg/L, placing the mixture in a sample dialysis tube with the molecular weight cutoff of 30000, centrifuging at 6000rpm for 20min to remove unreacted HPV16, and collecting the cutoff solution;
(3) step (2) was repeated 3 times, and the retentate in the dialysis tubing was HPV16 capture probe conjugate solution (concentration about 0.2 nm/. mu.L).
[ example 5 ]
HPV16 quality control probe conjugates: the difference from the example 4 lies in that the HPV16 capture probe (nucleotide sequence: 5'-cca aca aat gcc-3' -Biotin) is replaced by an HPV16 quality control probe (nucleotide sequence: Biotin-5'-caa cta ttt gtt-3').
[ example 6 ]
Preparing a test strip: sticking the nitrocellulose membrane on a bottom plate, and then fixing the combination pad, the sample pad and the water absorption pad in sequence; the prepared marker nucleic acid probe solution was adsorbed onto a conjugate pad (example 3) in an amount of 10. mu.L/cm2(ii) a Spray-coating [ example 4 ] capture probe on detection line of nitrocellulose membraneThe amount of the solution of the conjugate was 1. mu.L, and the solution of the conjugate of the quality control probe was sprayed on the quality control line of the nitrocellulose membrane (example 5) in an amount of 1. mu.L.
Sample solution preparation: solutions with target DNA concentrations of 0, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM, 5000pM were prepared, respectively, and the solvent of the solution was a buffer (1/4 SSC).
The test strips were immersed in 100. mu.L of the above sample solutions, and the detection region and the quality control region were visually evaluated within 10 min. The detection structure is shown in fig. 6. In FIG. 6, the test strips from left to right are sequentially immersed in solutions of 0, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM, and 5000pM in the target DNA concentration. It can be seen that, in the test strip, the detection line display can be visually observed under the condition that the target DNA concentration is 1pM, and the depth of the detection line is increased along with the increase of the DNA concentration.
[ example 7 ]
Preparing a test strip: sticking the nitrocellulose membrane on a bottom plate, and then fixing the combination pad, the sample pad and the water absorption pad in sequence; the prepared marker nucleic acid probe solution was adsorbed onto a conjugate pad (example 3) in an amount of 5. mu.L/cm2(ii) a The capture probe conjugate solution (example 4) was sprayed onto the detection line of the nitrocellulose membrane at a spray rate of 1 μ L, and the quality control probe conjugate solution (example 5) was sprayed onto the quality control line of the nitrocellulose membrane at a spray rate of 1 μ L.
Sample solution preparation: respectively preparing solutions with target DNA concentrations of 0, 0.1pM, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM and 5000pM, wherein the solvent of the solution is a buffer solution, and the buffer solution is prepared by mixing 1/4SSC and serum according to the ratio of 1: 1.
The test strips were immersed in 100. mu.L of the above sample solutions, and the detection region and the quality control region were visually evaluated within 10 min. The detection structure is shown in FIG. 7. In FIG. 7, the test strips from left to right are sequentially immersed in a solution containing target DNA at concentrations of 0, 0.1pM, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM, 5000pM, and 10 nM. It can be seen that, in the test strip, the detection line display can be visually observed under the condition that the target DNA concentration is 1pM, and the depth of the detection line is increased along with the increase of the DNA concentration.
[ example 8 ]
Detecting a nucleotide sequence of a target microRNA 21: 5'-UAG CUUA UCA GAC UGA UGU UGA-3' are provided. Wherein the detection target microRNA21 is synthesized.
Preparation of microRNA21 marker nucleic acid probe: taking 500 mu L of the polypyrrole nanoparticle dispersion liquid of the example 2, adding 15 mu L of dATP with the concentration of 1mmol/L, oscillating for 30min at the temperature of 25 ℃, adding 7.5 mu L of lauryl sodium sulfate aqueous solution with the mass concentration of 1 wt% and oscillating for 10min at the temperature of 25 ℃, then adding 10uL of microRNA21 detection probe (the nucleotide sequence is 5 '-Thiol-CCCCCT AGA CAC CGT GTT CAA CATC AGT-3') with the concentration of 1.0OD/mL, and incubating for 4h in a shaking table at room temperature; then centrifuging at 8000rpm for 8min, collecting precipitate, washing with phosphate buffer solution for 3 times, and suspending in elution buffer to obtain microRNA21 labeled nucleic acid probe solution (with concentration of about 6 × 10)-4mg/. mu.L) was stored at 4 ℃ for later use.
[ example 9 ]
Preparing a microRNA21 capture probe: the difference from the example 4 lies in that the HPV16 capture probe (nucleotide sequence: 5'-cca aca aat gcc-3' -Biotin) is replaced by the microRNA21 capture probe (nucleotide sequence: 5 '-CTG ATA AGC TAC CCCC-Biotin-3').
[ example 10 ]
Preparing a microRNA21 quality control probe: the difference from the example 4 lies in that the HPV16 capture probe (nucleotide sequence: 5'-cca aca aat gcc-3' -Biotin) is replaced by a microRNA21 quality control probe (nucleotide sequence: 5 '-Biotin-ACACGG TGT CTA GGG GG-3').
[ example 11 ]
Preparing a test strip: sticking the nitrocellulose membrane on a bottom plate, and then fixing the combination pad, the sample pad and the water absorption pad in sequence; the prepared marker nucleic acid probe was adsorbed onto a conjugate pad (example 8) in an amount of 5. mu.L/cm2(ii) a The capture probe solution (example 9) was sprayed onto the detection line of the nitrocellulose membrane at a spray rate of 1. mu.L, and the quality control probe solution (example 10) was sprayed onto the quality control line of the nitrocellulose membrane at a spray rate of 1. mu.L.
Sample solution preparation: solutions with target microRNA21 nucleic acid concentrations of 0, 0.1pM, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM and 5000pM are respectively prepared, and the solvent of the solution is buffer solution (1/4 SSC). Wherein the microRNA21 is artificially synthesized.
The test strips were immersed in 100. mu.L of the above sample solutions, and the detection region and the quality control region were visually evaluated within 10 min. The detection structure is shown in figure 8. In FIG. 8, the test strips from left to right are sequentially immersed in solutions of 0, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM, and 5000pM in the target DNA concentration. It can be seen that, in the test strip, the detection line display can be visually observed when the concentration of the target nucleic acid is 1pM, and the depth of the detection line increases with the increase of the concentration of the nucleic acid.
[ example 12 ]
The method for extracting the target RNAmicrorRNA 21 from the cells comprises the following steps: at 37 deg.C, 5% CO2MDA-MB-231 cells were cultured in L-15 medium containing 10% fetal bovine serum (Gibco) and 1% antibiotics (100U/ml penicillin and 100mg/ml streptomycin sulfate) in an atmosphere. Under the same conditions, HeLa cells were seeded in DMEM medium containing 10% antibiotics. All cells were treated in the log phase and counted using a cell counter prior to treatment. The extract was obtained by extracting total RNA (including microRNA21) using RNA extraction kit (chinese tiangen) according to the manufacturer's instructions.
The test strip was prepared in the same manner as in example 11.
The following sample solutions were prepared, respectively: a sample solution with a target concentration of 0, a sample solution with a target concentration of 50pM (in which RNAMICRORNA 21 is artificially synthesized), a sample solution containing HeLa cell total RNA extract, a sample solution containing MDA-MB-231 cell extract.
In FIG. 9, from left to right, the test strips were immersed in a sample solution with a target concentration of 0, a sample solution with a target concentration of 50pM (in which microRNA21 was artificially synthesized), a sample solution containing HeLa cell total RNA extract, and a sample solution containing MDA-MB-231 cell extract, respectively. The test result in fig. 9 can show that the test strip of the present application has good applicability and can detect microRNA21 in cancerous cells.
The foregoing has described the general principles, essential features, and advantages of the application. It will be understood by those skilled in the art that the present application is not limited to the embodiments described above, which are merely illustrative of the principles of the application, but that various changes and modifications may be made without departing from the spirit and scope of the application, and these changes and modifications are intended to be within the scope of the application as claimed. The scope of protection claimed by this application is defined by the following claims and their equivalents.
Claims (10)
1. A marker nucleic acid probe comprises a marker and a detection probe loaded on the marker, and is characterized in that the marker is a polypyrrole nanoparticle.
2. The marker nucleic acid probe according to claim 1, wherein the polypyrrole nanoparticle has an average particle diameter of 10nm to 200nm, further wherein the polypyrrole nanoparticle has an average particle diameter of 30nm to 80nm, and further wherein the polypyrrole nanoparticle has an average particle diameter of 50nm to 60 nm.
3. The marker nucleic acid probe of claim 1, wherein the detection probe is complementary paired with a nucleotide sequence at a first end of a detection target, the detection probe is single-stranded DNA, the 5 'end of the detection probe is modified with a thiol, carboxyl, or amino group, and the detection probe is complementary paired with a nucleotide sequence at a 3' end of the detection target.
4. The method for preparing a marker nucleic acid probe according to any one of claims 1 to 3, comprising the steps of:
s1, providing a dispersion of the polypyrrole nanoparticles;
s2, adding deoxynucleotides, a first stabilizer and the detection probe into the dispersion liquid in sequence to carry out coupling reaction, so that the detection probe is loaded on the polypyrrole nano particles.
5. The method for preparing a marker nucleic acid probe according to claim 4, wherein the deoxynucleotide is selected from a mixture of one or more of: dATP, dCTP, dGTP, dTTP, the first stabilizer being selected from a mixture of one or more of: polyvinyl alcohol, sodium dodecyl sulfate, docusate sodium, polyethylene glycol, tween40, tween60 and polyvinylpyrrolidone.
6. The method according to claim 4 or 5, wherein in step S2, the mass fraction of the polypyrrole nanoparticles in the dispersion is 1.5 to 2.5 wt%, a solution of the deoxynucleotide at a concentration of 0.1 to 10mmol/L, a solution of the first stabilizer at a concentration of 0.1 to 10 wt%, and a solution of the detection probe at a concentration of 1 to 10 μ g/mL are added to the dispersion, and the volume ratio of the dispersion, the solution of the deoxynucleotide, the solution of the first stabilizer, and the solution of the detection probe is (400 to 600): (1-10): (5-15): (50-100).
7. A test strip comprising a substrate, and a sample pad, a conjugate pad, a test pad and a water absorbent pad sequentially disposed on the substrate, wherein the conjugate pad carries the marker nucleic acid probe of any one of claims 1 to 3 or the marker nucleic acid probe prepared by the method of any one of claims 4 to 6.
8. The test strip of claim 7, wherein the test pad has a detection line disposed thereon and a quality control line, the detection line is disposed adjacent to the conjugate pad, the quality control line is disposed adjacent to the absorbent pad, the detection line has a conjugate of a capture probe, the capture probe is complementary-paired with a nucleotide sequence at a second end of the detection target, the quality control line has a conjugate of a quality control probe disposed thereon, and the quality control probe is complementary-paired with the detection probe.
9. The test strip of claim 8, wherein the conjugate of the capture probe is a conjugate of the capture probe and streptavidin, the 3 'end of the capture probe is labeled with biotin, and the capture probe is complementarily paired with the nucleotide sequence at the 5' end of the detection target; the conjugate of the quality control probe is a conjugate of the quality control probe and streptavidin, and the 3' end of the quality control probe is labeled by biotin.
10. The application of the polypyrrole nanoparticles is characterized in that the polypyrrole nanoparticles are used as a marker to be applied to a nucleic acid chromatography test strip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111653244.6A CN114410751B (en) | 2021-12-30 | 2021-12-30 | Marker nucleic acid probe, preparation method thereof, test strip and application of polypyrrole nano particle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111653244.6A CN114410751B (en) | 2021-12-30 | 2021-12-30 | Marker nucleic acid probe, preparation method thereof, test strip and application of polypyrrole nano particle |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114410751A true CN114410751A (en) | 2022-04-29 |
| CN114410751B CN114410751B (en) | 2024-01-12 |
Family
ID=81269774
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111653244.6A Active CN114410751B (en) | 2021-12-30 | 2021-12-30 | Marker nucleic acid probe, preparation method thereof, test strip and application of polypyrrole nano particle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114410751B (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020004246A1 (en) * | 2000-02-07 | 2002-01-10 | Daniels Robert H. | Immunochromatographic methods for detecting an analyte in a sample which employ semiconductor nanocrystals as detectable labels |
| CN103777003A (en) * | 2014-01-27 | 2014-05-07 | 西南大学 | Portable electrochemical quantitative immunochromatography filtering paper as well as test paper and application thereof |
| JP2014209117A (en) * | 2013-03-29 | 2014-11-06 | 積水メディカル株式会社 | Color particles for immunochromato, and immunochromato reagent for diagnosis using the same |
| CN106662585A (en) * | 2014-07-01 | 2017-05-10 | 新日铁住金化学株式会社 | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, kit for assaying analyte, and test strip for lateral flow chromatography |
| CN109991202A (en) * | 2019-04-16 | 2019-07-09 | 南京医科大学 | A method of it is detected based on aptamer fluorescent optical sensor for multiple target objects |
| CN111398587A (en) * | 2020-04-02 | 2020-07-10 | 安徽科技学院 | Colloidal gold lateral chromatography test strip for detecting cervical cancer and preparation method thereof |
| CN112522368A (en) * | 2020-11-20 | 2021-03-19 | 安徽科技学院 | Immunochromatographic test strip for detecting DNA and preparation method thereof |
| CN113406329A (en) * | 2021-06-15 | 2021-09-17 | 江南大学 | Universal aptamer colloidal gold lateral chromatography test paper for detecting small molecular substances |
-
2021
- 2021-12-30 CN CN202111653244.6A patent/CN114410751B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020004246A1 (en) * | 2000-02-07 | 2002-01-10 | Daniels Robert H. | Immunochromatographic methods for detecting an analyte in a sample which employ semiconductor nanocrystals as detectable labels |
| JP2014209117A (en) * | 2013-03-29 | 2014-11-06 | 積水メディカル株式会社 | Color particles for immunochromato, and immunochromato reagent for diagnosis using the same |
| CN103777003A (en) * | 2014-01-27 | 2014-05-07 | 西南大学 | Portable electrochemical quantitative immunochromatography filtering paper as well as test paper and application thereof |
| CN106662585A (en) * | 2014-07-01 | 2017-05-10 | 新日铁住金化学株式会社 | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, kit for assaying analyte, and test strip for lateral flow chromatography |
| CN109991202A (en) * | 2019-04-16 | 2019-07-09 | 南京医科大学 | A method of it is detected based on aptamer fluorescent optical sensor for multiple target objects |
| CN111398587A (en) * | 2020-04-02 | 2020-07-10 | 安徽科技学院 | Colloidal gold lateral chromatography test strip for detecting cervical cancer and preparation method thereof |
| CN112522368A (en) * | 2020-11-20 | 2021-03-19 | 安徽科技学院 | Immunochromatographic test strip for detecting DNA and preparation method thereof |
| CN113406329A (en) * | 2021-06-15 | 2021-09-17 | 江南大学 | Universal aptamer colloidal gold lateral chromatography test paper for detecting small molecular substances |
Non-Patent Citations (1)
| Title |
|---|
| GRACIELA C PEDRO等: "A novel nucleic acid fluorescent sensing platform based on nanostructured films of intrinsically conducting polymers", 《ANAL CHIM ACTA》, vol. 1047, pages 214 - 224, XP085564982, DOI: 10.1016/j.aca.2018.10.010 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114410751B (en) | 2024-01-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7119194B2 (en) | Nucleic acid-bondable magnetic carrier and method for isolating nucleic acid using the same | |
| JP4220397B2 (en) | Bioconjugate-nanoparticle probe | |
| Kalvoda et al. | Adsorptive stripping voltammetry in trace analysis | |
| JP4146239B2 (en) | Bio barcode based on oligonucleotide modified particles | |
| US9308582B2 (en) | Solution stable and chemically reactive metallic nanoparticles | |
| US20230081589A1 (en) | Universal Aptamer-based Colloidal Gold Lateral Flow Test Strip for Detecting Small-molecule Substances | |
| US6274387B1 (en) | Magnetic carrier, preparation thereof, and method of extraction of nucleic acid | |
| EP1260595A2 (en) | Nucleic acid-bondable magnetic carrier and method for isolating nucleic acid using the same | |
| WO2007103558A2 (en) | Hybrid energy transfer for nucleic acid detection | |
| JP2003514227A (en) | Binding promotion method for analyte detection | |
| EP3193172B1 (en) | Solid phase carrier, ligand-bound solid phase carrier, method for detecting or separating target substance, and method for producing solid phase carrier | |
| CN109270135B (en) | Modified electrode for norfloxacin molecular imprinting electrochemical sensor and preparation method thereof | |
| WO2002004668A2 (en) | Improved capture and detection of target nucleic acid in dipstick assays | |
| Cheng et al. | Hairpin probes based click polymerization for label-free electrochemical detection of human T-lymphotropic virus types II | |
| CN114410751B (en) | Marker nucleic acid probe, preparation method thereof, test strip and application of polypyrrole nano particle | |
| EP3115384B1 (en) | Solid support, ligand-bound solid support, detection or separation method for target substance, solid support production method, and ligand-bound solid support production method | |
| WO2005062982A2 (en) | Signal amplification methods for analyte detection | |
| AU2020103548A4 (en) | A Test Strip Based on TdT Signal Amplification Technique and Its Preparation Method | |
| CN109097445A (en) | A method of the nanometer technology for gold based on silver staining enhancement detects nucleic acid | |
| WO2009129236A2 (en) | Method for manipulating samples with magnetic nucleation nanoparticles | |
| CN101932730A (en) | The method of concentrating nucleic acid molecules | |
| JP2009136238A (en) | Method for detecting gene | |
| CN112522368B (en) | Immunochromatography test strip for detecting DNA and preparation method thereof | |
| JPH0131931B2 (en) | ||
| JP3448776B2 (en) | Carrier for nucleic acid binding, method for preparing the same, and method for detecting target nucleic acid |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |