CN116411135A - Inductively coupled plasma mass spectrometry nucleic acid detection method based on DTN and CRISPR Cas12a - Google Patents
Inductively coupled plasma mass spectrometry nucleic acid detection method based on DTN and CRISPR Cas12a Download PDFInfo
- Publication number
- CN116411135A CN116411135A CN202310194513.XA CN202310194513A CN116411135A CN 116411135 A CN116411135 A CN 116411135A CN 202310194513 A CN202310194513 A CN 202310194513A CN 116411135 A CN116411135 A CN 116411135A
- Authority
- CN
- China
- Prior art keywords
- ssdna
- nucleic acid
- dtn
- hpv
- nanoparticles
- 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
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 63
- 238000001514 detection method Methods 0.000 title claims abstract description 43
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 42
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 42
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 title claims abstract description 28
- 108700004991 Cas12a Proteins 0.000 title description 10
- 108091033409 CRISPR Proteins 0.000 title description 9
- 238000010354 CRISPR gene editing Methods 0.000 title description 9
- 239000002082 metal nanoparticle Substances 0.000 claims abstract description 53
- 239000011324 bead Substances 0.000 claims abstract description 45
- 238000007885 magnetic separation Methods 0.000 claims abstract description 13
- 239000000758 substrate Substances 0.000 claims abstract description 10
- 238000005406 washing Methods 0.000 claims abstract description 9
- 239000002105 nanoparticle Substances 0.000 claims description 70
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 66
- 108020004414 DNA Proteins 0.000 claims description 59
- 239000010931 gold Substances 0.000 claims description 47
- 102000053602 DNA Human genes 0.000 claims description 35
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 34
- 229910052737 gold Inorganic materials 0.000 claims description 34
- 238000000034 method Methods 0.000 claims description 34
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 33
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 27
- 229910052697 platinum Inorganic materials 0.000 claims description 27
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 claims description 22
- 239000002086 nanomaterial Substances 0.000 claims description 22
- 239000006228 supernatant Substances 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 230000004048 modification Effects 0.000 claims description 15
- 238000012986 modification Methods 0.000 claims description 15
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 13
- 108010090804 Streptavidin Proteins 0.000 claims description 12
- 238000007306 functionalization reaction Methods 0.000 claims description 12
- 125000003396 thiol group Chemical class [H]S* 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 9
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical group [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 229910052709 silver Inorganic materials 0.000 claims description 8
- 239000004332 silver Substances 0.000 claims description 8
- 238000011282 treatment Methods 0.000 claims description 8
- 239000007795 chemical reaction product Substances 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 230000002194 synthesizing effect Effects 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- QZPSXPBJTPJTSZ-UHFFFAOYSA-N aqua regia Chemical compound Cl.O[N+]([O-])=O QZPSXPBJTPJTSZ-UHFFFAOYSA-N 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 5
- 239000006185 dispersion Substances 0.000 claims description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 5
- 229910052763 palladium Inorganic materials 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 239000002131 composite material Substances 0.000 claims description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 238000001819 mass spectrum Methods 0.000 claims description 3
- 239000000523 sample Substances 0.000 abstract description 8
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000004445 quantitative analysis Methods 0.000 abstract 2
- 239000002184 metal Substances 0.000 abstract 1
- 208000022361 Human papillomavirus infectious disease Diseases 0.000 description 57
- 239000000872 buffer Substances 0.000 description 10
- 241000341655 Human papillomavirus type 16 Species 0.000 description 8
- 239000006180 TBST buffer Substances 0.000 description 8
- 108020003215 DNA Probes Proteins 0.000 description 6
- 239000003298 DNA probe Substances 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 206010008342 Cervix carcinoma Diseases 0.000 description 4
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 4
- 230000032683 aging Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 201000010881 cervical cancer Diseases 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000001509 sodium citrate Substances 0.000 description 4
- 238000001308 synthesis method Methods 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 3
- 101710134784 Agnoprotein Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241000701806 Human papillomavirus Species 0.000 description 1
- 241000701828 Human papillomavirus type 11 Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 150000004687 hexahydrates Chemical class 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000009616 inductively coupled plasma Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 150000004685 tetrahydrates Chemical class 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/708—Specific hybridization probes for papilloma
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an inductively coupled plasma mass spectrometry nucleic acid detection method based on DTN and CRISPRCas12a, which takes at least one HPV typed nucleic acid as a target, takes DTN functionalized magnetic beads as a carrier, takes CRISPR-Cas12a complex as a target recognition element, takes metal nano particles as a report probe, adopts spatially separated CRISPR-Cas12a to specifically recognize multiple target sequences and cut corresponding substrate probes, generates substrate probes with different concentrations corresponding to each target, and simultaneously acquires metal isotope signals on the surfaces of the magnetic beads by adopting ICP-MS after magnetic separation and washing, and realizes the multiple accurate quantitative analysis of HPV-DNA by researching the relationship between isotope signal intensity and HPV-DNA concentration. The invention can carry out multiple accurate quantitative analysis on HPV-DNA, and improves the accuracy and reliability of analysis results.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to an inductively coupled plasma mass spectrometry nucleic acid detection method based on DTN and CRISPR Cas12 a.
Background
Cervical cancer is the most common gynaecological malignancy, and is reported to be one of the leading causes of female death. Most cervical cancers are caused by persistent High-Risk Human papillomavirus (High Risk-Human PapillomaVirus, HR-HPV) infection. About 10 kinds of HR-HPVs (e.g., HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-52, HPV-58 and HPV-59) are reported to be closely related to precancerous lesions and cancers. HPV-16, HPV-52, HPV-18 and HPV-58 genotypes are most common in China. Early detection and genotyping of HR-HPV is extremely important for screening, diagnosis and treatment of cervical cancer. In recent years, the world health organization has used HPV-DNA detection as the method of choice for cervical cancer screening.
Although the conventional nucleic acid detection methods such as polymerase chain reaction (polymerase chain reaction, PCR) and other techniques are widely used for HPV-DNA detection and typing, the method requires amplification products, and a large amount of amplification products easily increase the risk of environmental pollution, easily generate false positive signals, and have complicated experimental procedures, thus greatly impeding wider clinical application. Therefore, there is an urgent need for new methods for genotyping HR-HPV-DNA that are simple to operate, highly sensitive, highly selective, multiplex-detectable, and do not require nucleic acid amplification.
CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-associated system) is a highly efficient gene editing tool that can achieve the identification, anchoring and cleavage of specific target nucleic acid sequences. Among other things, CRISPR-Cas12a systems have been applied in the field of in vitro diagnostics, which comprise gene-targeted crrnas and Cas proteins that, when forming ribonucleic acid complexes, can target specific nucleic acid sequences and activate the nuclease cleavage activity of Cas proteins. The high-efficiency enzyme cleavage property and the high-specificity sequence recognition property of the system enable the system to be combined with various spectrum analysis technologies such as a fluorescence method, a colorimetry method, a surface enhanced Raman scattering method and the like, so that the system has good detection performance of nucleic acid analytes, and is expected to replace traditional technologies such as PCR. However, the use of CRISPR-Cas12a based multiplex nucleic acid analyte detection is still very limited, especially for multiplex detection of HPV-DNA, due to the inherent non-specific single-stranded DNA (ssDNA) cleavage activity of CRISPR-Cas12a itself.
Disclosure of Invention
Aiming at the problems, the invention aims to provide an inductively coupled plasma mass spectrometry nucleic acid detection method based on DTN and CRISPR Cas12a, which combines a multiplex noble metal element marking technology of ICP-MS and a DNA tetrahedron nano structure (DTN) with CRISPR-Cas12a, and realizes amplification-free detection of high specificity and high sensitivity of multiplex HPV-DNA based on the CRISPR-Cas12a technology by utilizing the advantages and signal enhancement characteristics of ICP-MS (inductively coupled plasma mass spectrometry) that the interference of spectrum overlapping, scattering background and biological matrix substances is not limited and the characteristics of optimization of the conformation of a biological surface interface probe of a DNA tetrahedron nucleic acid framework structure.
The technical scheme of the invention is as follows:
an inductively coupled plasma mass spectrometry nucleic acid detection method based on DTN and CRISPR Cas12a, targeting at least one HPV typed nucleic acid, comprising the steps of:
s1: obtaining metal nano particles and magnetic beads, and modifying the metal nano particles by adopting mercapto functional ssDNA to obtain modified metal nano particles; adopting DTN to modify the magnetic beads to obtain DTN modified magnetic beads;
s2: mixing CRISPR-Cas12a protein corresponding to the target with crRNA to obtain a compound with a composite structure;
s3: adding HPV types with the same volume and different concentrations into the compound respectively, adding Linker substrates into each mixture, reacting for 30-60 min at 37 ℃, and heating to terminate the reaction to obtain a reaction product;
s4: mixing and incubating the reaction product with DTN modified magnetic beads, and then performing magnetic separation and washing;
s5: adding the modified metal nano particles into the product obtained in the step S4 for mixed incubation to form a sandwich structure;
s6: performing magnetic separation and washing on the sandwich structure to remove supernatant, digesting with aqua regia, and diluting with pure water to obtain a mass spectrum acquisition solution;
s7: performing inductively coupled plasma mass spectrometry detection on the mass spectrometry acquisition solution to obtain isotope signals of metal nano particles in the mass spectrometry acquisition solution;
s8: and researching the relation between the intensity of the isotope signal and the HPV typing concentration to obtain the HPV typing detection result.
Preferably, when a nucleic acid for HPV typing is used as a target, in step S1, gold nanoparticles are used as the metal nanoparticles;
when two HPV typed nucleic acids are used as targets, in the step S1, gold nanoparticles and silver nanoparticles are adopted as the metal nanoparticles;
when three HPV-typed nucleic acids are used as targets, in the step S1, gold nanoparticles, silver nanoparticles and platinum nanoparticles are adopted as the metal nanoparticles;
when four HPV-typed nucleic acids are used as targets, gold nanoparticles, silver nanoparticles, platinum nanoparticles, and palladium nanoparticles are used as the metal nanoparticles in step S1.
Preferably, in step S1, when the thiol-functionalized ssDNA is used to modify the metal nanoparticles, the DNA sequence shown as seq_1 is used to perform thiol functionalization, and then the gold nanoparticles are modified; performing sulfhydryl functionalization by adopting a DNA sequence shown as seq_2, and then modifying silver nano particles; thiol functionalization was performed using the DNA sequence shown as seq_3, followed by modification of the platinum nanoparticles.
Preferably, in step S1, when the DTN is used to modify the magnetic beads, the method specifically includes the following substeps:
s11: synthesizing a DNA tetrahedral nano structure;
s12: performing streptavidin functionalization on the magnetic beads to obtain streptavidin functionalized magnetic beads;
s13: mixing the DNA tetrahedron nano structure with the streptavidin functionalized magnetic beads in an equal volume, and vibrating and incubating for 1-2 h under the room temperature condition;
s14: and performing magnetic separation for multiple times, removing unreacted DNA tetrahedral nano structures, and then adding PBST buffer solution for dispersion to obtain the DTN modified magnetic beads.
Preferably, in step S11, the synthesis of the DNA tetrahedral nanostructure specifically comprises the following sub-steps:
uniformly mixing four equal amounts of single-stranded DNA in a TM buffer solution, annealing for 5-15 min at 90-95 ℃, and incubating for 2-4 h at 4 ℃ to obtain the DNA tetrahedron nano structure.
Preferably, the four single-stranded DNAs are biotin-modified ssDNA-A, biotin-modified ssDNA-B, biotin-modified ssDNA-C, ssDNA-D, respectively, which ssDNA-D is determined according to the metal nanoparticle and is selected from ssDNA-D Au 、ssDNA-D Ag 、ssDNA-D Pt ssDNA-D Pd The method comprises the steps of carrying out a first treatment on the surface of the The nucleic acid sequence of the ssDNA-A is shown as seq_4, the nucleic acid sequence of the ssDNA-B is shown as seq_5, the nucleic acid sequence of the ssDNA-C is shown as seq_6, and the ssDNA-D Au The nucleic acid sequence of said ssDNA-D is shown as seq_7 Ag The nucleic acid sequence of said ssDNA-D is shown in seq_8 Pt The nucleic acid sequence of (2) is shown as seq_9.
Preferably, in step S13, the amount of the DNA tetrahedral nanostructure is 1-2 uM, and the amount of the streptavidin-functionalized magnetic beads is 1-10 mg/mL.
Preferably, in step S2, the crRNA is determined according to the metal nanoparticle used, and when the metal nanoparticle is a gold nanoparticle, the crRNA is the crRNA shown in seq_10 Au The method comprises the steps of carrying out a first treatment on the surface of the When the metal nanoparticle is silver nanoparticle, the crRNA adopts crRNA as shown in seq_11 Ag When the metal nanoparticle is a platinum nanoparticle, the crRNA employs crRNA as shown in seq_12 Pt 。
Preferably, in step S3, the Linker substrate is selected from a Linker according to the metal nanoparticles Au 、Linker Ag 、Linker P 、Linker Pd The Linker Au The sequence of (2) is shown as seq_13, the Linker Ag The sequence of (2) is shown as seq_14, the Linker Pt The sequence of (2) is shown as seq_15.
Preferably, in step S3, when the reaction is terminated by heating, the reaction is heated to 65℃for 10 minutes.
The beneficial effects of the invention are as follows:
the invention realizes the combination of the CRISPR-Cas12a system based on spatial separation and the ICP-MS technology for the first time, performs multiple accurate diagnosis on HPV-DNA under the reinforcement effect of a DTN interface, and has universality on other multiple nucleic acid detection projects; according to the invention, DNA tetrahedron is modified on the magnetic bead surface interface, so that the orientation and distribution of the surface probe are optimized, the detection performance of the CRISPR-Cas12a system is further improved, and the HPV-DNA detection sensitivity of 50-100 pM is realized; the result of the sample detection after cervical test treatment is consistent with the result of capillary electrophoresis fragment analysis and gene sequencing, and has good clinical application prospect.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a schematic flow diagram of a method for inductively coupled plasma mass spectrometry nucleic acid detection based on DTN and CRISPR Cas12a according to an embodiment of the present invention;
FIG. 2 is a graph showing signal intensity results for different amounts of DTN and ssDNA according to one embodiment; fig. 2 (a) is a schematic diagram of a signal intensity result corresponding to DTN, and fig. 2 (b) is a schematic diagram of a signal intensity result corresponding to ssDNA;
FIG. 3 is a schematic diagram showing the transmission electron microscope result of gold nanoparticles according to one embodiment;
FIG. 4 is a graph showing the results of ultraviolet visible spectra before and after modifying ssDNA on gold nanoparticle surfaces according to one embodiment;
FIG. 5 is a graph showing particle size and potential results before and after surface modification of ssDNA of gold nanoparticles according to one embodiment;
FIG. 6 is a schematic diagram of a transmission electron microscope result of silver nanoparticles according to one embodiment;
FIG. 7 is a graph showing the results of UV-visible spectra before and after surface modification of ssDNA with silver nanoparticles according to one embodiment;
FIG. 8 is a graph showing particle size and potential results before and after surface modification of ssDNA with silver nanoparticles according to one embodiment;
FIG. 9 is a schematic diagram of a transmission electron microscope result of a platinum nanoparticle according to an embodiment;
FIG. 10 is a graphical representation of the results of UV-visible spectra before and after surface modification of ssDNA with platinum nanoparticles according to one embodiment;
FIG. 11 is a graph showing particle size and potential results before and after surface modification of ssDNA with platinum nanoparticles according to one embodiment;
FIG. 12 is a graph showing the relationship between HPV16 concentration and Au element signal in one embodiment, wherein the carrier is DTN-modified magnetic beads;
FIG. 13 is a graph showing the relationship between HPV18 concentration and Ag element signal, and the carrier is DTN modified magnetic beads;
FIG. 14 is a graph showing the relationship between HPV52 concentration and Pt element signal, wherein the carrier is DTN modified magnetic beads;
FIG. 15 is a graph showing the relationship between HPV16 concentration and Au element signal, wherein the carrier is ssDNA modified magnetic beads.
Detailed Description
The invention will be further described with reference to the drawings and examples. It should be noted that, without conflict, the embodiments and technical features of the embodiments in the present application may be combined with each other. It is noted that all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs unless otherwise indicated. The use of the terms "comprising" or "includes" and the like in this disclosure is intended to cover a member or article listed after that term and equivalents thereof without precluding other members or articles.
The invention provides an inductively coupled plasma mass spectrometry nucleic acid detection method based on DTN and CRISPR Cas12a, which takes at least one HPV typed nucleic acid as a target, and comprises the following steps:
s1: obtaining metal nano particles and magnetic beads, and modifying the metal nano particles by adopting mercapto functional ssDNA to obtain modified metal nano particles; and adopting DTN to modify the magnetic beads to obtain the DTN modified magnetic beads.
In a specific embodiment, if a nucleic acid for HPV typing is used as the target, the metal nanoparticle is a gold nanoparticle; if two HPV typed nucleic acids are used as targets, gold nanoparticles and silver nanoparticles are adopted as the metal nanoparticles; if three HPV typed nucleic acids are used as targets, gold nanoparticles, silver nanoparticles and platinum nanoparticles are adopted as the metal nanoparticles; if four HPV-typed nucleic acids are used as targets, gold nanoparticles, silver nanoparticles, platinum nanoparticles and palladium nanoparticles are adopted as the metal nanoparticles.
In a specific embodiment, the gold nanoparticles, silver nanoparticles, platinum nanoparticles, and palladium nanoparticles are synthesized using a reduction method. The reduction method of the gold nanoparticles, the silver nanoparticles and the platinum nanoparticles specifically comprises the following substeps: taking 1-2 ml of HAuCl respectively 4 ,AgNO 3 ,H 2 PtCl 6 ·6H 2 Adding the O solution (1% -8%) into 100mL of water for boiling, then adding 2mL of trisodium citrate solution, continuously heating for 20-40 min, and naturally cooling to form gold nanoparticles, silver nanoparticles and platinum nanoparticles, wherein the particle size distribution is 20-30 nanometers.
Besides the synthesis methods of the above embodiments, other synthesis methods in the prior art may be used or the gold nanoparticles, silver nanoparticles, platinum nanoparticles and palladium nanoparticles may be obtained directly by purchase.
In a specific embodiment, when the thiol-functionalized ssDNA is used to modify the metal nanoparticles, the DNA sequence shown as seq_1 is used to perform thiol functionalization, and then the gold nanoparticles are modified; performing sulfhydryl functionalization by adopting a DNA sequence shown as seq_2, and then modifying silver nano particles; thiol functionalization was performed using the DNA sequence shown as seq_3, followed by modification of the platinum nanoparticles.
Optionally, when the gold nanoparticles and the platinum nanoparticles are modified, a salt aging method is adopted for modification, and when the silver nanoparticles are modified, an acid method is adopted for modification.
The salt aging method specifically comprises the following substeps: au-ssDNA and Pt-ssDNA were first treated in 2-4 nM TCEP solution for 1h, then 60uL of 5-10 uM Au-ssDNA or Pt-ssDNA was added to 1ml of gold or platinum nanoparticle solution (containing 0.1% Tween 20 and 10mM Tris solution), stirred at room temperature for 12h, and 20uL of 3M NaCl solution was added 5 times at 1h intervals. Finally, the reaction is carried out for 24 hours at room temperature, a PEG2000 solution with 0-4 uM is added for 2 hours, finally, the centrifugation is carried out for 15 minutes at 12000r, the redundant DNA probe and PEG in the supernatant are removed, the washing is carried out for 3 times by using TBST buffer solution, and finally, the dispersion is carried out in 1mL TBS buffer solution.
The acid method specifically comprises the following substeps: ag-ssDNA was first treated in 2-4 nM TCEP solution for 1h, 600uL of silver nanoparticle solution was added, then 12uL of sodium citrate buffer (500 mM, pH 3) was added in two portions, and after incubation for 30min, 90. Mu. LHEPES buffer (500 mM, pH 7.6) was added to bring the pH back to neutral. Then, the supernatant was centrifuged at 12000rpm for 15min, and the excess DNA probe was removed, and then washed 3 times with TBST buffer, and then dispersed into 1ml TBS buffer, and then reacted with 4. Mu.M PEG for 2 hours to form a block. Finally, the supernatant was centrifuged at 12000rpm for 15min, excess PEG was removed, and then washed 3 times with TBST buffer, and then dispersed into 1ml TBS buffer for the next step.
In a specific embodiment, when the DTN is used for modifying the magnetic beads, the method specifically comprises the following substeps: s11: synthesizing a DNA tetrahedral nano structure; s12: performing streptavidin functionalization on the magnetic beads to obtain streptavidin functionalized magnetic beads; s13: mixing the DNA tetrahedron nano structure with the streptavidin functionalized magnetic beads in an equal volume, optionally, the dosage of the DNA tetrahedron nano structure is 1-2 uM, the dosage of the streptavidin functionalized magnetic beads is 1-10 mg/mL, and vibrating and incubating for 1-2 h at room temperature; s14: and performing magnetic separation for multiple times, removing unreacted DNA tetrahedral nano structures, and then adding PBST buffer solution for dispersion to obtain the DTN modified magnetic beads.
In a specific embodiment, the synthesis of DNA tetrahedral nanostructures specifically comprises the following sub-steps: uniformly mixing four equal amounts of single-stranded DNA in a TM buffer solution, annealing for 5-15 min at 90-95 ℃, and incubating for 2-4 h at 4 ℃ to obtain the DNA tetrahedron nano structure.
Alternatively, the four single-stranded DNAs are biotin-modified ssDNA-A, biotin-modified ssDNA-B, biotin-modified ssDNA-C, ssDNA-D, respectively, which are determined according to the metal nanoparticle and are selected from ssDNA-D Au 、ssDNA-D Ag 、ssDNA-D Pt ssDNA-D Pd (i.e., different metal nanoparticles are selected from DTN, and when gold nanoparticles are used only, biotin-modified ssDNA-A, biotin-modified ssDNA-B, and biotin-modified ssDNA-C, ssDNA-D are used) Au Synthesizing a DNA tetrahedral nano structure; when three metal nanoparticles of gold nanoparticles, silver nanoparticles and platinum nanoparticles are adopted, biotin-modified ssDNA-A, biotin-modified ssDNA-B and biotin-modified ssDNA-C, ssDNA-D are respectively adopted Au Biotin-modified ssDNA-A, biotin-modified ssDNA-B, biotin-modified ssDNA-C, ssDNA-D Ag Biotin-modified ssDNA-A, biotin-modified ssDNA-B, biotin-modified ssDNA-C, ssDNA-D P Synthesizing three kinds of DNA tetrahedral nano structures; the nucleic acid sequence of the ssDNA-A is shown as seq_4, the nucleic acid sequence of the ssDNA-B is shown as seq_5, the nucleic acid sequence of the ssDNA-C is shown as seq_6, and the ssDNA-D Au The nucleic acid sequence of said ssDNA-D is shown as seq_7 Ag The nucleic acid sequence of said ssDNA-D is shown in seq_8 Pt The nucleic acid sequence of (2) is shown as seq_9.
It should be noted that, the present invention mainly uses the spatial characteristics of the DNA tetrahedral nanostructure to optimize the orientation and distribution of the surface probe and enhance the signal, and other synthesis methods in the prior art besides the synthesis method of the above embodiment may be applied to the present invention.
S2: and mixing the CRISPR-Cas12a protein corresponding to the target with crRNA to obtain a complex with a composite structure.
In a specific embodiment, the crRNA is determined according to the metal nanoparticles used, and when the metal nanoparticles are gold nanoparticles, the crRNA is the crRNA shown in seq_10 Au The method comprises the steps of carrying out a first treatment on the surface of the When the metal nanoparticle is silver nanoparticle, the crRNA adopts crRNA as shown in seq_11 Ag When the metal nanoparticle is a platinum nanoparticle, the crRNA employs crRNA as shown in seq_12 Pt 。
In a specific embodiment, when three HPV types of different types are targeted, 20-50 nM Cas12a protein and three 50-150 nM crrnas are incubated in different PCR tubes at 37 ℃ for 15-30 min, respectively, to obtain the complex.
S3: HPV types with the same volume and different concentrations are respectively added into the compound, linker substrates are added into each mixture, and after reaction is carried out for 30-60 min at 37 ℃, the reaction is stopped by heating, thus obtaining reaction products.
In a specific embodiment, the Linker substrate is selected from the group consisting of Linker based on the metal nanoparticles Au 、Linker Ag 、Linker P 、Linker Pd The Linker Au The sequence of (2) is shown as seq_13, the Linker Ag The sequence of (2) is shown as seq_14, the Linker Pt The sequence of (2) is shown as seq_15.
Optionally, a buffer is added to the complex for adjusting the pH value of the solution in addition to the Linker substrate and HPV; when the reaction was terminated by heating, the reaction was maintained at 65℃for 10 minutes. In the case of terminating the reaction by heating, the enzyme protein is mainly inactivated by heating, and other temperatures and times may be used in addition to those used in this example, as long as the enzyme protein can be inactivated.
S4: and mixing and incubating the reaction product with DTN modified magnetic beads, optionally, mixing and incubating for 30-60 min, and then performing magnetic separation and washing.
S5: and (3) adding the modified metal nano particles into the product obtained in the step (S4) for mixed incubation to form a sandwich structure.
In a specific embodiment, when the modified metal nanoparticle is added, the modified metal nanoparticle is diluted 5-10 times based on the concentration of the nanoparticle modified by the original ssDNA.
S6: and performing magnetic separation and washing on the sandwich structure to remove supernatant, digesting with aqua regia, and diluting with pure water to obtain a mass spectrum acquisition solution.
In a specific example, 100 to 200uL of aqua regia is used for digestion and 4 to 8mL of pure water is used for dilution.
S7: and performing inductively coupled plasma mass spectrometry detection on the mass spectrometry acquisition solution to obtain isotope signals of the metal nano particles.
In a specific embodiment, the isotope of gold nanoparticle is selected from 197 Isotopes of Au and silver nanoparticles are selected from 107 Isotopes of Ag and platinum nanoparticles are selected from 195 Pt。
S8: and researching the relation between the intensity of the isotope signal and the HPV typing concentration to obtain the HPV typing detection result.
In a specific embodiment, as shown in FIG. 1, the inductively coupled plasma mass spectrometry nucleic acid detection method based on DTN and CRISPR Cas12a is used for detecting HPV-DNA, and in the implementation, three nucleic acids of HPV-16, HPV-18 and HPV-52 are used as targets.
The main reagents include: dynabeads commercial magnetic beads (Sieimer's fly); enGenLba Cas12a (Cpf 1) enzyme (New England); primer sequences (Biotechnology Co., ltd.); RNase inhibitors (bioengineering stock, inc.); tetrachloroauric acid (III) tetrahydrate (HAuCl) 4 ·4H 2 O), silver nitrate (AgNO) 3 99.0% or more), chloroplatinic acid hexahydrate (H) 2 PtCl 6 ·6H 2 O), trisodium citrate (. Gtoreq.99.0%) (Sigma-Aldrich).
The experimental instrument comprises: a constant temperature oscillator, an inductively coupled plasma mass spectrometer.
The detection method specifically comprises the following steps:
(1) Preparing DTN functionalized magnetic beads (MBs-DTN) and ssDNA functionalized magnetic beads (MBs-ssDNA);
100uL of 10mg/mL streptavidin functionalized commercial magnetic beads are repeatedly washed three times by using a TBS buffer, 30, 50, 100, 150, 200 and 300uL of 1uM DTN structure is added, and after incubation for 1h, the redundant DTN is removed by magnetic separation and redissolved in TBST.
The ssDNA was functionalized as above, except that 200, 300, 400, 500, 600, 700uL was used when the ssDNA was added.
As shown in FIG. 2, with the addition of different amounts of DTN and ssDNA, the resulting signal intensities were different, and 150uL and 500uL were finally selected as optimal parameters, respectively, for subsequent steps.
(2) Modifying ssDNA probes on surfaces of gold, silver and platinum nano particles;
the modification of ssDNA on the surface of the gold nano-particle is realized by a salt aging method, and specifically comprises the following substeps: after the ssDNA was treated in 4nM TCEP solution for 1h, 60uL of 8uM Au-ssDNA was added to 1ml of gold nanoparticle solution (containing 0.1% Tween 20 and 10mM Tris solution), stirred at room temperature for 12h, and 20uL of 3M NaCl solution was added 5 times at 1h intervals. Finally, the reaction is carried out for 24 hours at room temperature, a PEG2000 solution with 0uM is added for 2 hours, finally, the centrifugation is carried out for 15 minutes at 12000 revolutions, the redundant DNA probe in the supernatant is removed, the supernatant is washed for 3 times by using TBST buffer solution, and finally, the supernatant is dispersed in 1mL of TBS buffer solution, and the characterization result is shown in figures 3-5.
The modification of ssDNA on the surface of the silver nanoparticle is realized by an acid method, and specifically comprises the following substeps: ag-ssDNA was first treated with 4nM of TCEP solution for 1h, 600uL of silver nanoparticle solution was added, then 12uL of sodium citrate buffer (500 mM, pH 3) was added in two portions, and after 30min incubation, 90 uL of HEPES buffer (500 mM, pH 7.6) was added to restore the pH to neutral. Then, the supernatant was centrifuged at 12000rpm for 15min, and the excess DNA probe was removed, and then washed 3 times with TBST buffer, and then dispersed into 1ml TBS buffer, and then reacted with 4. Mu.M PEG for 2 hours to form a block. Finally, centrifugation at 12000rpm for 15min, removal of excess PEG from the supernatant, washing 3 times with TBST buffer, and further dispersion into 1ml TBS buffer for the next step, and characterization results are shown in FIGS. 6-8.
Modification of ssDNA on the surface of the platinum nano particle is realized by a salt aging method, and the method specifically comprises the following substeps: after 1h of treatment of ssDNA in 4nM TCEP solution, 60uL of 8uM Pt-ssDNA was added to 1ml of platinum nanoparticle solution (containing 0.1% Tween 20 and 10mM Tris), stirred at room temperature for 12h, and 20uL of 3M NaCl solution was added 5 times at 1h intervals. Finally, the reaction is carried out for 24 hours at room temperature, 4uM of PEG2000 solution is added for 2 hours, finally, the centrifugation is carried out for 15 minutes at 12000 revolutions, the redundant DNA probe and PEG in the supernatant are removed, the supernatant is washed for 3 times by using TBST buffer solution, and finally, the supernatant is dispersed in 1mL of TBS buffer solution, and the characterization result is shown in figures 9-11.
(3) Incubating and detecting;
50nM of Cas12a protein and three 150nM crRNAs were incubated in different PCR tubes at 37℃for 30min, respectively. 1uM Linker substrate, including Linker, was added to each of the three EP tubes Au 、Linker Ag 、Linker Pt Then, HPV mixtures (HPV sequence target concentrations of 0.1pM, 0.5pM, 1pM, 5pM, 10pM, 50pM, 100pM, 500pM, 1000pM, 5000pM, respectively) and buffer were simultaneously added to three EP tubes, the reaction was started at 37℃for 60min, and then the temperature was raised to 65℃for 10min to inactivate the enzyme proteins.
Three spatially independent CRISPR reaction products were mixed together and incubated with 40ug of DTN functionalized commercial magnetic beads for 30min at 37 ℃, and the supernatant removed by three magnetic separations for use. Three DNA probe functionalized gold, silver and platinum nano-particles are then added to the obtained product, and incubated at 37 ℃ for 30min. Removing supernatant from the product obtained after incubation by three times of magnetic separation, digesting with 200uL aqua regia, adding 8mL of pure water for dilution, and testing the product by ICP-MS to obtain 197 Au、 107 Ag、 195 The signal intensity of the Pt signal was correlated with the concentration of the three nucleic acid sequences by a calibration curve and a number. The results are shown in FIGS. 12-15.
As can be seen from fig. 12 to 15, the standard curves are respectively:
y=0.1342lgC+0.2319 (R 2 =0.9976) (1)
y=0.1557lgC+0.2113 (R 2 =0.9545) (2)
y=0.1692lgC+0.1898(R 2 =0.9832) (3)
for ssDNA modified magnetic beads:
y=0.1205lgC+0.1383(R 2 =0.9798) (4)
according to formulas (1) to (4), calculation was performed by dividing the slope by the 3-fold SD value, which is the standard deviation value of 10 blank samples, and the calculated minimum detection limits were 53fM, 79fM, 85fM and 837fM, respectively. Therefore, the invention can realize HPV-DNA detection sensitivity of 50-100 fM, and the detection scheme based on the DTN structure magnetic beads is lower than that of the detection line obtained by the conventional ssDNAc magnetic beads, and the signal effect is stronger.
It should be noted that in addition to the above examples for detecting three types of HPV, HPV-16, HPV-18 and HPV-52, the present invention has also succeeded in multiplex detection of other types of HPV, such as triple detection of HPV-58, HPV-56 and HPV-39, double detection of HPV-16 and HPV-18, quadruple detection of HPV-6, HPV-11, HPV-16 and HPV-18, etc.
In conclusion, the invention can realize amplification-free detection of multiplex HPV-DNA with high specificity and high sensitivity based on CRISPR-Cas12a technology. Compared with the prior art, the invention has obvious progress.
The present invention is not limited to the above-mentioned embodiments, but is intended to be limited to the following embodiments, and any modifications, equivalents and modifications can be made to the above-mentioned embodiments without departing from the scope of the invention.
Claims (10)
1. An inductively coupled plasma mass spectrometry nucleic acid detection method based on DTN and CRISPRCas12a, which is characterized by taking at least one HPV typed nucleic acid as a target, comprising the following steps:
s1: obtaining metal nano particles and magnetic beads, and modifying the metal nano particles by adopting mercapto functional ssDNA to obtain modified metal nano particles; adopting DTN to modify the magnetic beads to obtain DTN modified magnetic beads;
s2: mixing CRISPR-Cas12a protein corresponding to the target with crRNA to obtain a compound with a composite structure;
s3: adding HPV types with the same volume and different concentrations into the compound respectively, adding Linker substrates into each mixture, reacting for 30-60 min at 37 ℃, and heating to terminate the reaction to obtain a reaction product;
s4: mixing and incubating the reaction product with DTN modified magnetic beads, and then performing magnetic separation and washing;
s5: adding the modified metal nano particles into the product obtained in the step S4 for mixed incubation to form a sandwich structure;
s6: performing magnetic separation and washing on the sandwich structure to remove supernatant, digesting with aqua regia, and diluting with pure water to obtain a mass spectrum acquisition solution;
s7: performing inductively coupled plasma mass spectrometry detection on the mass spectrometry acquisition solution to obtain isotope signals of metal nano particles in the mass spectrometry acquisition solution;
s8: and researching the relation between the intensity of the isotope signal and the HPV typing concentration to obtain the HPV typing detection result.
2. The method for detecting inductively coupled plasma mass spectrometry nucleic acid based on DTN and crisp 12a according to claim 1, wherein in step S1, gold nanoparticles are used as the metal nanoparticles when nucleic acid of one HPV type is targeted;
when two HPV typed nucleic acids are used as targets, in the step S1, gold nanoparticles and silver nanoparticles are adopted as the metal nanoparticles;
when three HPV-typed nucleic acids are used as targets, in the step S1, gold nanoparticles, silver nanoparticles and platinum nanoparticles are adopted as the metal nanoparticles;
when four HPV-typed nucleic acids are used as targets, gold nanoparticles, silver nanoparticles, platinum nanoparticles, and palladium nanoparticles are used as the metal nanoparticles in step S1.
3. The method for detecting inductively coupled plasma mass spectrometry nucleic acid based on DTN and crisp rcas12a according to claim 2, wherein in step S1, when thiol-functionalized ssDNA is used to modify the metal nanoparticles, thiol-functionalization is performed by using a DNA sequence shown as seq_1, and then gold nanoparticles are modified; performing sulfhydryl functionalization by adopting a DNA sequence shown as seq_2, and then modifying silver nano particles; thiol functionalization was performed using the DNA sequence shown as seq_3, followed by modification of the platinum nanoparticles.
4. The method for detecting nucleic acid by inductively coupled plasma mass spectrometry based on DTN and crisp rcas12a according to claim 2, wherein in step S1, when the magnetic beads are modified by DTN, the method specifically comprises the following sub-steps:
s11: synthesizing a DNA tetrahedral nano structure;
s12: performing streptavidin functionalization on the magnetic beads to obtain streptavidin functionalized magnetic beads;
s13: mixing the DNA tetrahedron nano structure with the streptavidin functionalized magnetic beads in an equal volume, and vibrating and incubating for 1-2 h under the room temperature condition;
s14: and performing magnetic separation for multiple times, removing unreacted DNA tetrahedral nano structures, and then adding PBST buffer solution for dispersion to obtain the DTN modified magnetic beads.
5. The method for inductively coupled plasma mass spectrometry nucleic acid detection based on DTN and crisp rcas12a of claim 4, wherein in step S11, synthesizing DNA tetrahedral nanostructures specifically comprises the sub-steps of:
uniformly mixing four equal amounts of single-stranded DNA in a TM buffer solution, annealing for 5-15 min at 90-95 ℃, and incubating for 2-4 h at 4 ℃ to obtain the DNA tetrahedron nano structure.
6. The method for inductively coupled plasma mass spectrometry nucleic acid detection based on DTN and CRISPRCas12a according to claim 5, wherein the four single stranded DNAs are biotin-modified ssDNA-A, biotin-modified ssDNA-B, biotin-modified ssDNA-C, ssDNA-D, respectively, the ssDNA-D being determined based on the metal nanoparticle and selected from ssDNA-D Au 、ssDNA-D Ag 、ssDNA-D Pt ssDNA-D Pd The method comprises the steps of carrying out a first treatment on the surface of the The nucleic acid sequence of the ssDNA-A is shown as seq_4, the nucleic acid sequence of the ssDNA-B is shown as seq_5, the nucleic acid sequence of the ssDNA-C is shown as seq_6, and the ssDNA-D Au The nucleic acid sequence of said ssDNA-D is shown as seq_7 Ag The nucleic acid sequence of said ssDNA-D is shown in seq_8 Pt The nucleic acid sequence of (2) is shown as seq_9.
7. The method for detecting nucleic acid by inductively coupled plasma mass spectrometry based on DTN and CRISPRCas12a according to claim 4, wherein in the step S13, the amount of the DNA tetrahedral nano structure is 1-2 uM, and the amount of the streptavidin functionalized magnetic beads is 1-10 mg/mL.
8. The method for inductively coupled plasma mass spectrometry nucleic acid detection based on DTN and crisp rcas12a according to claim 2, wherein in step S2, the crRNA is determined according to the metal nanoparticles used, and when the metal nanoparticles are gold nanoparticles, the crRNA is determined according to the crRNA as shown in seq_10 Au The method comprises the steps of carrying out a first treatment on the surface of the When the metal nanoparticle is silver nanoparticle, the crRNA adopts crRNA as shown in seq_11 Ag When the metal nanoparticle is a platinum nanoparticle, the crRNA is as ScrRNA shown in eq_12 Pt 。
9. The method for inductively coupled plasma mass spectrometry nucleic acid detection based on DTN and crisp rcas12a of claim 8, wherein in step S3, the Linker substrate is selected from the group consisting of Linker according to the metal nanoparticle Au 、Linker Ag 、Linker P 、Linker Pd The Linker Au The sequence of (2) is shown as seq_13, the Linker Ag The sequence of (2) is shown as seq_14, the Linker Pt The sequence of (2) is shown as seq_15.
10. The method for detecting nucleic acid by inductively coupled plasma mass spectrometry based on DTN and crisp as12a according to claim 1, wherein in step S4, when the reaction is terminated by heating, the reaction is heated to 65 ℃ for 10min.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310194513.XA CN116411135B (en) | 2023-03-03 | 2023-03-03 | Inductively coupled plasma mass spectrum nucleic acid detection method based on DTN and CRISPR CAS a |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310194513.XA CN116411135B (en) | 2023-03-03 | 2023-03-03 | Inductively coupled plasma mass spectrum nucleic acid detection method based on DTN and CRISPR CAS a |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116411135A true CN116411135A (en) | 2023-07-11 |
CN116411135B CN116411135B (en) | 2024-07-16 |
Family
ID=87050582
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310194513.XA Active CN116411135B (en) | 2023-03-03 | 2023-03-03 | Inductively coupled plasma mass spectrum nucleic acid detection method based on DTN and CRISPR CAS a |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116411135B (en) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112595764A (en) * | 2020-12-31 | 2021-04-02 | 山东理工大学 | Aptamer sensor based on pyramid-shaped nanostructure and detection method thereof |
CN112816450A (en) * | 2021-01-07 | 2021-05-18 | 青岛农业大学 | Detection of aflatoxins B1Kit and detection of aflatoxin B1Method (2) |
CN113774114A (en) * | 2021-09-13 | 2021-12-10 | 上海交通大学 | Nucleic acid analysis method and application |
CN113960136A (en) * | 2021-09-09 | 2022-01-21 | 江苏大学 | Preparation method and application of fumonisin B1 electrochemical sensor with adjustable dynamic range |
CN113984726A (en) * | 2021-10-20 | 2022-01-28 | 上海大学 | Method for detecting mercury ions by amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA |
CN115144578A (en) * | 2022-06-09 | 2022-10-04 | 华南理工大学 | Fluorescent biosensor based on CRISPR/Cas12a integrated MXenes and preparation method and application thereof |
-
2023
- 2023-03-03 CN CN202310194513.XA patent/CN116411135B/en active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112595764A (en) * | 2020-12-31 | 2021-04-02 | 山东理工大学 | Aptamer sensor based on pyramid-shaped nanostructure and detection method thereof |
CN112816450A (en) * | 2021-01-07 | 2021-05-18 | 青岛农业大学 | Detection of aflatoxins B1Kit and detection of aflatoxin B1Method (2) |
CN113960136A (en) * | 2021-09-09 | 2022-01-21 | 江苏大学 | Preparation method and application of fumonisin B1 electrochemical sensor with adjustable dynamic range |
CN113774114A (en) * | 2021-09-13 | 2021-12-10 | 上海交通大学 | Nucleic acid analysis method and application |
CN113984726A (en) * | 2021-10-20 | 2022-01-28 | 上海大学 | Method for detecting mercury ions by amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA |
CN115144578A (en) * | 2022-06-09 | 2022-10-04 | 华南理工大学 | Fluorescent biosensor based on CRISPR/Cas12a integrated MXenes and preparation method and application thereof |
Non-Patent Citations (5)
Title |
---|
MENGDI BAO ET AL.: "Magnetic Bead-Quantum Dot (MB-Qdot) Clustered Regularly Interspaced Short Palindromic Repeat Assay for Simple Viral DNA Detection", 《APPL. MATER. INTERFACES》, vol. 12, pages 43435 - 43443, XP055945679, DOI: 10.1021/acsami.0c12482 * |
SHAOCHENG LIU ET AL.: "DNA Tetrahedron-Based MNAzyme for Sensitive Detection of microRNA with Elemental Tagging", 《APPL. MATER. INTERFACES》, vol. 13, pages 59076 - 59084 * |
SHIXI ZHANG ET AL.: "Multiplex DNA Assay Based on Nanoparticle Probes by Single Particle Inductively Coupled Plasma Mass Spectrometry", 《ANALYTICAL CHEMISTRY》, no. 86, pages 3541 - 3547 * |
XIAOHUI ZHAN ET AL.: "DNA tetrahedron-based CRISPR bioassay for treble-self-amplified and multiplex HPV-DNA detection with elemental tagging", 《BIOSENSORS AND BIOELECTRONICS》, 15 March 2023 (2023-03-15), pages 1 - 9 * |
顾大勇等: "以纳米金为报告系统的病原体快速检测基因芯片的研制", 《中华医院感染学》, vol. 17, no. 2, pages 143 - 147 * |
Also Published As
Publication number | Publication date |
---|---|
CN116411135B (en) | 2024-07-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Larguinho et al. | Gold and silver nanoparticles for clinical diagnostics—from genomics to proteomics | |
Mao et al. | Colorimetric detection of hepatitis B virus (HBV) DNA based on DNA-templated copper nanoclusters | |
CN108414758B (en) | Preparation method and application of SERS biosensor for detecting tumor marker miRNA-141 | |
JP2009523406A (en) | SERS-based method for detection of bioagents | |
CN108359715B (en) | Poly A mediated adjustable nano gold probe and preparation and application thereof | |
Yu et al. | Highly sensitive DNA detection using cascade amplification strategy based on hybridization chain reaction and enzyme-induced metallization | |
Wang et al. | Recent advances in the rapid detection of microRNA with lateral flow assays | |
US20090311798A1 (en) | Se(r)rs displacement assay | |
Chen et al. | Recent advancements in nanobioassays and nanobiosensors for foodborne pathogenic bacteria detection | |
Lu et al. | Plasmon-enhanced biosensors for microRNA analysis and cancer diagnosis | |
Xiang et al. | Magnetic microparticle-based multiplexed DNA detection with biobarcoded quantum dot probes | |
Tao et al. | Metal nanoclusters combined with CRISPR-Cas12a for hepatitis B virus DNA detection | |
JP2007525651A (en) | Methods for detecting analytes based on evanescent illumination and scattering-based detection of nanoparticle probe complexes | |
Wu et al. | A novel recyclable surface-enhanced Raman spectroscopy platform with duplex-specific nuclease signal amplification for ultrasensitive analysis of microRNA 155 | |
US20090162888A1 (en) | Sample control for correction of sample matrix effects in analytical detection methods | |
AU2021100190A4 (en) | CRISPR-Cas12a-based MOF-DNA Hydrogel Colorimetric Assay Kit and Method | |
Hsu et al. | Gold nanoparticle-based inductively coupled plasma mass spectrometry amplification and magnetic separation for the sensitive detection of a virus-specific RNA sequence | |
Jimenez et al. | Gold nanoparticles-modified nanomaghemite and quantum dots-based hybridization assay for detection of HPV | |
Zhu et al. | Highly sensitive and specific mass spectrometric platform for miRNA detection based on the multiple-metal-nanoparticle tagging strategy | |
CN113736853A (en) | Surface-enhanced Raman spectroscopy detection method for gene based on CRISPR/Cas12a protein | |
Yu et al. | SERS-based genetic assay for amplification-free detection of prostate cancer specific PCA3 mimic DNA | |
Xu et al. | A homogeneous nucleic acid assay for simultaneous detection of SARS-CoV-2 and influenza A (H3N2) by single-particle inductively coupled plasma mass spectrometry | |
Chen et al. | Sensitive CVG-AFS/ICP-MS label-free nucleic acid and protein assays based on a selective cation exchange reaction and simple filtration separation | |
Xiong et al. | Ultrasensitive visualization of virus via explosive catalysis of an enzyme muster triggering gold nano-aggregate disassembly | |
Zhan et al. | DNA tetrahedron-based CRISPR bioassay for treble-self-amplified and multiplex HPV-DNA detection with elemental tagging |
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 |