CN113801921A - Method for detecting miRNA (micro ribonucleic acid) by utilizing nuclear satellite structure multiplex - Google Patents
Method for detecting miRNA (micro ribonucleic acid) by utilizing nuclear satellite structure multiplex Download PDFInfo
- Publication number
- CN113801921A CN113801921A CN202111001495.6A CN202111001495A CN113801921A CN 113801921 A CN113801921 A CN 113801921A CN 202111001495 A CN202111001495 A CN 202111001495A CN 113801921 A CN113801921 A CN 113801921A
- Authority
- CN
- China
- Prior art keywords
- mirna
- reaction
- satellite structure
- washing
- maldi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 17
- 229920002477 rna polymer Polymers 0.000 title description 3
- 238000001514 detection method Methods 0.000 claims abstract description 35
- 239000010931 gold Substances 0.000 claims abstract description 28
- 229910052737 gold Inorganic materials 0.000 claims abstract description 28
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 claims abstract description 25
- 238000005406 washing Methods 0.000 claims abstract description 25
- 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 abstract description 22
- 108091070501 miRNA Proteins 0.000 claims abstract description 18
- -1 alkyl mercaptan Chemical group 0.000 claims abstract description 17
- 239000011324 bead Substances 0.000 claims abstract description 15
- 239000002679 microRNA Substances 0.000 claims abstract description 15
- 150000001356 alkyl thiols Chemical class 0.000 claims abstract description 13
- 238000006073 displacement reaction Methods 0.000 claims abstract description 12
- 229960002685 biotin Drugs 0.000 claims abstract description 11
- 235000020958 biotin Nutrition 0.000 claims abstract description 11
- 239000011616 biotin Substances 0.000 claims abstract description 11
- 239000006228 supernatant Substances 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 238000012360 testing method Methods 0.000 claims description 13
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000012491 analyte Substances 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 230000003595 spectral effect Effects 0.000 claims description 4
- QYKSUHRPPSCIFK-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-(11-sulfanylundecoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound OCCOCCOCCOCCOCCOCCOCCCCCCCCCCCS QYKSUHRPPSCIFK-UHFFFAOYSA-N 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 238000007605 air drying Methods 0.000 claims description 3
- 239000011159 matrix material Substances 0.000 claims description 3
- 238000000074 matrix-assisted laser desorption--ionisation tandem time-of-flight detection Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 230000036632 reaction speed Effects 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 229910001220 stainless steel Inorganic materials 0.000 claims description 3
- 239000010935 stainless steel Substances 0.000 claims description 3
- UWHCKJMYHZGTIT-UHFFFAOYSA-N tetraethylene glycol Chemical compound OCCOCCOCCOCCO UWHCKJMYHZGTIT-UHFFFAOYSA-N 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 238000004949 mass spectrometry Methods 0.000 abstract description 9
- 238000005119 centrifugation Methods 0.000 abstract description 4
- 238000009987 spinning Methods 0.000 abstract 1
- 108700011259 MicroRNAs Proteins 0.000 description 32
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- 108091033773 MiR-155 Proteins 0.000 description 13
- 239000000126 substance Substances 0.000 description 8
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 238000001819 mass spectrum Methods 0.000 description 5
- 108091027943 miR-16 stem-loop Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 239000002105 nanoparticle Substances 0.000 description 4
- 150000002343 gold Chemical class 0.000 description 3
- 238000001869 matrix assisted laser desorption--ionisation mass spectrum Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 102000011727 Caspases Human genes 0.000 description 2
- 108010076667 Caspases Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 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 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 108091023818 miR-7 stem-loop Proteins 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002077 nanosphere Substances 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000000758 substrate Substances 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
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a method for detecting miRNA by utilizing a nuclear satellite structure multiplex, which comprises the following steps: (1) the beads were removed and washed with 12.5mM Mg2+After adding Biotin DNA and spinning on a spinner, the column was removed with 12.5mM Mg2+Washing and fixing the volume; (2) gold spheres of modified DNA were mixed with three alkyl thiols at a concentration ratio of 1:3000 and reacted on a shaker at 20 c, and then excess unattached alkyl thiol molecules were removed by centrifugation in a centrifuge for 1 hour and the supernatant was aspirated; (3) adding a gold ball modified with alkyl mercaptan molecule mass tag into the magnetic beads, reacting for a period of time, and performing MALDI detection; (4) and (4) carrying out a strand displacement reaction. According to the invention, through MALDI mass spectrometry, the type of miRNA contained in the sample can be detected, and the type and content of 3 types of miRNA can be simultaneously and independently detected.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting miRNA (micro ribonucleic acid) by utilizing a nuclear satellite structure.
Background
The mass spectrum as a label-free analysis technology can directly give the intrinsic information of the molecular weight of the substance to be detected. Meanwhile, the accurate molecular weight information given by the mass spectrum not only improves the qualitative accuracy, but also effectively avoids the spectral peak overlapping interference which may occur in the simultaneous detection of multiple targets. The powerful molecular information acquisition and multi-component analysis capability of the mass spectrometry technology provides possibility for accurate determination of multiple targets in a complex system, and the possibility is also matched with the requirement of intracellular multi-enzyme activity evaluation. In recent years, many researchers have proposed schemes for detecting various small molecule substances using mass spectrometry techniques. In 2008, a mass bar code strategy is proposed first by a teaching group of Vincent Rotello, and by utilizing the efficient desorption ionization capacity of alkyl mercaptan molecules assembled on the surfaces of gold nanoparticles under laser irradiation, the accurate molecular weights of different alkyl mercaptans are used as bar codes to specifically indicate the correspondingly modified gold nanoparticles, and the mass spectrometry technology is utilized for the first time to carry out quantitative analysis on the cell uptake of different nanoparticles. (Zheng-Jiang Zhu, Partha S.Ghosh, Oscar R.Miranda, Richard W.Vachet, Vincent M.Rotello.J.am.chem.Soc.2008, 130, 14139-
Based on this, the professor of zhjunjie university of Nanjing in 2020 reported a protocol to reveal the activity of cascaded caspases in apoptosis in a multiplex and quantitative manner, a series of mass-labeled modified gold nanoparticles (AuNPs) were tethered to magnetic Fe3O4 nanospheres via linkers containing the protease substrate peptides of interest, forming a "one-to-many" core-satellite structure. The nanostructures are internalized into the cell, undergo an enzymatic reaction within the cell, and undergo a post-reaction Mass Spectrometry (MS) interrogation after magnetic separation from the cell. (Hongmei Xu, Xiaodan Huang, Zhenzhen Zhuang, Xuemeng Zhuang, Qianhao Min and Jun-Jie Zhu, chem. DOI:10.1039/d0sc01534b3)
Most of the detection based on MALD mass spectrometry is carried out on certain proteins in human bodies, for example, the activity of caspase in apoptosis is tested in background information, nucleic acid structures for regulating and controlling gene expression in human bodies are rarely reported, and actually, MicroRNA can also finely regulate and control gene expression, so that a simple and rapid scheme is necessary for testing MicroRNA.
MicroRNA (miRNA) is an endogenous, small RNA of about 20-24 nucleotides in length that has a number of important regulatory roles within the cell. Each miRNA may have multiple target genes, and several mirnas may also regulate the same gene. The complex regulatory network can regulate the expression of multiple genes through one miRNA or can finely regulate the expression of a certain gene through the combination of several miRNAs. It is speculated that mirnas regulate one third of the genes in humans.
At present, a method for detecting miRNA by utilizing nuclear satellite structure multiplex detection is lacked.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a method for detecting miRNA by utilizing a nuclear satellite structure multiplex.
In order to achieve the purpose, the invention adopts the following technical scheme: the invention discloses a method for detecting miRNA by utilizing a nuclear satellite structure multiplex, which comprises the following steps:
(1) taking out 20-40 mu L of magnetic beads, sucking supernatant, and using 12.5mM Mg2+Washing, adding 2-4 mu L Biotin DNA, rotating on a rotating instrument for 2-4 h, repeating the washing operation, and fixing the volume to 20-40 mu L;
(2) gold spheres of modified DNA were mixed with three alkyl thiols at a concentration ratio of 1: mixing at a ratio of 2000-1: 3000, arranging miRNA chains on a shaking bed, reacting at 20 ℃, at a reaction speed of 1250-1500 rpm for 6-12 h, centrifuging at 18000-20000 rcf to remove excessive unconnected alkyl mercaptan molecules, and washing with 0.1M PBS buffer for 2-3 times;
(3) adding excessive gold balls of modified alkyl mercaptan molecule mass tag into the magnetic beads of modified DNA, reacting on a shaker at 37 ℃, the reaction speed is 1050-1250 rpm, the reaction time is 6-12 h, washing supernate after the reaction is finished, and carrying out MALDI detection;
(4) strand displacement reaction: according to the Biotin DNA, the replacement strand is 1: 0.05-1: 1, adding a displacement chain, namely a miRNA chain to be detected, arranging the miRNA chain on a shaking table, reacting at the temperature of 25 ℃, at the rotating speed of 400-600 rpm for 3-6 h, washing the supernatant after the reaction is finished, and performing MALDI detection;
(5) MALDI detection: mu.L of analyte was mixed with 0.5. mu.L of DHB matrix solution, then 1. mu.L of this mixture was deposited on a 384-well stainless steel target plate, which was placed into a 4800Plus MALDI-TOF/TOF mass spectrometer for testing and corresponding mass spectral data were obtained after air drying.
Further, the 0.1M PBS phosphate buffered saline was 0.1M NaCl, 10mM phosphate.
Further, the three alkyl thiols are respectively Mass tag 1[ HS- (CH)2)11(OCH2CH2)3OH]((11-mercaptoundecyl) tris (ethylene glycol)), mass tag 2[ HS- (CH)2)11(OCH2CH2)4OH]((11-mercaptoundecyl) tetra (ethylene glycol)) mass tag 3[ HS- (CH)2)11(OCH2CH2)6OH]((11-mercaptoundecyl) hexa (ethylene glycol)); the relative molecular masses were 693, 781, 957, respectively.
Further, in the step (2), the reaction time was 12 hours.
Further, in the step (2), the reaction time was 12 hours.
Further, in the step (4), the reaction rate was 500 rpm.
Has the advantages that: according to the invention, through MALDI mass spectrometry, the type of miRNA contained in the sample can be detected, and the type and content of 3 types of miRNA can be simultaneously and independently detected. The experimental result is explored to obtain that the detection limit concentration of miRNA can reach pM level, and the sensitivity is extremely high.
(1) The invention modifies the assembly between the gold balls (3 types in the invention) of different alkyl mercaptan molecules (mass tag) and the magnetic beads, and the magnetic bead-gold ball assembly is the 'nuclear-satellite structure' in the invention; replacement of miRNA: each miRNA can replace a corresponding satellite gold ball in a nuclear-satellite structure, and the existence or the strength of the corresponding signal can be used for judging the existence or the quantity of the miRNA in the subsequent MALDI mass spectrometry detection; MALDI detection: the MALDI assay requires the introduction of a control group, i.e., a control group that does not have miRNA added to perform the strand displacement reaction.
(2) The invention aims to develop a miRNA responsive nuclear-satellite structure mass bar code (mass tag) nano probe to detect the type and content of miRNA in human cells, which can be reflected by the existence and the strength of mass spectrum signals.
Drawings
FIG. 1 is a MALDI mass spectrometry test chart of the present invention; when the detection object is a gold ball modified with mass tag 3, a MALDI mass spectrogram has a signal peak with an abscissa of 957 (957 is the relative molecular mass of mass tag 3, and similarly, 693 and 781 are the relative molecular masses of mass tag 1 and mass tag 2, respectively), the abscissa indicates the type of mass tag, and the ordinate indicates the intensity of the signal, and the stronger the signal, the higher the content.
Fig. 2 is a detection diagram of mirnas of the present invention. The detection reagent comprises a detection reagent, a miR-155 reagent, a miR-16 reagent and a miR-7 reagent, wherein (A) the detection reagent is not added, (B) the detection reagent only contains the miR-155 reagent, (C) the detection reagent contains the miR-155 reagent and the miR-16 reagent, and (D) the detection reagent contains the miR-155 reagent, the miR-16 reagent and the miR-let7 reagent.
Note that: after different miRNAs are added, corresponding gold spheres are replaced, and the types of the mass tags connected to the gold spheres are fixed, so that the replacement reaction of different gold spheres is the deletion of the mass tag signals in mass spectrum signals.
FIG. 3 is a graph showing the effect of miR-155 content in the test substance of the present invention on mass spectrum signals; the ratio q of the signal of 957 to the signal of the added internal standard 869 is used for calibration; (A) a control group to which no test substance was added, q was 100% (B), the miR-155 concentration in the test substance was 0.5 μ M, q was 0%, (C) the miR-155 concentration in the test substance was 1nM, q was 82%, (D) the miR-155 concentration in the test substance was 500pM, q was 100%.
FIG. 4 is a schematic diagram of the assembly between the magnetic ball and the gold ball decorated with different mass tags according to the present invention; wherein the miR-let7 can replace and modify a gold ball with a 693 signal; miR-16 can replace and modify a gold ball with a 781 signal; miR-155 can replace a gold ball modified with a 957 signal.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1
The structure is as follows: 1 μm diameter magnetic bead, 10nm diameter gold ball, miRNA-155, miRNA-let7, miRNA-16, alkyl thiol molecule (mass tag);
the invention discloses a method for detecting miRNA by utilizing a nuclear satellite structure multiplex, which comprises the following steps:
(1) remove 20. mu.L of magnetic beads, aspirate the supernatant and use 12.5mM Mg2+Washing, adding 3 mu L Biotin DNA, rotating on a rotator for 2h, repeating the washing operation and metering to 20 mu L;
(2) gold spheres of modified DNA were mixed with three alkyl thiols at a concentration ratio of 1: 2500 ratio mixing and reaction on a shaker at 20 ℃, 1400rpm for 9h, then by 19000rcf speed centrifugation to remove excess unlinked alkyl thiol molecules and 0.1M PBS buffer washing 2.5 times;
(3) taking magnetic beads of modified DNA, adding excessive gold balls of modified alkyl mercaptan molecule mass tag, reacting on a shaker at 37 ℃ and 1000rpm for 12h, washing supernate after the reaction is finished, and carrying out MALDI detection;
(4) strand displacement reaction: according to the Biotin DNA, the replacement strand is 1: adding a displacement chain, namely a miRNA chain to be detected, in a proportion of 0.05, reacting for 5 hours on a shaking table at 25 ℃ and 500rpm, washing supernate after the reaction is finished, and carrying out MALDI detection;
(5) MALDI detection: mu.L of analyte was mixed with 0.5. mu.L of DHB matrix solution, then 1. mu.L of this mixture was deposited on a 384-well stainless steel target plate, which was placed into a 4800Plus MALDI-TOF/TOF mass spectrometer for testing and corresponding mass spectral data were obtained after air drying.
Example 2
Example 2 differs from example 1 in that: the invention discloses a method for detecting miRNA by utilizing a nuclear satellite structure multiplex, which comprises the following steps:
in step (1), 40. mu.L of magnetic beads were removed, and the supernatant was aspirated and washed with 12.5mM Mg2+Washing for several times, adding 4 μ L Biotin DNA, rotating on a rotator for 3h, repeating the washing operation just before, and metering to 40 μ L;
in step (2), the DNA-modified gold beads were mixed with three alkylthiols at a concentration ratio of 1:3000 and reacted on a shaker at 20 ℃ for 6 hours at 1500rpm, followed by removal of excess unattached alkylthiol molecules by centrifugation at 18000rcf and washing 3 times with 0.1M PBS buffer;
in the step (3), taking the magnetic beads of the modified DNA, adding excessive gold balls of the modified alkyl mercaptan molecule mass tag, reacting on a shaking table at 37 ℃ and 1250rpm for 6h, washing supernate after the reaction is finished, and carrying out MALDI detection;
in step (4), strand displacement reaction: according to the Biotin DNA, the replacement strand is 1: adding a replacement strand, namely a miRNA strand to be detected, in the proportion of 1, reacting for 6h on a shaker at 25 ℃ and 600rpm, washing supernate after the reaction is finished, and carrying out MALDI detection.
Example 3
Example 3 differs from example 1 in that: the invention discloses a method for detecting miRNA by utilizing a nuclear satellite structure multiplex, which comprises the following steps:
in step (1), 30. mu.L of magnetic beads were removed, and the supernatant was aspirated and washed with 12.5mM Mg2+Washing for several times, adding 2 μ L Biotin DNA, rotating for 4h on a rotator, repeating the just washing operation, and metering to 30 μ L;
in step (2), the gold spheres of the modified DNA were mixed with three alkyl thiols at a concentration ratio of 1: 2000 and reacted for 12h at 20 ℃ 1250rpm on a shaker, then excess unattached alkanethiol molecules were removed by centrifugation at 20000rcf and washed 2 times with 0.1M PBS buffer;
in the step (3), taking the magnetic beads of the modified DNA, adding excessive gold balls of the modified alkyl mercaptan molecule mass tag, reacting on a shaking table at 37 ℃ and 1050rpm for 9h, washing supernate after the reaction is finished, and carrying out MALDI detection;
in step (4), strand displacement reaction: according to the Biotin DNA, the replacement strand is 1: adding a displacement strand, namely the miRNA strand to be detected, in a proportion of 0.08, reacting for 3h on a shaking table at 25 ℃ and 400rpm, washing the supernatant after the reaction is finished, and performing MALDI detection.
Test examples
As shown in FIG. 1, the existence and amount of the analyte can be reflected from the MALDI mass spectrum, and FIG. 1 shows that gold spheres modified with mass tag 3 are directly tested, a signal peak with an abscissa of 957 appears in the MALDI mass spectrum, the abscissa 957 represents the mass tag 3, and the ordinate represents the signal intensity, the stronger the signal is, the higher the analyte content is.
MALDI mass spectrum signal interpretation: in this example, a total of 3 characteristic peaks appear in the MALDI mass spectrogram, the abscissa thereof is 693(mass tag 1), 781(mass tag 2), 957(mass tag 3) in sequence, which represents the type of mass tag modified on the surface of the gold sphere, the ordinate is the peak value, the higher the peak value is, the stronger the signal is, which indicates the higher the mass tag content in the detection object, fig. 2A shows the MALDI mass spectrogram of the nuclear-satellite structure, when the detection object is not added, three different signals of 693, 781, and 957 appear, which indicates that the gold sphere modified with 3 different mass tags is successfully connected with the magnetic sphere, the 693, 781 signal appears in fig. 2B, which indicates that the detection object contains miR-155, the successful chain displacement reaction replaces the gold sphere modified with 957mass tag from the nuclear-satellite structure, which results in the absence of 957 signal, and the same principle, the absence of the 693 and the detection object in fig. 2C contains miR-155 and miR-7-89let, the absence of all three signals in FIG. 2D indicates that the test substance contains three miRNAs of miR-155, miR-16 and miR-let 7;
FIG. 3 shows the detection limit of miRNA, which is calibrated by the ratio q of the signal of 957 to the signal of the internal standard 869, by adding the same amount of mass tag 4(869) to all samples before MALDI test. Fig. 3A shows that after an internal standard 869 is added to a nuclear-satellite structure, four signals of 693, 781, 957 and 869 appear in a MALDI mass spectrogram, at this time, q is defined as 100%, fig. 3B,3C and 3D show that the concentration of miR-155 is 0.5 μ M, 1nM, and q is 0%, 82% and 100% at 500pM, respectively, and the detection limit is 500pM, that is, when an analyte contains miRNA of 500pM or more, the type and relative content of miRNA can be obtained from the mass spectrogram.
The 0.1M PBS phosphate buffer solution is 0.1M NaCl, 10mM phosphate.
The three alkyl thiols are respectively Mass tag 1[ HS- (CH)2)11(OCH2CH2)3OH]((11-mercaptoundecyl) tris (ethylene glycol)), mass tag 2[ HS- (CH)2)11(OCH2CH2)4OH]((11-mercaptoundecyl) tetra (ethylene glycol)) mass tag 3[ HS- (CH)2)11(OCH2CH2)6OH]((11-mercaptoundecyl) hexa (ethylene glycol)); the relative molecular masses were 693, 781, 957, respectively.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (6)
1. A method for detecting miRNA by utilizing nuclear satellite structure multiplex detection is characterized by comprising the following steps:
(1) taking out 20-40 mu L of magnetic beads, sucking supernatant, and using 12.5mM Mg2+Washing, adding 2-4 mu L Biotin DNA, rotating on a rotating instrument for 2-4 h, repeating the washing operation, and fixing the volume to 20-40 mu L;
(2) gold spheres of modified DNA were mixed with three alkyl thiols at a concentration ratio of 1: mixing at a ratio of 2000-1: 3000, arranging miRNA chains on a shaking bed, reacting at 20 ℃, at a reaction speed of 1250-1500 rpm for 6-12 h, centrifuging at 18000-20000 rcf to remove excessive unconnected alkyl mercaptan molecules, and washing with 0.1M PBS buffer for 2-3 times;
(3) adding excessive gold balls of modified alkyl mercaptan molecule mass tag into the magnetic beads of modified DNA, reacting on a shaking table at 37 ℃, wherein the rotating speed of the reaction is 1050-1250 rpm, the reaction time is 6-12 h, and washing supernate and carrying out MALDI detection after the reaction is finished;
(4) strand displacement reaction: according to the Biotin DNA, the replacement strand is 1: 0.05-1: 1, adding a displacement chain, namely a miRNA chain to be detected, arranging the miRNA chain on a shaking table, reacting at the temperature of 25 ℃, at the rotating speed of 400-600 rpm for 3-6 h, washing the supernatant after the reaction is finished, and performing MALDI detection;
(5) MALDI detection: mu.L of analyte was mixed with 0.5. mu.L of DHB matrix solution, then 1. mu.L of this mixture was deposited on a 384-well stainless steel target plate, which was placed into a 4800Plus MALDI-TOF/TOF mass spectrometer for testing and corresponding mass spectral data were obtained after air drying.
2. The method for detecting miRNA by using nuclear satellite structure multiplex as claimed in claim 1, wherein: the 0.1M PBS phosphate buffer solution is 0.1M NaCl, 10mM phosphate.
3. The method for detecting miRNA by using nuclear satellite structure multiplex as claimed in claim 1, wherein: the three alkyl thiols are respectively Mass tag 1[ HS- (CH)2)11(OCH2CH2)3OH]((11-mercaptoundecyl) tris (ethylene glycol)), mass tag 2[ HS- (CH)2)11(OCH2CH2)4OH]((11-mercaptoundecyl) tetra (ethylene glycol)) mass tag 3[ HS- (CH)2)11(OCH2CH2)6OH]((11-mercaptoundecyl) hexa (ethylene glycol)); the relative molecular masses were 693, 781, 957, respectively.
4. The method for detecting miRNA by using nuclear satellite structure multiplex as claimed in claim 1, wherein: in the step (2), the reaction time is 12 h.
5. The method for detecting miRNA by using nuclear satellite structure multiplex as claimed in claim 1, wherein: in the step (2), the reaction time is 12 h.
6. The method for detecting miRNA by using nuclear satellite structure multiplex as claimed in claim 1, wherein: in step (4), the rotational speed of the reaction was 500 rpm.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111001495.6A CN113801921A (en) | 2021-08-30 | 2021-08-30 | Method for detecting miRNA (micro ribonucleic acid) by utilizing nuclear satellite structure multiplex |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111001495.6A CN113801921A (en) | 2021-08-30 | 2021-08-30 | Method for detecting miRNA (micro ribonucleic acid) by utilizing nuclear satellite structure multiplex |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113801921A true CN113801921A (en) | 2021-12-17 |
Family
ID=78894326
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111001495.6A Pending CN113801921A (en) | 2021-08-30 | 2021-08-30 | Method for detecting miRNA (micro ribonucleic acid) by utilizing nuclear satellite structure multiplex |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113801921A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114214461A (en) * | 2021-12-26 | 2022-03-22 | 南京大学 | Isothermal HIV nucleic acid detection kit and detection method |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090131354A1 (en) * | 2007-05-22 | 2009-05-21 | Bader Andreas G | miR-126 REGULATED GENES AND PATHWAYS AS TARGETS FOR THERAPEUTIC INTERVENTION |
CN109486914A (en) * | 2018-12-26 | 2019-03-19 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of nucleic acid visible detection method |
-
2021
- 2021-08-30 CN CN202111001495.6A patent/CN113801921A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090131354A1 (en) * | 2007-05-22 | 2009-05-21 | Bader Andreas G | miR-126 REGULATED GENES AND PATHWAYS AS TARGETS FOR THERAPEUTIC INTERVENTION |
CN109486914A (en) * | 2018-12-26 | 2019-03-19 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of nucleic acid visible detection method |
Non-Patent Citations (2)
Title |
---|
HONGMEI XU ET AL.: ""Protease-responsive mass barcoded nanotranslators for simultaneously quantifying the intracellular activity of cascaded caspases in apoptosis pathways"", 《CHEMICAL SCIENCE》, vol. 11, pages 5280 - 5288 * |
TIAN LI ET AL.: ""A simple and non-amplification platform for femtomolar DNA and microRNA detection by combining automatic gold nanoparticle enumeration with target-induced strand-displacement"", 《BIOSENSORS AND BIOELECTRONICS》, vol. 105, pages 137 - 142, XP085343458, DOI: 10.1016/j.bios.2018.01.034 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114214461A (en) * | 2021-12-26 | 2022-03-22 | 南京大学 | Isothermal HIV nucleic acid detection kit and detection method |
CN114214461B (en) * | 2021-12-26 | 2024-03-26 | 南京大学 | Isothermal HIV nucleic acid detection kit and detection method |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Liu et al. | Advancing single-cell proteomics and metabolomics with microfluidic technologies | |
Liu et al. | “Development and application of analytical detection techniques for droplet-based microfluidics”-A review | |
JP2017519983A (en) | Quantitative measurement method for measuring targets based on atmospheric pressure | |
CN102676508A (en) | Small molecule probe based on nano-gold and aptamer and preparation method of small molecule probe | |
CN104312914B (en) | A kind of protein molecule electronic device based on nano-pore structure | |
Wu et al. | Identification of proteins and bacteria based on a metal ion–gold nanocluster sensor array | |
US20080156646A1 (en) | Nanostructured electrochemical biosensor with aptamer as molecular recognition probe | |
CN113801921A (en) | Method for detecting miRNA (micro ribonucleic acid) by utilizing nuclear satellite structure multiplex | |
CN113976195A (en) | Microfluidic chip for exosome separation and enrichment and method for analyzing exosome surface protein | |
CN101782570A (en) | Biomolecule competition analysis method and application thereof | |
CN109613095A (en) | Terminal enzyme (DNA) electrochemica biological sensor preparation method and application based on i-motif change of configuration | |
Cui et al. | An intelligent, autocatalytic, DNAzyme biocircuit for amplified imaging of intracellular microRNAs | |
Hu et al. | Polymerase-Driven Logic Signal Amplification for the Detection of Small Extracellular Vesicle Surface Proteins and the Identification of Breast Cancer | |
CN112893864A (en) | Silver nanocluster prepared based on hairpin template and application of silver nanocluster in chloramphenicol detection | |
CN110553991B (en) | Biological/chemical detection reagent and detection method based on hollow gold nanoparticle-DNA compound | |
CN101130814A (en) | Nucleic acid ligand group chip and manufacturing method thereof | |
CN115436335B (en) | Method for detecting thrombin based on perylene derivative probe without marking | |
Cui et al. | Smart Engineering of a Self-Powered and Integrated Nanocomposite for Intracellular MicroRNA Imaging | |
CN114113287A (en) | Serum protein preparation method and serum proteome mass spectrum detection method | |
CN107219208A (en) | A kind of double fluorescence probes based on silicon nanoparticle and aptamer and its preparation method and application | |
Fan et al. | Single microentity analysis-based ultrasensitive bioassays: Recent advances, applications, and perspectives | |
US20040235028A1 (en) | Electrical readout of the binding of analyte molecules to probe molecules | |
CN108531592A (en) | A kind of DNA encoding technology and cancer markers detection method associated with nano-pore technology | |
Wang et al. | A CRISPR/Cas12a-SERS platform for amplification-free detection of African swine fever virus genes | |
CN113311152B (en) | Triple signal amplification magnetic relaxation sensing immunoassay method |
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 |