CN113801921A - A method for multiplex detection of miRNA using nuclear satellite structure - Google Patents
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Abstract
本发明公开了一种利用核卫星结构多联检测miRNA的方法，包括如下步骤：(1)取出磁珠，用12.5mM Mg2+加入Biotin DNA并在旋转仪上旋转后，取下用12.5mM Mg2+洗涤，并定容；(2)已修饰DNA的金球与三种烷基硫醇，按照浓度比1：3000的比例混合并在摇床上以20℃反应，然后通过在离心机中离心1小时后吸取上清液来去除过量未连接的烷基硫醇分子；(3)加入已修饰烷基硫醇分子mass tag的金球于磁珠中并反应一段时间后，进行MALDI检测；(4)链置换反应。本发明通过MALDI质谱测试，可以检测出样品中含有的miRNA的种类，并且可以同时独立的检测3种miRNA的种类与含量多少。
The invention discloses a method for multiplexed detection of miRNA using nuclear satellite structure. Mg 2+ was washed, and the volume was fixed; (2) the DNA-modified gold spheres were mixed with three alkyl thiols in a concentration ratio of 1:3000 and reacted on a shaker at 20 °C, and then passed through in a centrifuge. After centrifugation for 1 hour, the supernatant was aspirated to remove excess unconnected alkyl thiol molecules; (3) MALDI detection was performed after adding the mass-tagged gold spheres of modified alkyl thiol molecules to the magnetic beads and reacting for a period of time; (4) Strand displacement reaction. The present invention can detect the types of miRNA contained in the sample through MALDI mass spectrometry test, and can simultaneously and independently detect the types and contents of three miRNAs.
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.
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| CN114214461A (en) * | 2021-12-26 | 2022-03-22 | 南京大学 | Isothermal HIV nucleic acid detection kit and detection method |
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| CN114214461B (en) * | 2021-12-26 | 2024-03-26 | 南京大学 | Isothermal HIV nucleic acid detection kit and detection method |
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