CN116926168A

CN116926168A - microRNA detection method for secondary signal amplification and application

Info

Publication number: CN116926168A
Application number: CN202311202461.2A
Authority: CN
Inventors: 胡瑞彬; 张博
Original assignee: Southwest University of Science and Technology
Current assignee: Southwest University of Science and Technology
Priority date: 2023-09-18
Filing date: 2023-09-18
Publication date: 2023-10-24
Anticipated expiration: 2043-09-18
Also published as: CN116926168B

Abstract

The application discloses a microRNA detection method for secondary signal amplification and application thereof. In a first aspect of the present application, there is provided a nucleic acid molecule detection composition comprising: a nucleic acid probe for specifically binding to a nucleic acid molecule; tailing enzyme and ATP, wherein the tailing enzyme is used for catalyzing ATP to form polyA tail at the 3' -end of the nucleic acid molecule; the detection kit comprises a polyT and a detection label, wherein the polyT is used for specifically binding to a polyA tail, and a plurality of detection labels are introduced on the polyA tail, and the detection label is modified on the polyT or can be modified on the polyT. The application develops a method capable of realizing high-sensitivity detection without depending on PCR amplification technology by utilizing the characteristic that a nucleic acid molecule is subjected to terminal modification of polyA under the action of polymerase and hybridized with biotinylated short-segment polyT and introducing a plurality of fluorescent molecules as signal amplification in a streptavidin-biotin mode.

Description

microRNA detection method for secondary signal amplification and application

Technical Field

The application relates to the technical field of in-vitro diagnosis, in particular to a microRNA detection method for secondary signal amplification and application thereof.

Background

microRNA (miRNA) is a non-coding RNA (ncRNA) of approximately 19 to 25 nucleotides in length and plays a role in posttranscriptional regulation of gene expression. As an important disease marker molecule, mirnas are widely distributed in body fluids such as blood, urine, sweat, and the like. The body fluid sample containing miRNA is obtained, and the expression abundance of the miRNA can be quantitatively measured, so that a new idea can be provided for early diagnosis, treatment and prognosis monitoring of diseases. miRNA has the following characteristics: (1) The in vivo content is extremely low, and a great deal of researches show that the miRNA content capable of being combined with the RNA-induced silencing complex to regulate and control gene expression is only at the fmol/L even lower level; (2) The fragment is small, and the length of miRNA is generally 19-25 nucleotide fragments, so that the false positive phenomenon often occurs when an amplification primer is designed, and the accurate judgment of the result is affected; (3) Homologous sequences are highly similar, and there may be miRNA members in one miRNA family that differ only by a single base, such as the Let-7 family. Furthermore, the occurrence of a disease is often accompanied by aberrant expression abundance of multiple mirnas; the abnormal abundance of the same miRNA appears in various disease tissues. Therefore, the application of miRNA in the accurate diagnosis of clinical diseases is urgently needed to establish a new method capable of simultaneously analyzing the miRNA with high sensitivity, high specificity and multiple targets.

Traditional miRNA detection methods include Northern blot hybridization, microarray technology and real-time quantitative PCR technology (qRT-PCR). However, the Northern blot hybridization technique requires complicated procedures such as transfer, and has a large sample requirement and low sensitivity. The chips used in Microarray technology are costly, and sample requirements are also large and time consuming. The qRT-PCR technology has higher sensitivity, accuracy and practicability, and is a gold standard for quantitative detection of genes. However, because of the short miRNA fragment, it is necessary to design universal primers to aid in its amplification, which inevitably results in false positives, and the equipment and consumables are expensive. In addition, the sequencing technology plays an important role in miRNA detection, but the operation steps are complicated, equipment and consumables are expensive, the sequencing cost is high, and the wide application of the sequencing technology is limited.

Therefore, there is a need to provide a miRNA detection product that combines high sensitivity, high specificity and high throughput.

Disclosure of Invention

The present application aims to solve at least one of the technical problems existing in the prior art. Therefore, the application provides a microRNA detection method for secondary signal amplification and application thereof, and the detection by using the detection composition provided by the method can achieve high sensitivity, specificity and high flux.

In a first aspect of the present application, there is provided a nucleic acid molecule detection composition comprising:

a nucleic acid probe for specifically binding to the nucleic acid molecule;

a tailing enzyme and ATP, the tailing enzyme for catalyzing the ATP to form a polyA tail at the 3' end of the nucleic acid molecule;

a polyT for specifically binding to the polyA tail and introducing a plurality of the detection tags on the polyA tail, the detection tags being modified or capable of being modified at the polyT.

In some embodiments of the application, the nucleic acid probe comprises a sequence of length 10-50 nt complementary to a nucleic acid molecule, for example, 10 nt, 15 nt, 20 nt, 25 nt,30 nt, 35 nt, 40 nt, 45 nt, 50 nt complementary sequences.

In some embodiments of the application, the tailing enzyme isE. coliPoly (A) Polymerase (PAP enzyme).

In some embodiments of the application, the tailing enzyme forms a polyA tail at the 3' end of the nucleic acid molecule that is 20-300 nt in length, e.g., 20 nt,30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt, 110 nt, 120 nt, 130 nt, 140 nt, 150 nt, 160 nt, 170 nt, 180 nt, 190 nt, 200 nt, 210 nt, 220 nt, 230 nt, 240 nt, 250 nt, 260 nt, 270 nt, 280 nt, 290 nt, 300 nt.

In some embodiments of the present application, the length of the polyT is 3-80 nt, for example, 3 nt, 5 nt, 6 nt, 8 nt, 10 nt, 20 nt,30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt. In the embodiment of the application, at least one mode of the tail adding time, the polyT length and the like of the nucleic acid molecules is controlled, so that a plurality of polyT are hybridized on the polyA tail, and a plurality of detection marks are introduced, thereby realizing signal amplification. In some embodiments, the length of the polyT is 20~60 nt,30~50 nt.

In some embodiments of the application, the length of the polyT is less than 1/2 of the length of the polyA tail, e.g., 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10.

In some embodiments of the application, the detection label comprises at least one of a chromogenic label, a fluorescent label, and the like.

In some embodiments of the application, the fluorescent label comprises at least one of a fluorescent dye, a fluorescent protein.

In some embodiments of the application, the fluorescent label comprises a near infrared fluorescent label.

In some embodiments of the application, the fluorescent label comprises a near infrared fluorescent dye.

In some embodiments of the application, the near infrared fluorescent dye comprises at least one of MPA, IRDye800, cy7, cy7.5, cy 5.5.

In some embodiments of the application, modification of the detection tag to or capable of modification to the polyT refers to pre-forming a complex of the detection tag and the polyT or modification of a separate detection tag to the polyT during detection to form a complex.

In some embodiments of the application, the detection label and the polyT are modified by means of streptavidin-biotin. The modification in this way has extremely strong affinity, is stable in biological environment and has low cost.

In some embodiments of the application, the detection composition further comprises a solid support to which the nucleic acid probes are immobilized.

In some embodiments of the application, the nucleic acid probes comprise a plurality of nucleic acid probes that target different nucleic acid molecules, the plurality of different nucleic acid probes being immobilized at different locations on the solid support. Thus, whether the corresponding nucleic acid molecule exists or not can be determined according to the detection marks at different positions, or the level of the nucleic acid molecule can be further determined according to the strength of the detection marks. For example, there are 1 or more, 10 or more, 100 or more, 1000 or more, 10000 or more, 100000 or more, 1000000 or more nucleic acid probes; the solid phase carrier has immobilized thereon 1 or more, 2 or more, 3 or more, 5 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more nucleic acid probes.

In some embodiments of the application, a plurality of different nucleic acid probes are immobilized at different locations on a solid support to form a probe array.

In some embodiments of the application, the solid support comprises a plasmonic chip.

In some embodiments of the application, a plasmonic chip includes a substrate and a metal film.

In some embodiments of the present application, the metal film is a film layer formed by at least one of simple substances and alloys of gold, silver, platinum, copper, aluminum, titanium, nickel and chromium. In some of these embodiments, the metal film is a gold film. In some of these embodiments, the metal film is a nano-metal film.

In some embodiments of the application, the substrate comprises at least one of glass, nitrocellulose, cellulose acetate, silicone wafers, nylon films, polypropylene films, magnetic beads, latex, resin, plastic.

In some embodiments of the application, the plasmonic chip is a plasmonic gold chip.

In some embodiments of the application, the surface of the solid support has a self-assembled layer to which the nucleic acid probes are coupled.

In some embodiments of the application, the self-assembled layer comprises a modifying molecule having a bifunctional group at the end.

In some embodiments of the application, the functional group is selected from at least one of amino, carboxyl, mercapto, double bond, triple bond, halo, hydroxy, ether bond, aldehyde, ketone, cyano, nitro, sulfo, maleimide groups.

In some embodiments of the application, the modifying molecule is selected from the group consisting of 4-aminophenylthiophenol, 4-hydroxyphenyl thiophenol, 2-carboxythiophenol, mercaptoethylamine, mercaptohexylamine, mercaptododecylamine, mercaptohexadecylamine, mercapto- (PEG) n-NH ₂ At least one of a protein (e.g., BSA); n is 10 to 10000.

In some embodiments of the application, the nucleic acid probe is coupled to the self-assembled layer by a coupling agent.

In some embodiments of the application, the coupling agent is at least one of succinimidyl 6- (maleimido) hexanoate, 2- [ [2- (Fmoc-amino) ethoxy ] acetic acid, succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, sodium sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, amino-heptapolyethylene glycol-azide.

In some embodiments of the application, the end of the nucleic acid probe is modified with at least one of a thiol, amino, carboxyl, double bond, triple bond, halo, hydroxy, ether bond, aldehyde, ketone, cyano, nitro, sulfo, tartaric, malic, citric, maleimide group.

In some embodiments of the application, the nucleic acid probe has 0 to 4 repeated bases, e.g., 0, 1, 2, 3, 4 repeated bases at the end attached to the solid support.

In some embodiments of the application, the repeated base at the end of the nucleic acid probe attached to the solid support is a.

In some embodiments of the application, the nucleic acid molecule to be tested is RNA.

In some embodiments of the application, the nucleic acid molecule to be tested is microRNA.

It will be appreciated that in some cases, the individual components of the detection compositions described above are independent of each other and are only mixed while participating in the respective reactions. In other embodiments, some of the components are premixed.

In some embodiments of the application, the individual components of the detection compositions described above may be in any form selected from a dry powder or solution to participate in the reaction.

In a second aspect of the application, there is provided a kit comprising a detection composition of any of the foregoing.

In a third aspect of the present application, there is provided a detection method using the aforementioned detection composition or the aforementioned kit, the detection method comprising the steps of:

mixing and incubating the to-be-detected sample with tailing enzyme and ATP to obtain a to-be-detected sample with a polyA tail at the 3' -end;

mixing a to-be-detected sample with a polyA tail added to the 3' -end with a nucleic acid probe to capture a to-be-detected nucleic acid molecule therein;

adding polyT and detection markers, so that the nucleic acid molecules to be detected introduce a plurality of detection markers on the polyA tail;

detecting the detection mark.

In some embodiments of the application, further comprising immobilizing the nucleic acid probe on a solid support.

In some embodiments of the application, further comprising immobilizing the nucleic acid probe on a plasmonic chip.

In some embodiments of the application, immobilizing a nucleic acid probe on a solid support comprises modifying the solid support with a first group, adding the nucleic acid probe, and incubating to immobilize the nucleic acid probe on the solid support. Wherein the nucleic acid probe is modified with a second group capable of forming a chemical bond with the modified first group on the solid support. Wherein the first group and the second group are at least one selected from the group consisting of amino, carboxyl, mercapto, double bond, triple bond, halogen, hydroxyl, ether bond, aldehyde group, ketone group, cyano, nitro, sulfo, maleimide group.

In some embodiments of the application, immobilizing the nucleic acid probe on the solid support comprises modifying the solid support with a Sulfo-SMCC and incubating the thiol-modified nucleic acid probe after the reducing agent treatment with the solid support to immobilize the nucleic acid probe on the solid support. In some embodiments, the reducing agent comprises at least one of TCEP, DTT.

In some embodiments of the application, the nucleic acid probe is incubated with the solid support at a concentration of 1 to 10. Mu.M, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Mu.M. In some embodiments, the concentration of the nucleic acid probe is 2 to 8. Mu.M, 4 to 6. Mu.M.

In some embodiments of the application, the incubation time of the sample to be tested with the tailing enzyme and ATP is 10-180 min, for example 10, 20, 30, 40, 60, 90, 120, 150, 180 min. In some embodiments, the test article is incubated with the tailing enzyme and ATP in combination for a period of 20~120 min,20~40 min.

In some embodiments of the application, the hybridization is performed by adding the polyT and the detection label for a period of 0.1 to 12 hours, for example, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, h. In some embodiments, the hybridization time is 0.5-8, 1-6, 2-5, 3-5 hours with the addition of the polyT and the detection label.

In some embodiments of the present application, detecting the detection marker includes determining whether a corresponding detection signal is present, and if so, indicating that a corresponding target miRNA is present in the test article, the level of the target miRNA in the test article may be further determined according to the strength of the detection signal.

In some embodiments of the application, detecting the detection marker includes the fluorescence imaging device detecting the presence or absence of a corresponding fluorescence signal.

The embodiment of the application also provides application of the detection composition or the kit or the detection method in early diagnosis, treatment and prognosis monitoring of diseases or preparation of products for early diagnosis, treatment and prognosis monitoring of diseases.

In some embodiments of the application, the disease comprises cancer.

In some embodiments of the application, the cancer comprises at least one of a solid tumor and a non-solid tumor. The solid tumor comprises at least one of brain cancer, head and neck cancer, lung cancer, liver cancer, breast cancer, gastric cancer, colon cancer, rectal cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, lymphoma, and skin cancer, and the non-solid tumor comprises leukemia.

The embodiment of the application has the following beneficial effects:

aiming at the problem that the sensitivity, the specificity and the flux of miRNA detection are difficult to be integrated, the signal enhancement effect of a plasmon material on a near infrared region fluorescent molecule is utilized as primary signal amplification, the characteristic of modifying polyA at the tail end of miRNA under the action of polymerase is utilized, the miRNA is hybridized with biotinylated short-segment polyT, a plurality of fluorescent molecules are introduced in a streptavidin-biotin mode to serve as secondary signal amplification, and a method capable of realizing high-sensitivity miRNA detection without depending on a PCR amplification technology is developed through the two signal amplification. In addition, probe molecules aiming at different target miRNAs are loaded on the surface of the plasmon chip through expanding two-dimensional space information, so that a high-density DNA microarray is formed, and the detection of multiple miRNAs of a single fluorescent channel is realized. After the multiple miRNA analysis model system is adopted, the measurement result of miRNA which is differentially expressed in different cancer cells is consistent with the qRT-PCR result.

The concrete explanation is as follows: referring to fig. 1, a certain chemical modification is performed on the surface of a plasmonic chip, so that a nucleic acid probe molecule immobilized on the surface of the plasmonic chip is about 100 nanometers away from the surface, and an optimal condition is provided for the subsequent plasmon enhancement of fluorescent signals. Nucleic acid probes aiming at different target miRNAs are immobilized on different areas of a chip in the form of an array to form a DNA microarray. These nucleic acid probe molecules are capable of complementary hybridization to a target miRNA modified by terminal polyA to form a miRNA/DNA complex. After introducing polyA at the tail end of miRNA, adding biotin-modified short polyT to the surface of the treated chip, so that the short polyT is hybridized and complemented with the polyA modified at the tail end of the miRNA, and forming a stable miRNA/DNA complex with a plurality of biotin modifications. On the basis of the complex with a plurality of biotin modifications at the end constructed above, streptavidin of a fluorescent molecule labeled in advance is added thereto, and a plurality of fluorescent molecules are introduced by means of specific binding of streptavidin to biotin. And scanning fluorescent signals on the constructed chip loaded with a large number of fluorescent molecules by using a high-resolution fluorescent scanning imager, and establishing a dynamic response relation between the chip and the corresponding miRNA concentration according to the intensity of the fluorescent signals. According to the same mode, for the DNA microarray of the probe, a plurality of miRNAs aiming at different targets are immobilized in different areas by expanding two-dimensional space information of the chip, so that simultaneous determination of the miRNAs of the targets is realized on the same chip, the miRNA detection flux is increased, and the detection of multiple miRNAs of a single fluorescent channel is realized.

Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.

Drawings

FIG. 1 is a schematic diagram of the reaction principle in the example of the present application.

FIG. 2 shows the surface structure of a plasmonic chip and the verification result of the method in the embodiment of the application. Wherein a in fig. 2 is a surface scanning electron microscope imaging diagram of the plasmonic chip, and a scale is 1 mu m; b in fig. 2 is a schematic diagram of modification of polyA at the end of miRNA and agarose gel images before and after modification of polyA; lane 1 is the product of the modified poly adenine at the end of miRNA21, lane 2 is the miRNA21 standard, and lane 3 is marker; c in fig. 2 is a graph of chip fluorescence results of different combinations of different mirnas 21-P1, mirnas 21-polyA and polyT40 to verify the feasibility of the method; d in FIG. 2 is the mean value of the fluorescence signal obtained from the chip statistics in c, and the error bars represent the standard deviation of the mean value of the fluorescence signal.

FIG. 3 is the results of an optimization experiment for different parameters in the examples. In fig. 3, a is a graph of an optimized experimental result of the number of probe miRNA21-P1 terminal-linked polyA; b in fig. 3 is a graph of the optimized experimental results of the concentration of the probe miRNA 21-P1; c in FIG. 3 is a graph of optimized experimental results of the length of the polyT fragment; d in FIG. 3 is a graph of optimized experimental results of the concentration of polyT 40; e in fig. 3 is a graph of the optimized experimental results of time for the hydroxyl end of miRNA21 to join a; f in FIG. 3 is a graph of the results of the optimization experiment of hybridization time. In the above optimization experiments, the miRNA21 concentration was 50 pM. The average signal intensity for each parameter is obtained by subtracting the blank signal average signal intensity from five parallel signal points. Error bars represent standard deviations of the average signal intensities.

Fig. 4 is a graph showing the results of analysis performance study of multiple mirnas developed based on plasmonic chips in the examples. Wherein a in fig. 4 is a fluorescence signal imaging diagram of miRNA21 concentration from 5 nM to 5 fM; b in fig. 4 is a graph of the linear response between fluorescence signal intensity and serial gradient diluted target miRNA concentration established from graph a statistics in fig. 4; c in fig. 4 is a selective fluorescence imaging map; the mixture contains miRNA21, miRNA141 and miRNA155;1-mismatch miRNA21 (single base mismatch), 2-mismatch miRNA21 (double base mismatch), 3-mismatch miRNA21 (three base mismatch); d in FIG. 4 is a plot of fluorescence signal intensity obtained from c in FIG. 4. In the above experiments, the miRNA concentrations were 50 pM. The average signal strength for each parameter was obtained by subtracting the average signal strength of the blank signal from fifteen parallel signal points. Error bars represent standard deviations of the average signal intensities.

Figure 5 is the result of analysis of multiple target mirnas from several cell lines in the examples. A in fig. 5 is a fluorescent signal imaging graph of differentially expressed mirnas in MCF-7, a 549. The positive signal group (Pos-ctrl) was used as a positive reference for normalizing the fluorescent signal. Every three parallel points represent one sample (n=3). FIG. 5 b is a graph of the proposed method for assessing differentially expressed miRNAs in MCF-7 cells compared to the results of the qRT-PCR method, and FIG. 5 c is a graph of the proposed method for assessing differentially expressed miRNAs in A549 cells compared to the results of the qRT-PCR method. From about 5X 10 ⁶ miRNAs were extracted from MCF-7, A549, MCF-10A cell lines and these differentially expressed miRNAs were assayed using qRT-PCR. In the detection method of the embodiment, the relative expression level is calculated by normalizing the average fluorescence signal intensity. The relative expression level of qRT-PCR method is obtained by comparing Ct (DeltaDeltaCt), i.e. ΔΔct= delta Ct (experiment) group) - Δct (control group); delta ct=ct (target gene) -Ct (reference gene). Error bars were obtained from standard deviations of triplicate experiments.

Detailed Description

The conception and the technical effects produced by the present application will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present application. It is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present application based on the embodiments of the present application.

The following detailed description of embodiments of the application is exemplary and is provided merely to illustrate the application and is not to be construed as limiting the application.

In the description of the present application, the meaning of a plurality means one or more, the meaning of a plurality means two or more, and the meaning of greater than, less than, exceeding, etc. is understood to exclude the present number, and the meaning of above, below, within, etc. is understood to include the present number, and the meaning of about means within the range of ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% etc. of the present number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.

In the description of the present application, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The application is illustrated below with reference to specific examples.

Example 1: reaction system of miRNA (micro ribonucleic acid) and polyA tail

Referring to fig. 2 b, the principle of adding a polyA tail to the end of miRNA in this example is shown, and ATP in the reaction system is converted into AMP by PAP polymerase and sequentially attached to the 3' -hydroxyl end of miRNA to form a polyA tail. The longer the enzymatic reaction, the longer the length of the polyA tail attached to the 3' hydroxyl end. Since miRNA21 is up-regulated in expression in a variety of cancer cells, miRNA21 was selected as the target for reaction and detection in this example, as well as in other examples below.

The experimental procedure was as follows:

HPLC grade miRNA21 standard was dissolved with 1×te buffer and diluted to 100 pM. 10 mu L of diluent is added into the mixed solution shown in the following table 1, and after being uniformly mixed, the mixed solution is placed at a constant temperature of 37 ℃ to react for 30 min, and then the reaction is terminated after heat treatment at 95 ℃ for 5 min. And then the reaction product miRNA21-polyA is put in a refrigerator at the temperature of minus 20 ℃ for standby.

TABLE 1 composition of the mixture

The reaction product and the standard were verified by 2% agarose gel electrophoresis experiments, as shown in fig. 2 b, the band length of the reaction product was significantly longer than that of the standard, indicating that the polyA tail was added to the end of miRNA21 by the above reaction.

Example 2: immobilization of probes

Referring to a of fig. 2, scanning electron microscope imaging results show that the surface of the plasmonic chip has compact nano gold islands and rich nano ravines, and Near Infrared (NIR) fluorescence enhancement can be realized by using the structure. Therefore, it is considered that the immobilization of the probe onto the plasmonic chip achieves the primary signal amplification of the detection signal.

The experimental procedure was as follows:

a plasma gold chip (pGOLD) from Nirmidas Biotech was immersed in a 20% BSA solution and placed at room temperature protected from light for 2 h. After washing the surface of the chip with double distilled water to remove excess BSA, it was immersed in double distilled water containing 5 mg of Sulfo-SMCC and left at room temperature under light-shielding conditions for 1 h. The superfluous sulfoSMCC on the chip surface is washed by double distilled water and the surface is dried.

5. Mu.M thiol-modified DNA probe 1 was treated with 25 mM TCEP at room temperature for h, and the DNA probe of the treated miRNA21 was spray-printed on the surface of the Sulfo-SMCC-treated chip using a high-precision 3D spotting apparatus to form a DNA probe array. After spotting, the chip was placed in an incubation box with a humidity of about 80%, and placed in a dark place at room temperature for 2 h. The chip was then placed in an aqueous solution containing 0.1% β -mercaptohexanol and left in the dark at room temperature for 30 min, blocking the sites not occupied by the probes. The residual β -mercaptohexanol on the surface was then washed off with 1×te buffer. Obtaining the chip immobilized with the probe miRNA 21-P1.

Example 3: detection of miRNA

The chip with the probe immobilized in example 2 was placed on a FAST frame mold, and the mixed solution as in table 2 was added thereto, and reacted at room temperature in a dark place 2 h. After the hybridization reaction was completed, the excess reaction on the surface was washed off with 1×te buffer. Then 2 nM IRDye800-labeled streptavidin solution was added to the surface and incubated for 30 min at room temperature in the dark. Excess IRDye 800-labeled streptavidin solution on the surface was washed away and the chip was dried. The chip surface fluorescence signal is scanned using a fluorescence scanning imager.

TABLE 2 detection of the composition of the mixture

The results of the control group without at least one of the probe miRNA21-P1, the modified target miRNA21-polyA and the poly T40 are respectively provided, and as shown in the c and d of the figure, the result is that only when the probe miRNA21-P1, the modified target miRNA21-polyA and the poly T40 exist simultaneously, a strong fluorescent signal appears on the chip.

Example 4: condition optimization experiment

1. miRNA-P1

The experiment is carried out by referring to examples 1-3, and the intensity of fluorescent signals under the condition that the number of the terminal bases of the miRNA-P1 is different under the same experimental condition is compared. As a result, as shown in FIG. 3 a, it can be seen from the graph that the background signal was almost zero when the number of thiol-terminated polyA bases of the probe miRNA21-P1 was 0. When the number of thiol-terminated polyA bases of the probe miRNA21-P1 is 6 or 12, although the fluorescent signal of the experimental group is enhanced, the background signal is also significantly enhanced at the same time, probably due to hybridization of polyT used for hybridization of polyA on miRNA with polyA on the probe. Therefore, in order to improve the sensitivity, the probe miRNA-P1 with the thiol end free of adenine is selected for the next optimization.

In example 2, probe miRNA-P1 with different concentrations was selected for immobilization, and as shown in b of FIG. 3, it can be seen from the graph that the fluorescence signal intensity is strongest when the probe concentration is 5 [ mu ] M. The possible reasons are that the fluorescence signal intensity cannot reach saturation (e.g. 1 μm, 2 μm) at low probe concentrations, steric hindrance exists and the fluorescence intensity is easily quenched (e.g. 10 μm) when the probe concentration is excessive. Therefore, in order to improve the sensitivity, a probe miRNA-P1 of 5 mu M is selected for the next optimization.

2. polyT

Biotinylated polyT is a critical step in the introduction of the fluorescent signal IRDye 800. For this, the fragment length and concentration of the polyT are optimized with reference to examples 1-3. As a result, as shown in FIGS. 3 c and d, the fluorescence signal intensity was maximized when the length of the polyT was 40 nt. The possible reason is that hybridization with polyA is unstable when the length of the polyT fragment is less than 40 nt, and the signal intensity is less than optimal. When the length of the polyT fragment is more than 40 and nt, the fragment is too long, so that the number of signal molecules introduced after hybridization is insufficient, and the optimal state is not achieved. Furthermore, when the concentration of polyT40 is 1 μm, its signal intensity is significantly higher than other combinations. Therefore, the optimal polyT length is 40 nt with a concentration of 1 μm.

3. miRNA-polyA

In order to further improve the fluorescence signal intensity, the time for generating polyA tail at the hydroxyl end of miRNA21 and the hybridization process are optimized according to reference examples 1-3. As shown in fig. 3 e and f, the fluorescence signal increases rapidly and gradually towards saturation for the first 30 min when the hydroxyl ends of miRNA21 are linked to form a polyA tail, and further extension of time does not further enhance the fluorescence signal. And when the hybridization process of miRNA-polyA and polyT is continued for 4 h, the signal intensity tends to be saturated, and the plateau is reached within the range of 4-6 h. Therefore, the optimal tailing time was 30 min and the hybridization time was 4 h.

Example 5: sensitivity experiment

According to the optimal conditions of example 4, the synthesized miRNA21 was diluted to 5 nM to 5 fM in sequence, and fluorescence signals thereof were detected, respectively, to examine the detection sensitivity and dynamic interval of the detection method. The result is shown in fig. 4 a, where the fluorescence signal intensity and the miRNA concentration are positively correlated. When the miRNA21 concentration is 5 nM, the fluorescence signal intensity tends to saturate. Furthermore, referring to fig. 4 b, there is a certain dynamic response relationship between fluorescence signal intensity and miRNA21 concentration. The limit of detection (LOD) was as low as 5 fM based on the calculation of the blank mean plus three standard deviations. Moreover, as shown by the experimental results, when the concentration of the miRNA21 is 5 fM, the error value corresponding to the signal intensity is still small, which indicates that the proposed multi-target miRNA analysis method has higher stability and consistency.

Example 6: specificity experiments

According to the optimal optimization conditions of example 4, the selectivity of the method was tested under the same experimental conditions using miRNA mixtures (including miRNA21, miRNA141 and miRNA 155), target miRNA21, single base mismatched miRNA21, double base mismatched miRNA21, three base mismatched miRNA21 and random RNAs. The results are shown in figures 4 c and d, which show that when the target miRNA21 is present, the corresponding fluorescent signal intensities are significantly stronger than the other combinations. When single base mismatch occurs in the target, the fluorescence signal intensity is reduced by 93% compared with that of the target molecule; the fluorescence signal intensity was almost identical to the blank signal when the target showed double base mismatch, three base mismatch and random RNA. The experimental results show that the method provided by the embodiment can be used for distinguishing base mismatch and has good selectivity. Moreover, there is no obvious difference between the signal intensity of the three mirnas mixed together and the signal intensity in the presence of only one target miRNA molecule, indicating that the method provided by the embodiment has the capability of specifically recognizing the target miRNA molecule from a complex sample.

Example 7: multiplex detection

With reference to the foregoing embodiments, probes targeting different mirnas, including miRNA21, miRNA141, miRNA155, miRNA29b-2, miRNA181a, let-7a, miRNA16-2, miRNA205, miRNA16-1, miRNA25, etc., are jet printed at different positions of the plasmonic chip by the high-precision 3D spotting device.

MCF-7, A549 and MCF-10A cells were inoculated into DMEM medium containing 1% streptomycin-penicillin and 10% FBS, respectively, and placed in 5% CO ₂ Is placed in a constant temperature and humidity incubator. After the cells had grown to the flask, the medium was discarded and washed three times with 1 XPBS buffer, after which the cells were scraped off and collected in a 1.5 mL centrifuge tube. Extracting miRNA from the cells according to the instructions of the miRNA extraction kit, and using the method according to the previous embodimentAnd detecting and analyzing the plasmonic chip immobilized with a plurality of different probes. Wherein, the fluorescence signal intensity of the target miRNAs in MCF-10A normal breast cells is used as a control. As a result, as shown in fig. 5, the relative expression of some mirnas including miRNA29b-2, miRNA181a and Let-7a was significantly higher than miRNA16-2, miRNA205, miRNA16-1 and miRNA25 in the MCF-7 breast cancer cell line; in A549 lung cancer cells, the relative expression of miRNA16-2 and miRNA205 was significantly higher than that of miRNA29b-2, let-7a and miRNA16-1. Furthermore, the relative expression of miRNA141, miRNA29b-2, miRNA181a, let-7a, miRNA16-2 and miRNA205 in these two tumor cell lines was also significantly different. These results are consistent with those reported previously. Notably, miRNA21 and miRNA155 were highly expressed in both breast cancer MCF-7 and lung cancer a549 cell lines. This is consistent with the conclusion reported in the literature that both mirnas are highly expressed in most cancer species cells.

The results of qRT-PCR verification on miRNAs extracted from the cells are shown in fig. 5 b and c, and the relative expression amounts of the miRNAs detected by the two methods in the two tumor cells are basically the same, which shows that the results of the miRNA detection methods provided by the examples have good consistency with the qRT-PCR results.

The results show that the detection method provided by the embodiment of the application can analyze the multi-target miRNA in the complex matrix with high sensitivity and high specificity, and has important biological significance in early diagnosis, treatment and prognosis monitoring of diseases.

The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.

Claims

1. A composition for detecting a nucleic acid molecule comprising:

a nucleic acid probe for specifically binding to the nucleic acid molecule;

2. The detection composition of claim 1, wherein the detection composition further comprises a solid support to which the nucleic acid probe is immobilized.

3. The detection composition of claim 2, wherein the nucleic acid probes comprise a plurality of nucleic acid probes that target different nucleic acid molecules, a plurality of different of the nucleic acid probes being immobilized at different locations on the solid support.

4. The detection composition of claim 2, wherein the solid support comprises a plasmonic chip.

5. The detection composition according to claim 2, wherein the nucleic acid probe has 0 to 4 repeated bases at the end attached to the solid support.

6. The detection composition according to claim 1, wherein the polyT has a length of 20 to 60 nt.

7. The detection composition of claim 1, wherein the nucleic acid molecule is microRNA.

8. Kit comprising a detection composition according to any one of claims 1 to 7.

9. Detection method using the detection composition according to any one of claims 1 to 7 or the kit according to claim 8, characterized by comprising the steps of:

mixing and incubating the to-be-detected product with tailing enzyme and ATP to obtain a to-be-detected product with a polyA tail at the 3' -end;

adding a polyT and a detection label, so that a nucleic acid molecule to be detected introduces a plurality of detection labels on the polyA tail;

and detecting the detection mark.

10. The method of claim 9, further comprising immobilizing the nucleic acid probe on a plasmonic chip.