阅读说明：本技术 基于自组装核酸探针信号放大法检测dnmt1的方法 (Method for detecting DNMT1 based on self-assembly nucleic acid probe signal amplification method ) 是由 于斐 巩芳芳 吴拥军 刘利娥 何磊良 王艺琳 万珍珍 张咪咪 宋露露 杜梦思 于 2020-12-30 设计创作，主要内容包括：本发明提供了一种基于自组装核酸探针信号放大法检测DNMT1的方法,主要利用EDC/sulfo-NHS活化法构建免疫磁珠,且免疫磁珠上的McAb-(DNMT1)能特异性捕获样品中的DNMT1；依次形成双抗夹心免疫磁珠和生物素化的双抗夹心免疫磁珠；利用核酸自组装技术构建信号放大探针Biotin-聚FAM；通过链霉亲和素能够将所述Biotin-聚FAM与所述生物素化的双抗夹心免疫磁珠偶联在一起形成荧光复合物；利用所述荧光复合物能发出荧光的特性且荧光强度和DNMT1的浓度之间的线性关系,建立检测DNMT1水平的方法,该方法具有操作简单,准确性好,灵敏度较高,可适用于生物样品中DNMT1水平的检测等特点。(The invention provides a method for detecting DNMT1 based on a self-assembly nucleic acid probe signal amplification method, which mainly utilizes an EDC/sulfo-NHS activation method to construct immunomagnetic beads, and McAb on the immunomagnetic beads DNMT1 Can specifically capture DNMT1 in a sample; sequentially forming double-antibody sandwich immunomagnetic beads and biotinylated double-antibody sandwich immunomagnetic beads; constructing a signal amplification probe Biotin-poly FAM by utilizing a nucleic acid self-assembly technology; the Biotin-poly FAM and the biotinylated double-antibody sandwich immunomagnetic beads can be coupled together through streptavidin to form a fluorescent complex; the characteristic that the fluorescent complex can emit fluorescence and the linear relation between the fluorescence intensity and the concentration of DNMT1 are utilized to establish the detection of the DNMT1 levelThe method has the characteristics of simple operation, good accuracy, high sensitivity, suitability for detecting the DNMT1 level in a biological sample and the like.)

1. A method for detecting DNMT1 based on a self-assembly nucleic acid probe signal amplification method, comprising the steps of:

preparation of a Signal amplification Probe biotinylated Polyfluorescein was prepared by mixing biotinylated DNA strand S1 and fluorescein-labeled DNA strand S2, wherein the biotinylated DNA strand S1 is abbreviated as: S1-Biotin, the fluorescein-labeled DNA strand S2 is abbreviated as: S2-FAM, the biotinylated fluorescein being abbreviated as: biotin-poly FAM;

construction of immunomagnetic beads Using carbon dioxideFixing DNMT1 mouse anti-human monoclonal antibody on the surface of carboxyl magnetic beads by an imine hydrochloride/N-hydroxysuccinimide activation method to form immunomagnetic beads, wherein the DNMT1 mouse anti-human monoclonal antibody is abbreviated as: McAb_DNMT1；

Forming double-antibody sandwich immunomagnetic beads, and mixing the immunomagnetic beads, DNMT1 solution and DNMT1 polyclonal antibody to form double-antibody sandwich immunomagnetic beads, wherein the DNMT1 solution is a sample to be tested containing DNMT1 or a standard solution containing DNMT1, and the DNMT1 polyclonal antibody is abbreviated as: PcAb_DNMT1；

Generating biotinylated double-antibody sandwich immunomagnetic beads, and reacting the double-antibody sandwich immunomagnetic beads with biotinylated goat anti-rabbit IgG to generate the biotinylated double-antibody sandwich immunomagnetic beads, wherein the biotinylated goat anti-rabbit IgG is abbreviated as: goat anti-rabbit IgG-Biotin;

forming a fluorescent compound, and connecting the Biotin-poly FAM with the biotinylated double-antibody sandwich immunomagnetic beads through streptavidin to form the fluorescent compound;

fluorescence detection and establishment of regression equation the regression equation for detecting the concentration of DNMT1 was established using a fluorometer to detect the relationship between the fluorescence intensity of the fluorescent complex, the fluorescence intensity and the concentration of DNMT 1.

2. The method for detecting DNMT1 of claim 1, wherein the step of preparing a signal amplification probe comprises: and mixing the S1-Biotin and the S2-FAM, heating in water bath at 80-100 ℃ for 3-5 min, taking out, cooling to room temperature, and carrying out vortex hybridization for 1-3 h to carry out self-assembly to form the Biotin-poly FAM.

3. The method for detecting DNMT1 of claim 2, wherein the step of constructing immunomagnetic beads comprises: firstly, suspending the carboxyl magnetic beads in 100 mu L of MES buffer solution, and then adding N-hydroxysuccinimide solution and carbodiimide hydrochloride solution to carry out room temperature oscillation reaction and magnetic separation treatment to obtain activated magnetic beads; subsequently adding the McAb to the activated magnetic beads_DNMT1Shaking with PBS buffer solution at room temperatureThe McAb should be_DNMT1Coupling with the carboxyl magnetic beads, and performing magnetic separation treatment to remove supernatant to obtain coupled magnetic beads; and blocking the coupled magnetic beads by using BSA solution with the mass fraction of 0.5% to form the immunomagnetic beads.

4. The method of claim 3, wherein the step of forming double antibody sandwich immunomagnetic beads comprises: adding DNMT1 solution with the concentration of 0-200 ng/mL into the immunomagnetic beads for incubation and magnetic separation in sequence, and then adding PcAb_DNMT1Sequentially carrying out incubation, magnetic separation and cleaning treatment on the diluent to prepare the double-antibody sandwich immunomagnetic bead, wherein the PcAb_DNMT1The diluted solution is used for diluting PcAb with BSA solution with the mass fraction of 0.5%_DNMT1And (4) preparing.

5. The method of detecting DNMT1 of claim 4, wherein the step of generating biotinylated double antibody sandwich immunomagnetic beads comprises: and (2) diluting the goat anti-rabbit IgG-Biotin with the BSA solution with the mass fraction of 0.5% according to the proportion of 1: 500-1: 1000 to prepare a goat anti-rabbit IgG-Biotin diluent, adding the goat anti-rabbit IgG-Biotin diluent into the double-antibody sandwich immunomagnetic bead, carrying out incubation treatment, and then carrying out magnetic separation and cleaning treatment to prepare the biotinylated double-antibody sandwich immunomagnetic bead.

6. The method of detecting DNMT1 of claim 5, wherein the step of forming a fluorescent complex comprises: and adding streptavidin with the concentration of 0.5-8 mu g/mL into the generated biotinylated double-antibody sandwich immunomagnetic beads, performing magnetic separation and washing, then adding the Biotin-poly FAM probe with the concentration of 300-800 nmol/L, and performing magnetic separation and cleaning treatment again to obtain the fluorescent compound.

7. The method of detecting DNMT1 of claim 6, wherein the steps of fluorescence detection and regression equation establishment comprise: combining the fluorescent complexDissolving in hybridization buffer solution to form fluorescent compound solution, and detecting the fluorescence intensity F of the fluorescent compound solution and the blank test fluorescence intensity F by adopting a fluorometer₀(ii) a At DNMT1 concentration C_DNMT1Establishing a linear regression equation for detecting DNMT1 in a concentration range of 2-200 nmol/L: y =0.28814X +0.01782, wherein X is lgC_DNMT1Y is lg (F/F)₀) Correlation coefficient ofr=0.9962。

8. The method for detecting DNMT1 of claim 7, wherein the detection limit of the concentration of DNMT1 is 0.05 nmol/L.

Technical Field

The invention relates to the field of biotechnology detection, in particular to a method for detecting DNMT1 based on a self-assembly nucleic acid probe signal amplification method.

Background

DNA methylation is an important component of epigenetics and plays an important role in cell proliferation, aging of the body, the development of cancer and other vital activities. DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) are modification enzymes important in the methylation process, and mainly play a role in transferring methyl groups of S-adenosylmethionine (SAM) to cytosine or adenine of a corresponding DNA sequence so as to achieve DNA methylation. DNMTs are also involved in important biological activities such as maintenance of chromosome stability, embryonic development, cell differentiation, etc. Animal studies have shown that DNMT1^-/-Mice develop slowly and die in mid-pregnancy, DNMT3a^-/-Mice died around after birth, whereas DNMT3b^-/-Mice died before birth.

Compared with DNMT3a and DNMT3b, DNMT1 is the most important and the earliest studied, and can maintain normal DNA methylation, can also be methylated de novo, and has the capacity of regulating the cell cycle and regulating the expression of tumor suppressor genes. Overexpression of DNMT1 can lead to abnormal DNA methylation, which is closely associated with the development of cancer. Lin et al found DNOverexpression of MT1 can cause abnormal regulation of the p53/Sp1 pathway and lead to hypermethylation of multiple TSG promoters, resulting in the occurrence and development of non-small cell lung cancer and poor prognosis.These results demonstrate that overexpression of DNMT1 and DNMT3b may be associated with different pathological symptoms of gastric cancer, and that co-expression of DNMT1 or DNMT3b or both has a significantly different effect on hypermethylation of tumor promoter-associated genes, wherein co-expression of both is closely related to hypermethylation of RAR-beta 2. Peng et al showed that the expression level of DNMT1 protein was elevated at many stages of the pancreatic cancer course, whereas overexpression of DNMT1 was associated with the invasiveness of pancreatic cancer. In addition, type 2 diabetes, Alzheimer's disease, epilepsy and the like are also closely related to the abnormal expression level of DNMT 1. The abnormal expression level of DNMT1 usually occurs earlier than other symptoms of malignant tumor, which indicates that it is possible to be used as a potential tumor biomarker and become a drug action target in various tumor diagnosis and treatment processes.

At present, the detection of DNMT1 in biological samples is mainly performed from both aspects of DNMT1 activity and DNMT1 level. A number of new detection methods have been applied to the detection of DNMT1 activity, such as electrochemistry, fluorescence, chemiluminescence, and colorimetry. However, the detection of DNMT1 activity in biological samples was performed ex vivo, and the results of this detection do not reflect the true activity of DNMT1 in vivo. In contrast, detection of the level of DNMT1 is more meaningful. In one aspect, the level of DNMT1 is measured by directly measuring DNMT1 to explore the relationship between the level of DNMT1 and the development of disease, rather than by measuring the activity of DNMT1 by measuring the catalytic or other related products of DNMT 1. On the other hand, the biological samples tested were derived from each organism and were more convincing because they could truly reflect the level of DNMT1 in the samples. However, neither DNMT1 activity nor the level of DNMT1 was included in the indicator for cancer diagnosis. Therefore, it is of great importance to determine a DNMT1 marker for cancer diagnosis or treatment to fill the current clinical gap.

Disclosure of Invention

In view of the above, the present invention provides a method for detecting DNMT1 based on a self-assembly nucleic acid probe signal amplification method, which mainly detects the level of DNMT1 by using a traditional Polymerase Chain Reaction (PCR) bridging medium, a streptavidin-binding antigen-antibody specific binding system, and a nucleic acid self-assembly technology, so as to solve the above problems.

The invention provides a method for detecting DNMT1 based on a self-assembly nucleic acid probe signal amplification method, which comprises the following steps:

preparation of Signal amplification Probe

Biotinylated DNA strand S1 and fluorescein-labeled DNA strand S2 were mixed to prepare biotinylated fluorescein, wherein the biotinylated DNA strand S1 is abbreviated as: S1-Biotin, the fluorescein-labeled DNA strand S2 is abbreviated as: S2-FAM, the biotinylated fluorescein being abbreviated as: biotin-poly FAM;

construction of immunomagnetic beads DNMT1 mouse anti-human monoclonal antibodies are immobilized on the surface of carboxyl magnetic beads by using a carbodiimide hydrochloride/N-hydroxysuccinimide activation method to form immunomagnetic beads, wherein the DNMT1 mouse anti-human monoclonal antibodies are abbreviated as: McAb_DNMT1；

Forming double-antibody sandwich immunomagnetic beads, and mixing the immunomagnetic beads, DNMT1 solution and DNMT1 polyclonal antibody to form double-antibody sandwich immunomagnetic beads, wherein the DNMT1 solution is a sample to be detected containing DNMT1 or a standard solution containing DNMT 1;

generating biotinylated double-antibody sandwich immunomagnetic beads, and reacting the double-antibody sandwich immunomagnetic beads with biotinylated goat anti-rabbit IgG to generate the biotinylated double-antibody sandwich immunomagnetic beads, wherein the biotinylated goat anti-rabbit IgG is abbreviated as: goat anti-rabbit IgG-Biotin;

forming a fluorescent compound, and connecting the Biotin-poly FAM with the biotinylated double-antibody sandwich immunomagnetic beads through streptavidin to form the fluorescent compound;

fluorescence detection and establishment of regression equation the regression equation for detecting the concentration of DNMT1 was established using a fluorometer to detect the relationship between the fluorescence intensity of the fluorescent complex, the fluorescence intensity and the concentration of DNMT 1.

Based on the above, the step of preparing the signal amplification probe includes: and mixing the S1-Biotin and the S2-FAM, heating in water bath at 80-100 ℃ for 3-5 min, taking out, cooling to room temperature, and carrying out vortex hybridization for 1-3 h to carry out self-assembly to form the Biotin-poly FAM.

Based on the above, the step of constructing the immunomagnetic beads comprises: firstly, suspending the carboxyl magnetic beads in 100 mu L of MES buffer solution, and then adding a sulfo-NHS solution and an EDC solution to carry out room temperature oscillation reaction and magnetic separation treatment to obtain activated magnetic beads; subsequently adding the McAb to the activated magnetic beads_DNMT1Shaking reaction with PBS buffer at room temperature_DNMT1Coupling with carboxyl magnetic beads, and performing magnetic separation treatment to remove supernatant to obtain coupled magnetic beads; and blocking the coupled magnetic beads by using BSA solution with the mass fraction of 0.5% to form the immunomagnetic beads.

Based on the above, the step of forming the double-antibody sandwich immunomagnetic bead comprises: adding DNMT1 solution with the concentration of 0-200 ng/mL into the immunomagnetic beads for incubation and magnetic separation in sequence, and then adding PcAb_DNMT1Sequentially carrying out incubation, magnetic separation and cleaning treatment on the diluent to prepare the double-antibody sandwich immunomagnetic bead, wherein the PcAb_DNMT1The diluted solution is used for diluting PcAb with BSA solution with the mass fraction of 0.5%_DNMT1And (4) preparing.

Based on the above, the step of generating the biotinylated double-antibody sandwich immunomagnetic bead comprises: and (2) diluting the goat anti-rabbit IgG-Biotin with the BSA solution with the mass fraction of 0.5% according to the proportion of 1: 500-1: 1000 to prepare a goat anti-rabbit IgG-Biotin diluent, adding the goat anti-rabbit IgG-Biotin diluent into the double-antibody sandwich immunomagnetic bead, carrying out incubation treatment, and then carrying out magnetic separation and cleaning treatment to prepare the biotinylated double-antibody sandwich immunomagnetic bead.

Based on the above, the step of forming a fluorescent complex comprises: and adding streptavidin with the concentration of 0.5-8 mu g/mL into the generated biotinylated double-antibody sandwich immunomagnetic beads, performing magnetic separation and washing, then adding the Biotin-poly FAM probe with the concentration of 300-800 nmol/L, and performing magnetic separation and cleaning treatment again to obtain the fluorescent compound.

Based on the above, the steps of fluorescence detection and regression equation establishment include: dissolving the fluorescent compound in a hybridization buffer solution to form a fluorescent compound solution, and detecting the fluorescence intensity F of the fluorescent compound solution and the blank test fluorescence intensity F by adopting a fluorometer₀(ii) a At DNMT1 concentration C_DNMT1Establishing a linear regression equation for detecting DNMT1 in a concentration range of 2-200 nmol/L: y is 0.28814X +0.01782, wherein X is lgC_DNMT1Y is lg (F/F)₀) The correlation coefficient r is 0.9962.

Based on the above, the detection limit of the concentration of DNMT1 was 0.05 nmol/L.

The invention provides a method for detecting DNMT1 based on a self-assembly nucleic acid probe signal amplification method, which mainly utilizes a carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (sulfo-NHS) activation method to construct immunomagnetic beads, and McAb on the immunomagnetic beads_DNMT1Can specifically capture DNMT1 in a sample; sequentially forming double-antibody sandwich immunomagnetic beads and biotinylated double-antibody sandwich immunomagnetic beads; constructing a signal amplification probe biotinylated poly-fluorescein (Biotin-poly-FAM) by utilizing a nucleic acid self-assembly technology; the Biotin-poly FAM and the biotinylated double-antibody sandwich immunomagnetic beads can be coupled together to form a fluorescent complex through a bridging medium, namely streptavidin; the method for detecting the DNMT1 level is established by utilizing the characteristic that the fluorescent compound can emit fluorescence and the linear relation between the fluorescence intensity and the concentration of DNMT1, and has the characteristics of simple operation, good accuracy, high sensitivity, suitability for detecting the DNMT1 level in a biological sample and the like. Therefore, the method for detecting DNMT1 based on the self-assembly nucleic acid probe signal amplification method provided by the invention mainly utilizes magnetic nanoparticles as solid phase carriers, and simultaneously establishes a new method for detecting the DNMT1 level by combining the nucleic acid self-assembly signal amplification method, the method has the characteristics of low detection limit and strong specificity, and the method can be applied to the detection of the DNMT1 level in actual sample plasma, so as to provide a new idea for the early diagnosis of tumors.

Drawings

FIG. 1 is a schematic diagram of the principle of detection of DNMT1 based on the self-assembly nucleic acid probe signal amplification method, wherein (A) the principle of construction of immunomagnetic beads, (B) the principle of formation of signal amplification probe Biotin-poly FAM, and (C) the principle of detection of DNMT1 level by the self-assembly nucleic acid probe signal amplification technology are shown in the figure.

FIG. 2 is an agarose gel electrophoresis of Biotin-poly FAM constructed in accordance with the present invention.

FIG. 3 is a scanning electron micrograph of carboxyl magnetic beads used in an example of the present invention.

FIG. 4 is a scanning electron micrograph of immunomagnetic beads constructed according to an embodiment of the present invention.

FIG. 5 is a representation of ELISA using the immunomagnetic beads of FIG. 4 constructed according to an embodiment of the present invention.

FIG. 6 is a graph of fluorescence intensity versus dilution ratio for different goat anti-rabbit IgG-Bio provided in the examples of the present invention.

FIG. 7 is a graph of fluorescence intensity versus streptavidin concentration provided by an embodiment of the present invention.

FIG. 8 is a graph showing the relationship between the detection wavelength and the fluorescence intensity under different concentrations of Biotin-poly FAM according to the embodiment of the present invention.

FIG. 9 is a graph of fluorescence intensity versus DNMT1 concentration for different samples provided by an example of the present invention.

FIG. 10 is a standard graph of the concentration of detected DNMT1 constructed from the fluorescence intensity at different DNMT1 concentrations shown in FIG. 9.

FIG. 11 is a histogram of the specificity of the detection of DNMT1 concentration according to the standard curve shown in FIG. 10 provided by the present invention.

Detailed Description

The technical solution of the present invention is further described in detail by the following embodiments.

The instruments and equipment used in the examples of the present invention are shown in Table 1, the reagents and materials used are shown in Table 2, the nucleic acids used are all purchased from Shanghai Biotechnology engineering Co., Ltd, and the nucleic acid sequences are specifically shown in Table 3; the experimental waters were Milli-Q water (resistivity greater than 18.2M Ω cm).

TABLE 3 instrumentation used in embodiments of the invention

Name of instrument	Type and manufacturer
		Fluorescence spectrometer	FS5 Edinburgh instruments, UK
Electric heating constant temperature water tank	PK-8D, Shanghai sperm macro laboratory Equipment Co., Ltd
		PH meter	pH213, HANNA, Italy
Centrifugal machine	Centrifuge 5418, eppendorf, Germany
		Constant temperature oscillator	CHA-S, Changzhou Guohua electric appliances Co., Ltd
Magnetic separator	Wuxi Baimaige Biotech Co Ltd
		Ultraviolet spectrophotometer	UV-1601, Shimadzu Japan
Vortex mixing instrument	XH-C, Jintan City Danrui electric appliance factory
		Precision electronic balance	FA1204, King-Kong instruments & meters Ltd
Low-temperature refrigerator	BCD-215KS, Haier group of Qingdao Ltd
		Vertical ultra-low temperature storage box	DW-86L386, Qingdao Haier Special electric appliances Co., Ltd
Steam pot for high-pressure sterilization	HVE-50, HIRAYAMA, Japan
		Ultrapure water device	Millipore Inc. USA
Scanning electron microscope	Chuiss MERLIN Compact
		Electrophoresis apparatus	DYY-7C, six instruments factories in Beijing
Adjustable liquid-transfering gun	Thermo Scientific
		Ultra-micro ultraviolet spectrophotometer	Nano Drop 2000，Thermo Scientific
Microwave oven with a heat exchanger	P70D20TL-D4, Foshan mountainGuid zone grand Shi microwave oven appliances Co., Ltd
		Multifunctional microplate reader	SpectraMax M2e, Meigu molecular instruments Ltd
Gel imager	Bio-rad Co Ltd, USA

TABLE 2 reagents and materials used in the examples of the invention

TABLE 3 nucleic acid sequences employed in the examples of the invention

Various solutions used in the examples of the present invention

(1)0.01mol/L PBS (pH 7.4) buffer: dissolving a commercially available PBS powder bag in 1000 mM LLII-Q water, transferring into a 2L volumetric flask, washing with Milli-Q water for 3 times, transferring into the volumetric flask, diluting to 2L, mixing, labeling, and storing in a refrigerator at 4 deg.C for later use.

(2) Hybridization buffer: 2.4228g Tris, 5.8440g NaCl, 2.0330g MgCl were weighed out accurately₂·6H₂O was dissolved in 900mL of Milli-Q water, adjusted to pH 7.4 with 0.1mol/L HCl, made up to 1000mL and used after autoclaving.

(3)0.01mol/L MES: 1.0000g of morpholine ethanesulfonic acid monohydrate was accurately weighed, dissolved in autoclaved Milli-Q water, and adjusted to 500mL with KOH to pH 6.0, and stored in a refrigerator at 4 ℃ for use.

(4) PBST washing solution: 0.01mol/L PBS buffer contains 0.05% Tween-20.

(5)0.59mol/L sulfo-NHS solution: 0.0260g of sulfo-NHS was dissolved in 200. mu.L of MES buffer and used as it was.

(6)0.31mol/L EDC solution: 0.0120g EDC was weighed out and dissolved in 200. mu.L MES buffer for use.

(7) 0.5% BSA solution: 0.0500g BSA was weighed accurately and dissolved in 10mL 0.05mol/L CBS and stored in a refrigerator at 4 ℃ for 1 day.

The embodiment of the invention provides a method for detecting DNMT1 based on a self-assembly nucleic acid probe signal amplification method, and the corresponding principle of the method for detecting DNMT1 is shown in FIG. 1. As can be seen from fig. 1: according to the embodiment of the invention, carboxyl magnetic beads are used as a solid phase carrier, firstly EDC/sulfo-NHS activation method is used for activating the carboxyl magnetic beads on the surfaces of the magnetic beads to generate a semi-stable amino reaction active NHS ester intermediate, and then McAb is added_DNMT1，McAb_DNMT1to-NH of₂Reacting with NHS ester intermediate on the surface of the magnetic bead to generate amide compound, thereby reacting McAb_DNMT1Coupling with carboxyl magnetic beads; after magnetic separation and washing, the sites of the carboxyl magnetic beads which are not coupled with the antibody are blocked by 0.5% BSA solution to form immunomagnetic beads. McAb when DNMT1 is present in the sample to be tested_DNMT1DNMT1 that will specifically capture the sample, after magnetic separation and washing, PcAb was added_DNMT1Forming double-antibody sandwich immunomagnetic beads, washing unbound PcAb after magnetic separation_DNMT1(ii) a Adding goat anti-rabbit IgG-Bio, and magnetically separating to wash away unbound goat anti-rabbit IgG-Biotin; adding bridging medium streptavidin, adding a detection probe Biotin-poly FAM after magnetic separation and washing, connecting the Biotin-modified Biotin-poly FAM with double-antibody sandwich immunomagnetic beads captured with DNMT1 by the streptavidin, and removing the unbound detection probe Biotin-poly FAM by magnetic separation and washing; and (3) detecting the fluorescence intensity of the system on a fluorimeter after being dissolved in the hybridization buffer solution, and realizing the quantitative detection of the expression level of the DNMT1 according to the linear relation between the fluorescence intensity and the concentration of the DNMT 1.

Specifically, the method for detecting DNMT1 provided by the embodiment of the present invention includes the following steps:

preparing a signal amplification probe, mixing S1-Biotin and S2-FAM with equal volumes according to a molar ratio of 1:1, heating in a water bath at 95 ℃ for 5min, taking out and cooling to room temperature, carrying out vortex hybridization for 2h, and carrying out self-assembly on two nucleic acid chains at room temperature to form Biotin-poly FAM;

constructing immunomagnetic beads, taking 1.5mL of EP tube, and washing with PBST once; adding 100 μ L of 50mg/mL carboxylated magnetic beads, magnetically separating, discarding the supernatant, washing with 500 μ L MES buffer solution for 3 times, and resuspending in 100 μ L MES; respectively adding 100 mu L of in-situ prepared 0.59mol/L sulfo-NHS solution and 0.01mol/L EDC solution, oscillating and reacting for 10min at room temperature, and magnetically separating and discarding the supernatant; to the activated beads, 8. mu.L of 10mg/mL mouse anti-human McAb was added_DNMT1And 595 mu L of PBS buffer solution, shaking and reacting for 2h at room temperature, and magnetically separating and discarding the supernatant; adding 500 mu L of 0.5% BSA solution into the magnetic beads, performing vortex reaction at room temperature for 30min, and performing magnetic separation to remove supernatant; washing the sealed immunomagnetic beads with PBS buffer solution for 3 times, redissolving the immunomagnetic beads in 500 mu L PBS buffer solution to obtain 10mg/mL immunomagnetic beads modified with DNMT1 mouse anti-human monoclonal antibody, and storing the immunomagnetic beads in a refrigerator at 4 ℃ for later use;

adding 50 mu L of 1mg/mL double-antibody sandwich immunomagnetic beads into a low-adsorption centrifuge tube which is washed for 1 time by PBST washing liquid, and magnetically separating and discarding the supernatant; then adding 20 mu L of DNMT1 solution with different concentrations, mixing uniformly, oscillating in a constant temperature oscillation box at 37 ℃ for 1h, magnetically separating, discarding supernatant, and washing with PBST washing solution for 3 times; then, 100. mu.L of PcAb was added dropwise_DNMT1Carrying out oscillation reaction for 1h in a constant-temperature oscillation box at 37 ℃ to form double-antibody sandwich immunomagnetic beads, and carrying out magnetic separation to obtain a centrifugal tube with the double-antibody sandwich immunomagnetic beads;

generating biotinylated double-antibody sandwich immunomagnetic beads, diluting the goat anti-rabbit IgG-Biotin with BSA solution with the mass fraction of 0.5% according to the proportion of 1: 500-1: 1000 to prepare goat anti-rabbit IgG-Biotin diluent, adding 100 mu L of goat anti-rabbit IgG-Biotin into a centrifugal tube with the double-antibody sandwich immunomagnetic beads, carrying out oscillation reaction in a 37 ℃ constant temperature oscillation box for 1h to form the biotinylated double-antibody sandwich immunomagnetic beads, carrying out magnetic separation, discarding supernatant, and washing PBST washing liquid for 3 times to obtain the centrifugal tube with the biotinylated double-antibody sandwich immunomagnetic beads;

adding 100 mu L of streptavidin with the concentration of 0.5-8 mu g/mL into a centrifuge tube with biotinylated double-antibody sandwich immunomagnetic beads, performing oscillation reaction in a constant temperature oscillation box at 37 ℃ for 0.5h, adding 30 mu L of Biotin-poly FAM with the concentration of 300-800 nmol/L, performing oscillation reaction in the constant temperature oscillation box at 37 ℃ for 0.5h, performing magnetic separation, discarding supernatant, and washing with PBST for 3 times to obtain a fluorescent compound;

performing fluorescence detection and establishing a regression equation, and re-dissolving the fluorescent compound by adopting 200 mu L of hybridization buffer solution to form a fluorescent compound solution; and then, measuring the fluorescence intensity of the fluorescent compound solution under the excitation wavelength of 475nm by using a fluorometer, and establishing a regression equation for detecting the concentration of DNMT1 according to the linear relation between the fluorescence intensity and the concentration of DNMT1 to realize the quantitative detection of the expression level of DNMT 1.

Therefore, the method for detecting DNMT1 by the self-assembly nucleic acid probe signal amplification method mainly relates to the aspects of a nucleic acid self-assembly signal amplification method, immunomagnetic beads, bridging medium streptavidin, a fluorescence detection method and the like. The present invention will be further explained with respect to the effects of these aspects on the detection results.

1. Effect of Biotin-Poly FAM

Because the hybridization cycle times of the two nucleic acid single strands S1-Biotin and S2-FAM in the solution are different, the quantity of FAM in the formed double-strand Biotin-poly FAM is also different, and the more FAM on the Biotin-poly FAM, the stronger the signal amplification effect. Therefore, the step "preparation of signal amplification probe" in the method for detecting DNMT1 provided in the present invention was used to prepare the probe Biotin-polyfam, and 2% agarose gel electrophoresis characterization was performed to determine the DNA double helix structure of the self-assembled Biotin-polyfam. The agarose gel electrophoresis characterization result of the probe Biotin-poly FAM is shown in FIG. 2. Among them, lane 1 in FIG. 2 is 5. mu. mol/L S1-Biotin, lane 2 is 5. mu. mol/L S2-FAM; lane 3 is 5. mu. mol/L Biotin-poly FAM.

As can be seen from fig. 2: the hybridization of S1-Biotin and S2-FAM forms a DNA double-chain structure with a plurality of FAMs and different lengths, the longest length can reach 500bp, which indicates that each DNA double-chain in the Biotin-poly FAM formed by self-assembly contains a plurality of FAMs, and can preliminarily indicate that the Biotin-poly FAM can realize the signal amplification effect.

2. Influence of immunomagnetic beads

Scanning electron micrographs of hydroxylated magnetic beads and prepared immunomagnetic beads adopted by the step "construction of immunomagnetic beads" in the method for detecting DNMT1 provided by the embodiment of the invention are respectively shown in FIG. 3 and FIG. 4. Comparing fig. 3 and fig. 4, it can be seen that: McAb is present on the surface of the antibody-modified magnetic bead_DNMT1The protein is rough, which preliminarily shows that the antibody is modified on the surface of the carboxyl magnetic bead, and the construction of the immunomagnetic bead is successful.

The immunocompetence of the immunomagnetic beads has an important influence on the detection result of the method for detecting DNMT1 provided by the embodiment of the invention. The immunological activity test of the immunomagnetic beads comprises the following steps:

1) adding 300 μ L of 0.5% BSA solution into each well of an enzyme label plate, incubating at 37 ℃ for 60min, and washing with PBST washing solution for 3 times after magnetic separation;

2) taking the constructed immunomagnetic beads modified with DNMT1 mouse anti-human monoclonal antibody, diluting to 1mg/mL by PBS buffer solution, adding the immunomagnetic beads into the closed enzyme label plate respectively according to the dosage of 25, 50 and 75 mu L per hole, setting 3 multiple holes per dosage, and magnetically separating and discarding the supernatant;

3) diluting DNMT1 to different concentrations of 0, 25, 50 and 100ng/mL with PBS buffer solution, adding 50 μ L of DNMT1 to each well, 3 each concentration in parallel, incubating at 37 deg.C for 60min, magnetically separating and discarding supernatant, and washing with PBST for 3 times, 1min each time;

4) PcAb was incubated with 0.5% BSA solution_DNMT1Diluting by 2000 times, adding 100 mu L of the solution into an ELISA plate per well, incubating for 60min at 37 ℃ to form double-antibody sandwich immunomagnetic beads, magnetically separating and discarding supernatant, and washing with PBST washing solution for 3 times, 1min each time to obtain the ELISA plate with the double-antibody sandwich immunomagnetic beads;

5) diluting goat anti-rabbit IgG-HRP 8000 times by 0.5% BSA solution, adding 100 μ L of goat anti-rabbit IgG-HRP into an ELISA plate with the double-antibody sandwich immunomagnetic beads formed, incubating at 37 ℃ for 60min to form biotinylated double-antibody sandwich immunomagnetic beads, magnetically separating and discarding supernatant, washing PBST for 8 times, and washing for 1min each time to obtain the ELISA plate with the biotinylated double-antibody sandwich immunomagnetic beads;

6) adding 100 mu L of TMB single-component developing solution into each hole of an ELISA plate with biotinylated double-antibody sandwich immunomagnetic beads, and incubating at 37 ℃ in a dark place for 20min to perform a developing reaction;

7) 50 μ L of 2mol/L H was added to each well₂SO₄The reaction is stopped to obtain the ELISA plate with the fluorescent compound;

8) placing the ELISA plate with the formed fluorescent compound in a fluorometer, and detecting the OD value of the fluorescence intensity of the fluorescent compound at 450 nm.

By using the principle of antigen-antibody specific binding, ELISA characterization of the immunological activity of the immunomagnetic beads prepared by the step of "constructing immunomagnetic beads" provided by the embodiment of the present invention is performed, and the characterization result is shown in fig. 5. The absorbance value at the same DNMT1 concentration is gradually increased along with the increase of the dosage of the immunomagnetic beads; under the same dosage of immunomagnetic beads, the absorbance value is gradually increased along with the increase of the concentration of the target DNMT1, which indicates that the McAb_DNMT1The coupling agent is successfully coupled to the surface of the carboxyl magnetic bead, and the constructed immunomagnetic bead retains the activity of an antibody, can be specifically combined with a target, and can be used for subsequent experiments.

3. Optimization of parametric conditions

The amounts of goat anti-rabbit IgG-Bio, streptavidin, and Biotin-poly FAM used significantly affect the detection results of the method for detecting DNMT1 by self-assembly nucleic acid probe signal amplification provided in the present invention, and the following experiments were performed to optimize the parameter conditions according to the method for detecting DNMT1 provided in the present invention.

3.1 goat anti-rabbit IgG-Biotin dilution ratio

Goat anti-rabbit IgG-Biotin linked PcAb_DNMT1And streptavidin, if the dosage is too much, on one hand, the next step of cleaning is difficult, non-specific adsorption is caused, and false positive is generated; on the other hand, the consumption of the reagent is too low, which results in a low detected signal. Therefore, in the same conditions, 6 dilution ratios 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000 were set to perform the one-way optimization experiment on goat anti-rabbit IgG-Bio, and the experimental results are shown in FIG. 6.

As can be seen in fig. 6: with the reduction of the dilution ratio of the goat anti-rabbit IgG-Biotin, the fluorescence intensity tends to increase first and then decrease. When the dilution ratio is 1:500, the fluorescence intensity is lower than 1:600, mainly because when the dilution ratio is larger, the antibody concentration is larger, the antibody protein molecules are adhered to each other, and the combination of biotin and streptavidin can be shielded, so that the fluorescence signal value is lower; when the dilution ratio is large, the antibody concentration in the solution is too low, which affects the accuracy and sensitivity of the detection method provided by the embodiment of the invention. Therefore, the optimal dilution ratio of goat anti-rabbit IgG-Biotin is 1: 600.

3.2 streptavidin concentration

The streptavidin is used as a bridging medium to connect an antigen-antibody reaction system and a detection signal probe, the dosage of the streptavidin has great influence on an experimental result, and the detection signal strength of the experiment is directly related. Therefore, under the same conditions, 6 concentrations of 0.5, 1, 2, 4, 6, 8. mu.g/ml were set for single factor optimization experiments on streptavidin concentrations, and the results are shown in FIG. 7.

As seen in fig. 7: the fluorescence intensity gradually increases with the increase of the streptavidin concentration, and therefore, the streptavidin concentration of 8 μmol/L corresponding to the maximum fluorescence intensity in the experimental range is selected as the optimal concentration of streptavidin in the method provided by the embodiment of the invention.

3.3 Biotin-Poly FAM concentration optimization

The Biotin-poly FAM is a signal amplification substance, all fluorescence signals in the method provided by the embodiment of the invention are derived from the Biotin-poly FAM, and can directly reflect the real content of a substance DNMT1 to be detected in the method, so that under the same other conditions, 6 concentration gradients 300, 400, 500, 600, 700 and 800nmol/L are set to perform a single-factor optimization experiment on the Biotin-poly FAM, and the experiment result is shown in FIG. 8.

As can be seen in fig. 8: as the concentration of Biotin-poly FAM increases, the fluorescence intensity tends to decrease after increasing, and reaches the maximum at 700nmol/L, so the optimal concentration of Biotin-poly FAM is 700 nmol/L.

4. Evaluation of methodology

4.1 creation of Standard Curve

According to the method for detecting DNMT1 provided by the embodiment of the invention, under optimized conditions, the fluorescence intensities of different DNMT1 concentrations of 2, 4, 8, 16, 32, 64, 128 and 200nmol/L are measured, the fluorescence intensity detection results are shown in FIG. 9, and linear fitting is performed according to the experiment results, so that a standard curve for detecting the DNMT1 level shown in FIG. 10 is obtained.

As can be seen in fig. 10: the concentration of DNMT1 is in the range of 2-200 nmol/L, lg (F/F)₀) Has better linear relation with lgC, and the corresponding standard curve is that Y is 0.28814X +0.01782(X is lgC)_DNMT1Y is lg (F/F)₀))，r＝0.9962。

4.2 detection Limit

According to the method for detecting DNMT1 provided by the embodiment of the invention, under the optimized conditions, the fluorescence intensities of 4 solutions with different concentrations which are lower than the lower limit of the linear range of the DNMT1 solution are measured, the results are shown in Table 4, the fluorescence intensities of 4 blank solutions are simultaneously measured, and the average value of the fluorescence intensities of the blank solutions is calculatedThe standard deviation SD is 156 and,

table 4 test results of low concentration DNMT1 (n ═ 3)

As can be seen from table 4: when the concentration of DNMT1 is 0.05nmol/L, the corresponding fluorescence intensity value is greater thanTherefore, the detection limit of the DNMT1 level detection method established in the embodiment of the invention is 0.05 nmol/L.

4.3 precision and recovery from spiked samples

According to the method for detecting DNMT1 provided by the embodiment of the invention, the fluorescence intensity of the solution at the high, medium and low concentrations of 150, 25 and 5nmol/L is measured respectively, and then the SD value and RSD, namely the precision of the established method, are obtained. The recovery of the spiked sample was determined using the following equation. The results of experimental precision and recovery from spiking are shown in Table 5.

Table 5 precision and recovery of DNMT1 level (n ═ 3)

As can be seen from Table 5: the precision range of the standard curve established by the detection method provided by the embodiment of the invention is 4.89-16.27%, and the precision of the method is better at low concentration and high concentration; the recovery rates of the low, medium and high concentration are 140.0%, 94.0% and 101.3% respectively.

4.4 specificity

The components of the actual sample are complex, a plurality of proteins with similar structures to those of a substance to be detected DNMT1 exist, AluI, DNMT3a and DNMT3b are DNA methyltransferases with similar functions to DNMT1, the structures of the DNA methyltransferases are very similar to those of DNMT1, and therefore the established method is required to have very strong specificity. Therefore, in the examples of the present invention, AluI, DNMT3a, and DNMT3b were selected as detection targets in the specific experiments. The method for detecting DNMT1 established in the embodiment of the invention is adopted to detect the levels of 250U/ml, 500U/ml AluI methyltransferase, 40nmol/L DNMT1, 40nmol/L DNMT3a and 40nmol/L DNMT3b methyltransferase respectively, the fluorescence signal values of the detection solutions are compared, the specificity of the DNMT1 level is detected, and the experimental result is shown in FIG. 11.

As can be seen in fig. 11: with the increase of the AluI concentration, the fluorescence intensity is not changed basically, and the fluorescence signals of DNMT3a and DNMT3b with the same concentration as a target substance are lower; the fluorescence intensity value is obviously increased when the detection target substance is DNMT1, so that the selectivity of the method for detecting DNMT1 provided by the embodiment of the invention on AluI methyltransferase, DNMT3a and DNMT3b is low, and the effect of the method can be approximately ignored compared with DNMT1, so that the method for detecting the DNMT1 level established by the embodiment of the invention has stronger specificity.

5. Sample analysis

To further demonstrate the potential of the above-described method for detecting DNMT1 provided in the examples of the present invention in practical applications, the method was used for the detection of DNMT1 levels in human plasma. 20 μ L of human plasma samples (from first subsidiary Hospital of Zhengzhou university) containing DNMT1 at various concentrations were prepared, and then the human plasma samples were tested according to the above-described method for detecting DNMT1 provided in the examples of the present invention, and the recovery rate was calculated by comparing the determined DNMT1 content with the added amount of DNMT1, and the results are shown in Table 6.

TABLE 6 measurement of DNMT1 levels in plasma samples

Amount added (nmol/L)	Practical measurement (nmol/L)	Recovery (%)
			3	3.18±0.08	106.0
13	13.17±0.09	101.3
			90	91.41±3.68	101.6

From table 6, it can be seen that: the recovery rates of DNMT1 in the low, medium and high concentration plasma solutions were 106.0%, 101.3% and 101.6%, respectively, thus confirming the utility of the above detection method DNMT1 established in the examples of the present invention.

Therefore, the method for detecting DNMT1 based on the self-assembly nucleic acid probe signal amplification method provided by the embodiment of the invention mainly utilizes magnetic nanoparticles as solid phase carriers, and simultaneously establishes a new method for detecting the DNMT1 level by combining the nucleic acid self-assembly signal amplification method, the method has the characteristics of low detection limit and strong specificity, and the method is successfully applied to the detection of the DNMT1 level in the plasma of an actual sample, and is expected to provide a new index for clinical diagnosis and treatment of cancer.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.