CN112326637A

CN112326637A - Chemiluminescence biosensor for detecting 5-hydroxymethylcytosine and detection method and application thereof

Info

Publication number: CN112326637A
Application number: CN202011195224.4A
Authority: CN
Inventors: 张春阳; 胡娟; 申大忠; 李琛琛; 董月红
Original assignee: Shandong Normal University
Current assignee: Shandong Normal University
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2021-02-05
Anticipated expiration: 2040-10-30
Also published as: CN112326637B

Abstract

The invention provides a chemiluminescence biosensor for detecting 5-hydroxymethylcytosine and a detection method and application thereof, belonging to the technical field of molecular detection. The chemiluminescent biosensor for 5-hydroxymethylcytosine comprises T4 bacteriophage beta-glucosyltransferase, uridine diphosphate glucose, sodium periodate, aldehyde reaction probe ARP, terminal transferase, hemin and luminol solution. The hydroxymethyl cytosine-glycosylation, periodate oxidation and biotinylation-isothermal signal amplification strategies designed by the invention can detect 5hmC of any sequence in genome DNA without changing reaction temperature or special labeled nucleic acid probes or specific templates for signal amplification, do not relate to isotope labeling or specific antibodies, and eliminate radioactive hazards and erroneous interpretation of whole genome map data in antibody-based experiments. Making genome-wide analysis of 5hmC possible in a limited number of biological and clinical samples.

Description

Chemiluminescence biosensor for detecting 5-hydroxymethylcytosine and detection method and application thereof

Technical Field

The invention belongs to the technical field of molecular detection, and particularly relates to a chemiluminescent biosensor for detecting 5-hydroxymethylcytosine, and a detection method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Epigenetic modifications refer to heritable changes in gene expression that occur without altering the DNA sequence. 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major epigenetic modifications found in the mammalian genome. 5hmC is an oxidation intermediate in a 10-11 translocase (TET) mediated oxidation process of 5 mC. 5hmC is involved in many key cellular functions including gene regulation, embryonic development, embryonic stem cell differentiation, normal myelogenesis and zygote development. Aberrant DNA hydroxymethylation is associated with various diseases and is a predictive biomarker for various cancers, neurological abnormalities and risk disorders. Therefore, accurate detection of 5hmC in genomic DNA is crucial for epigenetic regulation of disease occurrence and related studies for early diagnosis of cancer.

Currently, the commonly used methods for identifying 5hmC are bisulfite oxidation sequencing and TET-assisted bisulfite sequencing. However, the inventors have found that these bisulfite treatment based methods result in significant sequencing bias under acidic and thermal conditions due to DNA degradation under selective and specific circumstances. In addition, there are also several new methods for genome-wide detection of 5hmC, such as liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography and immunoassay. These methods can effectively distinguish between 5hmC and 5mC, but they involve expensive and complex instrumentation, hazardous radioactive substrates and specific antibodies (such as anti-5 hmC antibodies and anti-cytosine-5-methylenesulfonate antibodies), where the sensitivity of the immunoassay is relatively poor. To improve detection sensitivity, some techniques introduce amplification strategies including boronic acid-mediated Polymerase Chain Reaction (PCR), peroxytungstate oxidation-mediated ligation PCR, ligase-mediated rolling circle amplification. They inevitably give rise to non-specific amplification and even false positives and require careful design of primers, labelled nucleic acid probes and specific templates. Furthermore, these methods only allow detection of 5hmC at specific locations and at specific fragments. Therefore, it is very necessary to develop a new 5hmC detection strategy.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a chemiluminescence biosensor for detecting 5-hydroxymethylcytosine and a detection method and application thereof. The hydroxymethyl cytosine-glycosylation, periodate oxidation and biotinylation-isothermal signal amplification strategy (hmC-GLIB-IAS) designed by the invention does not need to change the reaction temperature, does not need a specially-labeled nucleic acid probe or a specific template for signal amplification, can detect 5hmC of any sequence in genome DNA, does not relate to isotope labeling or specific antibodies, eliminates radioactive hazards and misinterpretation of whole genome map data in antibody-based experiments. Furthermore, this strategy is free of hydrogen sulfate involvement, it employs a combination of enzymatic and chemical reactions, without any DNA degradation, and is capable of isolating only one 5hmC DNA from 5mC and C, making possible genome-wide analysis of 5hmC in a limited number of biological and clinical samples. Therefore, it has good practical application value.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided a chemiluminescent biosensor for detecting 5-hydroxymethylcytosine, the chemiluminescent biosensor comprising at least:

t4 bacteriophage beta-glucosyltransferase (T4 beta-GT), uridine diphosphate glucose (UDP-Glc), sodium periodate, aldehyde reaction probe ARP (Aldehyde reactive probe), terminal transferase (TDT), hemin and luminol solution.

More specifically, the chemiluminescent biosensor comprises a glycosylation module, a periodate oxidation module, a biotinylation module, a TDT-assisted isothermal amplification module, and a chemiluminescent detection module.

In a second aspect of the invention, there is provided the use of a chemiluminescent biosensor as described above for the detection of 5-hydroxymethylcytosine;

the third aspect of the invention provides a chemiluminescence detection method of 5-hydroxymethylcytosine, the method comprises the steps of sequentially carrying out glycosylation, periodate oxidation and biotinylation treatment on a sample to be detected, adding streptavidin-coated Magnetic Beads (MB) for enrichment, carrying out magnetic separation, generating a large amount of long-chain G-rich sequences by the enriched biotinylated DNA under a terminal transferase (TDT) assisted isothermal amplification strategy, and then adding hemin to form a hemin-G quadruple nanostructure, wherein the structure catalyzes H to form a hemin-G quadruple nanostructure₂O₂The mediated luminol is oxidized to generate a chemiluminescent signal, thereby completing the detection.

Compared with the prior art, one or more technical schemes have the following beneficial technical effects:

1. the technical scheme does not need to use a specially-labeled nucleic acid probe or a special template for signal amplification, is ingenious in design and simple to operate, and provides a powerful platform for detecting 5hmC in an actual genome DNA sample.

2. The technical scheme does not use bisulfite, does not have any obvious DNA degradation, and improves the detection sensitivity.

3. The technical scheme is established on the basis of selective chemoenzymatic reaction, and can well distinguish 5hmC, 5mC and 5C.

4. The technical scheme does not relate to isotope labeling or specific antibodies, eliminates radioactive hazards and wrong interpretation of whole genome map data in antibody-based experiments, effectively reduces cost, and has high repeatability and accuracy.

5. According to the technical scheme, PCR is not needed, the reaction temperature is not needed to be changed, and the whole genome analysis of 5hmC at a constant reaction temperature is realized. Therefore, the method has great potential in biomedical research, disease diagnosis and drug discovery and has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a schematic diagram of a chemiluminescent detection method of the present invention.

FIG. 2A is a SYBR Gold stained PAGE analysis of TDT catalyzed amplification products in an example of the present invention; lane 1: 5hmC-DNA and TDT amplification product; lane 2: 5hmC-DNA, without TDT; FIG. 2B shows the measurement of chemiluminescence intensity in the absence of any one of the reagents (columns 1-6) and GLIB-IAS with 5hmC-DNA priming (column 7) in an example of the invention.

FIG. 3A is a graph showing the linear relationship between the chemiluminescence intensity and the logarithm of the concentration of 5hmC-DNA in the range of 238fM-23.8nM in the example of the present invention; FIG. 3B shows the change in chemiluminescence intensity when only reaction buffer (blank), C-DNA, 5mC-DNA and 5mC-DNA were used, respectively, in an example of the present invention; FIG. 3C is the correlation of the measured value with the actually input 5hmC level in a mixture of 5hmC-DNA and 5 mC-DNA.

FIG. 4A is a graph showing the chemiluminescence intensities of 5hmC in the genomic DNA of U-118MG cells, A549 cells, and HeLa cells quantified in the examples of the present invention; FIG. 4B is a linear relationship between chemiluminescence intensity and log U-118MG cell concentration in the range of 414 fg/. mu.L to 4.14 ng/. mu.L in the examples of the invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

As mentioned above, the existing detection for 5-hydroxymethylcytosine generally has the defects of complex operation, high cost, long time consumption, poor sensitivity and the like.

In view of the above, in one embodiment of the present invention, there is provided a chemiluminescent biosensor for detecting 5-hydroxymethylcytosine, the chemiluminescent biosensor comprising at least:

t4 bacteriophage beta-glucosyltransferase (T4 beta-GT), sodium periodate, aldehyde reaction probe ARP (Aldehydereactive probe), terminal transferase (TDT), hemin and luminol solution.

Wherein the glycosylation module comprises T4 phage beta-glucosyltransferase, NEBuffer 4, UDP-Glc;

the periodate oxidation module comprises sodium periodate, sodium phosphate and sodium sulfite;

the biotinylation module comprises an ARP;

the TDT auxiliary isothermal amplification module comprises TDT, dGTP and dATP;

the chemiluminescence detection module comprises hemin, luminol solution and hydrogen peroxide.

In yet another embodiment of the present invention, there is provided a use of the above chemiluminescent biosensor for detecting 5-hydroxymethylcytosine;

in another embodiment of the present invention, a chemiluminescent detection method for 5-hydroxymethylcytosine is provided, the method comprises the steps of sequentially performing glycosylation, periodate oxidation and biotinylation treatment on a sample to be detected, adding streptavidin-coated Magnetic Beads (MB) for enrichment, performing magnetic separation, generating a large amount of long-chain G-rich sequences from the enriched biotinylated DNA under a terminal transferase (TDT) assisted isothermal amplification strategy, and adding hemin to form hemin-G tetrabinnerRice structure catalyzing H₂O₂The mediated luminol is oxidized to generate a chemiluminescent signal, thereby completing the detection.

In another embodiment of the present invention, the glycosylation process is specifically: carrying out incubation reaction on a sample to be detected, T4 beta-GT and UDP-Glc in NEBuffer 4;

in another embodiment of the present invention, the incubation reaction conditions are as follows: reacting at 35-40 deg.C for 1-3h, preferably at 37 deg.C for 2 h; further, removing unreacted nucleotides and ions by using a QIAquick nucleotide excision purification column;

in another embodiment of the present invention, the periodate oxidation treatment is specifically: reacting the glycosylated sample in a sodium periodate and sodium phosphate system, and then reacting with sodium sulfite;

in another embodiment of the present invention, the reaction conditions in the sodium periodate and sodium phosphate system are as follows: reacting at 20-25 ℃ for 10-20h, preferably at 22 ℃ for 16 h; the reaction with sodium sulfite has the specific conditions that: reacting at room temperature for 5-20 min, preferably 10 min; further, a QIAquick nucleotide excision purification column is used for removing salt ions;

in another embodiment of the present invention, the biotinylation treatment is specifically: mixing and incubating the sample after periodate oxidation treatment with ARP; preferably, the specific conditions of the mixed incubation reaction are as follows: reacting at 35-40 deg.C for 0.5-2h, preferably at 37 deg.C for 1 h; further, removing redundant ARP by using a QIAquick nucleotide removal purification column;

in another embodiment of the present invention, the specific method of adding streptavidin-coated Magnetic Beads (MB) for enrichment and magnetic separation comprises: mixing the biotinylation processed sample with a streptavidin-coated Magnetic Bead (MBs) solution, uniformly mixing at room temperature, and incubating for 10-20 minutes, preferably 15 minutes, in a dark place; then carrying out magnetic separation; obtaining biotinylated 5 hmC-DNA;

in another embodiment of the present invention, the specific method for TDT-mediated isothermal amplification comprises: mixing the biotinylated 5hmC-DNA, TDT, dATP and dGTP for amplification reaction;

in another embodiment of the present invention, the specific conditions of the amplification reaction are: reacting at 35-40 deg.C for 0.5-2h, preferably at 37 deg.C for 1 h;

in another embodiment of the present invention, the chemiluminescence detection method comprises: incubating the mixture of luminol solution, hemin solution and the amplification reaction product to fold the G-rich polymer product into a G-quadruplex structure; then adding H to the mixture₂O₂Measuring the chemiluminescent signal;

in another embodiment of the present invention, the incubation reaction is performed under the following conditions: incubation is carried out at room temperature for 10-60 minutes, preferably 30 minutes.

In summary, the present invention provides a label-free and template-free chemiluminescent biosensor, and a method for sensitive detection of 5hmC in genomic DNA samples based on glycosylation, periodate oxidation, biotinylation, and terminal transferase (TDT) assisted isothermal amplification strategies. When 5hmC is present, T4 phage β -glucosyltransferase (T4 β -GT) transfers the glucose group in uridine diphosphate glucose (UDP-Glc) to the hydroxymethyl group of 5hmC for glycosylation. In contrast, 5mC and C cannot react with T4 β -GT because they have no hydroxymethyl group, and only 5hmC of genomic DNA can be covalently modified by glucosyl group to form 5ghmC, which can effectively distinguish 5hmC, 5mC and C. Subsequently, 5ghmC was oxidized with sodium periodate to convert the ortho-hydroxyl group to an aldehyde. 5ghmC was further modified by ARP to allow biotin (biotin) to be added to 5hmC, forming biotin-5 ghmC. After magnetic separation, biotinylated DNA was enriched and then incubated with 60% dGTP + 40% dATP + TDT to generate large amounts of long-chain G-rich sequences. In the presence of hemin (hemin), the G-rich product can combine with the cofactor hemin to form a hemin-G tetrad nanostructure that catalyzes H₂O₂Mediated luminol oxidation, produces a significant chemiluminescent signal. Notably, this approach is based on selective chemoenzymatic reactions, does not involve isotopic labeling or specific antibodies, eliminates potential radioactive hazards and misinterpretation of whole genome map data in antibody-based assays. This strategy is that of being PCR-free,no change in reaction temperature is required, nor is a specially labeled nucleic acid probe or a specific template for signal amplification required. Furthermore, this strategy is free of hydrogen sulfate involvement, it employs a combination of enzymatic and chemical reactions, without any DNA degradation, and is capable of isolating only one 5hmC DNA from 5mC and C, making possible genome-wide analysis of 5hmC in a limited number of biological and clinical samples.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

Preparation of biotinylated 5hmC-DNA product: the preparation of biotinylated 5hmC-DNA product involves three sequential steps, including glucosylation, high iodate oxidation and biotinylation. The glycosylation reaction was carried out in 30. mu.l of reaction solution containing double stranded DNA (dsDNAs), NEBuffer 4(50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT, pH 7.9), 2mM UDP-Glc and 4U T4. beta. -GT in a 37 ℃ water bath for 2h, followed by removal of unreacted nucleotides and ions using a QIAquick nucleotide excision purification column. The purified DNA was subjected to periodate oxidation in a 50. mu.l reaction system containing 20 mmol of sodium periodate and 100 mmol of sodium phosphate (pH 7.0) at 22 ℃ for 16 hours, followed by reaction with 40 mmol of sodium sulfite at room temperature for 10 minutes, and then salt ions were removed with a QIAquick nucleotide excision purification column. The biotinylation reaction was performed by placing the DNA in 30. mu.l of a solution containing 2mM ARP, incubating at 37 ℃ for 1h, and then removing excess ARP using a QIAquick nucleotide removal purification column.

And (3) enriching biotinylated 5hmC-DNA by streptavidin-coated magnetic beads: the synthesized biotinylated 5hmC-DNA product was mixed with 40 μ l of 5 μ g per μ l of streptavidin-coated Magnetic Bead (MBs) solution and incubated on a rotary homogenizer for 15 minutes at room temperature in the absence of light. Magnetic separation was then performed and the mixture was washed 3 times with 1x binding and washing buffer (5 mmol/l Tris-HCl (pH 7.5), 0.5 mmol/l EDTA, 1 mol/l NaCl) and the biotinylated 5hmC-DNA with magnetic beads conjugate (5hmC-DNA-MB) was resuspended in ultrapure water.

TDT-mediated isothermal amplification: the biotinylated 5hmC-DNA was reacted in 30. mu.l reaction solution containing 8U of TDT, 0.6. mu.l of dATP (10 mmol), 0.9. mu.l of dGTP (10 mmol), and 3. mu.l of 10 XTDT reaction buffer for 1h at 37 ℃ for amplification reaction.

And (3) chemiluminescence detection: a total of 30. mu.L of the newly configured luminol solution (0.1 mmole) and hemin solution (2. mu. mole) and reaction product mixture was incubated in 70. mu.L of reaction solution at room temperature for 30 minutes with 20. mu.L of HEPES buffer solution (40 mmole HEPES, 20 mmole potassium chloride, pH 8.0) to fold the G-rich polymeric product into a G-quadruplex structure. 80 microliters of H was added to the mixture₂O₂After (100 mmol) the chemiluminescent signal was measured using a GloMax 96 microplate luminometer with a time interval of 0.4 s.

Cell culture and extraction of genomic DNA: different cell lines including U-118MG cells (human glioblastoma cells), A549 cells (human lung adenocarcinoma cells) and HeLa cells (human cervical carcinoma cells) were placed in Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin, respectively, at 37 deg.C, 5% CO₂Is cultured in a humid atmosphere. Genomic DNA was extracted using QIAamp DNA mini kit according to the instructions. The concentration of genomic DNA was determined using a NanoDrop 2000 spectrophotometer. The genomic DNA was cut to 50-200bp using dsDNA fragment enzyme for 5hmC detection in whole genomic DNA.

The experimental principle of the method is shown in fig. 1. A5 hmC-DNA sequence (TCG ATG TAG TGC GTC AC) was designed^hmC GGA TGA TAG CTG TAG TCA) and a5 hydroxymethylcytosine modified at the 18 th base at the 5' end: (^hmC, fig. 1), T4 β -GT enzyme is capable of glycosylating 5hmC to 5ghmC (g hmC)^ghmC, figure 1), then treated by sodium sulfite to open the ring, and added with an ARP aldehyde reaction probe to introduce biotin (biotin-5ghmC, figure 1) to the 5hmC, and the 5hmC is separated by magnetic separation through the combination of streptavidin of magnetic beads and biotin, thus achieving the purpose of distinguishing 5hmC, 5mC and 5C and enriching 5 hmC. The TDT enzyme may be cleaved from the 3' end of the 5hmC-DNAInitial amplification to generate a large number of long-chain G-rich sequences (FIG. 1), and after adding hemin, the G-rich product can combine with cofactor hemin to form hemin-G quadruplex nanostructure which can catalyze H₂O₂Mediated luminol oxidation, produces a significant chemiluminescent signal.

1. Feasibility test

Gel electrophoresis experiments and chemiluminescence signal measurements were performed to investigate the feasibility of the proposed method for detecting 5hmC (fig. 2A). Amplification products were analyzed using native polyacrylamide gel electrophoresis (PAGE) and following the GLIB reaction, using 5hmC-DNA containing a 5hmC modification, biotinylated DNA was obtained to initiate TDT amplification, resulting in a long observed characteristic band and different molecular weights (fig. 2A, lane 1). In contrast, in the absence of TDT, only 36-bp dsDNA bands were detected, and no amplified band was observed (FIG. 2A, lane 2). The chemiluminescent signal was further measured under different experimental conditions (fig. 2B). GLIB-IAS induced by 5hmC-DNA resulted in a significant chemiluminescent signal being observed (FIG. 2B, column 7). In contrast, no significant chemiluminescent signal was observed due to the absence of one of the reactants (FIG. 2B, columns 1-6). These results indicate that only 5hmC-DNA can produce a significant chemiluminescent signal in the presence of all reagents.

2. Sensitive and specific detection

Under optimal experimental conditions, the chemiluminescence intensities of hydroxymethylated DNA at different concentrations were measured. FIG. 3A shows that the chemiluminescent signal increases significantly with increasing concentration of 5 hmC-DNA. Furthermore, the logarithm of the chemiluminescence intensity (I) versus the 5hmC-DNA concentration (C) shows a good linear relationship over a large dynamic range of 5 orders of magnitude from 238fM to 23.8 nM. The regression equation is that I is 9.04 multiplied by 10⁵+6.30×10⁴log₁₀C, the correlation coefficient is 0.991. The detection limit was calculated to be 2.07 x 10 by evaluating the average response of the blank area, plus three times the standard deviation^-13And M. Compared with a fluorescence method (0.167nM), a capillary electrophoresis fluorescence method (90pM), an electrochemical immunosensor method (0.032nM) and an electrochemiluminescence method (16.3pM), the sensitivity of 5hmC-GLIB-IAS is respectively improved by 806 times, 434 times, 154 times and 78.7 times. Further useThree oligonucleotide sequences containing 5hmC-, 5mC-, C (i.e., 5hmC-DNA, 5mC-DNA, and C-DNA) were used to assess the specificity of the strategy. The chemiluminescent intensity of 5mC-DNA C-DNA as shown in FIG. 3B remained at background level, and the 5hmC-DNA signal was 50.0 and 43.9 times higher than 5mC-DNA and C-DNA, respectively, indicating the high specificity and single base resolution of this approach to 5 hmC.

To investigate the feasibility of this method to accurately analyse the level of hydroxymethylation in a mixture, a series of artificial mixtures were prepared by mixing 5hmC-DNA and 5mC-DNA in different proportions. The measured 5hmC-DNA level (Y) is well linear with the input 5hmC-DNA level (X) (FIG. 3C). The regression equation is 0.0479+0.988X with a correlation coefficient of 0.993, which is effectively distinguishable even as low as 0.1% 5 hmC.

3. Detection of actual samples

In order to verify the feasibility of the strategy in 5hmC genome analysis, 3 cancer cell lines were measured, including 5hmC of human glioma cell line (U-118MG cell), human lung adenocarcinoma cell line (a549 cell) and human cervical cancer cell line (HeLa cell). According to previous researches, the content of 5hmC in different tissues is greatly different, the content of 5hmC in the brain is the highest, and the content of 5hmC in various human tumors is obviously reduced. In particular, there is no evidence of the presence of 5hmC in a549 and HeLa cells due to the limited sensitivity of the detection method. In this example, 300ng of genomic DNA extracted from the above cancer cells was analyzed using the hmC-GLIB-IAS method and the exact 5hmC content in different types of cells was successfully quantified (FIG. 4A). The 5hmC level of U-118MG cells was determined to be 0.179% of total nucleotides. Notably, the presence of 5hmC was successfully detected in a549 cells and HeLa cells, which were much less abundant than U-118MG cells (0.0240% for a549 cells and 0.0111% for HeLa cells).

Further, the relationship between the chemiluminescence intensity and the genomic DNA concentration was investigated using U-118MG cells as a model. As shown in FIG. 4B, the chemiluminescence intensity (I) increased with increasing genomic DNA concentration (C), and in the range of 414 fg/. mu.L-4.14 ng/. mu.L, the chemiluminescence intensity correlated linearly with the logarithm of the genomic DNA concentration. The regression equation is that I is 6.33 multiplied by 10⁵+1.04×10⁵log₁₀C(R²0.992). The detection limit was calculated to be 39.2 fg/. mu.L. The results show that the method has good accuracy in 5hmC whole genome analysis and wide application prospect.

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> university of Shandong Master

<120> chemiluminescence biosensor for detecting 5-hydroxymethylcytosine and detection method and application thereof

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 36

<212> DNA

<213> Artificial sequence

<400> 1

tcgatgtagt gcgtcachmcgg atgatagctg tagtca 36

Claims

1. A chemiluminescent biosensor for detecting 5-hydroxymethylcytosine comprising at least:

t4 bacteriophage beta-glucosyltransferase, uridine diphosphate glucose, sodium periodate, aldehyde reaction probe ARP, terminal transferase, hemin and luminol solution.

2. The chemiluminescent biosensor of claim 1 comprising a glycosylation module, a periodate oxidation module, a biotinylation module, a TDT-assisted isothermal amplification module, and a chemiluminescent detection module;

preferably, the glycosylation module comprises T4 phage β -glucosyltransferase, uridine diphosphate glucose, and NEBuffer 4;

preferably, the periodate oxidation module comprises sodium periodate, sodium phosphate and sodium sulfite;

preferably, the biotinylation module comprises ARP;

preferably, the TDT-assisted isothermal amplification module comprises TDT, dGTP, and dATP;

preferably, the chemiluminescence detection module comprises hemin, luminol solution and hydrogen peroxide.

3. Use of the chemiluminescent biosensor of any one of claims 1-2 for detecting 5-hydroxymethylcytosine.

4. A chemiluminescence detection method of 5-hydroxymethylcytosine is characterized by comprising the steps of sequentially carrying out glycosylation, periodate oxidation and biotinylation treatment on a sample to be detected, adding streptavidin-coated magnetic beads for enrichment, carrying out magnetic separation, generating a large number of long-chain G-rich sequences by enriched biotinylated DNA under a terminal transferase assisted isothermal amplification strategy, adding hemin to form a hemin-G quadruple nanostructure, and catalyzing H by the structure₂O₂Mediated luminol oxidation, resulting in a chemiluminescent signal.

5. The chemiluminescent detection method of claim 4 wherein the glycosylation process is specifically: carrying out incubation reaction on a sample to be detected, UDP-Glc and T4 beta-GT in NEBuffer 4;

preferably, the incubation reaction specific conditions are: reacting at 35-40 ℃ for 1-3h, and preferably at 37 ℃ for 2 h;

preferably, a QIAquick nucleotide excision clean-up column is used to remove unreacted nucleotides and ions.

6. The chemiluminescent detection method of claim 5 wherein the periodate oxidation treatment is specifically: reacting the glycosylated sample in a sodium periodate and sodium phosphate system, and then reacting with sodium sulfite;

preferably, the reaction conditions in the sodium periodate and sodium phosphate system are as follows: reacting for 10-20h at 20-25 ℃; further preferably reacting for 16h at 22 ℃; the reaction with sodium sulfite has the specific conditions that: reacting at room temperature for 5-20 min, preferably 10 min;

preferably, a QIAquick nucleotide excision clean-up column is used to remove salt ions.

7. The chemiluminescent detection method of claim 5 wherein the biotinylation treatment is specifically: mixing and incubating a sample subjected to periodate oxidation treatment with ARP; preferably, the specific conditions of the mixed incubation reaction are as follows: reacting at 35-40 deg.C for 0.5-2h, preferably at 37 deg.C for 1 h;

preferably, a QIAquick nucleotide removal purification column is used to remove excess ARP.

8. The chemiluminescent detection method of claim 5 wherein the specific method of adding streptavidin coated magnetic beads for enrichment and magnetic separation is: mixing the biotinylation processed sample with a streptavidin-coated magnetic bead solution, uniformly mixing at room temperature, and incubating for 10-20 minutes, preferably 15 minutes, in a dark place; then carrying out magnetic separation; biotinylated 5hmC-DNA was obtained.

9. The chemiluminescent detection method of claim 5 wherein the TDT-mediated isothermal amplification is performed by: mixing biotinylated 5hmC-DNA, TDT, dATP and dGTP for amplification reaction;

preferably, the specific conditions of the amplification reaction are as follows: the reaction is carried out for 0.5 to 2 hours at the temperature of between 35 and 40 ℃, and the reaction is preferably carried out for 1 hour at the temperature of between 37 ℃.

10. The chemiluminescence detection method according to claim 5, wherein the chemiluminescence detection method comprises: mixing luminol solution, hemin solution andincubating the mixture of the amplification reaction products to fold the G-rich polymer product into a G-quadruplex structure; then adding H to the mixture₂O₂Measuring the chemiluminescent signal;

preferably, the incubation reaction specific conditions are: incubation is carried out at room temperature for 10-60 minutes, preferably 30 minutes.