CN112326637B - Chemiluminescence biosensor for detecting 5-hydroxymethylcytosine and detection method and application thereof - Google Patents

Abstract

The invention provides a chemiluminescence biosensor for detecting 5-hydroxymethylcytosine, a detection method and application thereof, and belongs to the technical field of molecular detection. The chemiluminescent biosensor of 5-hydroxymethylcytosine includes T4 bacteriophage β-glucosyltransferase, uridine diphosphate glucose, sodium periodate, aldehyde group reaction probe ARP, terminal transferase, hemoglobin and luminol solution. The hydroxymethylcytosine-glycosylation, periodate oxidation, biotinylation-isothermal signal amplification strategy designed in the present invention does not need to change the reaction temperature, nor does it require a special labeled nucleic acid probe or a specific template for signal amplification , which can detect 5hmC of any sequence in genomic DNA and does not involve isotopic labels or specific antibodies, eliminating radioactive hazards and misinterpretation of genome-wide mapping data in antibody-based experiments. This enables genome-wide analysis of 5hmC in a limited number of biological and clinical samples.

Description

A chemiluminescent biosensor for detecting 5-hydroxymethylcytosine and its detection method and application

technical field

The invention belongs to the technical field of molecular detection, in particular to a chemiluminescence biosensor for detecting 5-hydroxymethylcytosine, a detection method and application thereof.

Background technique

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

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

The commonly used methods to identify 5hmC include oxidative bisulfite sequencing and TET-assisted bisulfite sequencing. However, the inventors found that these bisulfite treatment-based methods lead to significant sequencing biases under acidic and thermal conditions due to selective and environmental-specific DNA degradation. In addition, there are some 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 5hmC from 5mC, but they involve expensive and complicated instruments, harmful radioactive substrates, and specific antibodies (such as anti-5hmC antibodies and anti-cytosine-5-methylene sulfonate antibodies), where The sensitivity of immunoassays is relatively poor. In order to improve the detection sensitivity, some techniques have introduced a number of amplification strategies, including boronic acid-mediated polymerase chain reaction (PCR), peroxytungstate oxidation-mediated ligation PCR, and ligase-mediated rolling circle amplification . However, they inevitably have non-specific amplification and even false positives, and require carefully designed primers, labeled nucleic acid probes, and specific templates. Furthermore, these methods only allow detection of 5hmC at specific locations and fragments. Therefore, it is very necessary to develop new 5hmC detection strategies.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a chemiluminescence biosensor for detecting 5-hydroxymethylcytosine, and a detection method and application thereof. The hydroxymethylcytosine-glycosylation, periodate oxidation, biotinylation-isothermal signal amplification strategy (hmC-GLIB-IAS) designed in the present invention does not need to change the reaction temperature, nor does it require special labeled nucleic acid probes Or specific templates for signal amplification that can detect 5hmC of any sequence in genomic DNA and do not involve isotopic labels or specific antibodies, eliminating radioactive hazards and misinterpretation of genome-wide profiling data in antibody-based experiments. In addition, this strategy does not involve bisulfate, it uses a combination of enzymatic and chemical reactions without any DNA degradation, and is able to isolate DNA with only one 5hmC from 5mC and C, making it possible to isolate DNA with only one 5hmC in a limited number of biological and clinical samples. Genome-wide analysis of 5hmC becomes possible. Therefore, it has good practical application value.

For realizing the above-mentioned technical purpose, the technical scheme adopted in the present invention is as follows:

A first aspect of the present invention provides a chemiluminescence biosensor for detecting 5-hydroxymethylcytosine, the chemiluminescence biosensor at least comprising:

T4 phage β-glucosyltransferase (T4β-GT), uridine diphosphate glucose (UDP-Glc), sodium periodate, aldehyde reactive probe ARP (Aldehyde reactive probe), terminal transferase (TDT), Hemin and Luminol solution.

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

The second aspect of the present invention provides the application of the above chemiluminescence biosensor in detecting 5-hydroxymethylcytosine;

A third aspect of the present invention provides a chemiluminescence detection method for 5-hydroxymethylcytosine, the method comprising sequentially performing glycosylation, periodate oxidation, and biotinylation on a sample to be tested, and then adding Streptavidin-coated magnetic beads (MB) were used for enrichment. After magnetic separation, the enriched biotinylated DNA produced a large number of long-chain G-rich sequences under the terminal transferase (TDT)-assisted isothermal amplification strategy. Then hemin was added to form a hemin _- G four-chain nanostructure, which catalyzed the H2O2 _- mediated oxidation of luminol to generate a chemiluminescent signal, thereby completing the detection.

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

1. The above technical solution does not require the use of specially labeled nucleic acid probes or specific templates for signal amplification, and is ingenious in design and simple in operation, providing a powerful platform for the detection of 5hmC in actual genomic DNA samples.

2. The above technical solution does not use bisulfite, and there is no obvious DNA degradation, which improves the detection sensitivity.

3. The above technical scheme is based on the selective chemical enzymatic reaction, and can well distinguish 5hmC, 5mC and 5C.

4. The above technical solution does not involve isotope labeling or specific antibodies, eliminates radioactive hazards and erroneous interpretation of genome-wide map data in antibody-based experiments, effectively reduces costs, and has high reproducibility and accuracy.

5. The above technical solution does not require PCR and does not need to change the reaction temperature, and realizes the whole genome analysis of 5hmC at a constant reaction temperature. Therefore, it has great potential in biomedical research, disease diagnosis and drug discovery, and has good practical application value.

Description of drawings

The accompanying drawings forming a part of the present invention are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation of the present invention.

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

Figure 2A shows the analysis of TDT-catalyzed amplification products by SYBR Gold staining PAGE method in the embodiment of the present invention; Lane 1: 5hmC-DNA and TDT amplification products; Lane 2: 5hmC-DNA without TDT; Figure 2B shows the present invention Chemiluminescence intensities were measured in the examples in the absence of any of these reagents (columns 1-6) and GLIB-IAS with 5hmC-DNA initiation (column 7).

Fig. 3A shows a linear relationship between the chemiluminescence intensity and the logarithm of the concentration of 5hmC-DNA in the range of 238 fM-23.8 nM in the embodiment of the present invention; Fig. 3B shows that only the reaction buffer (blank), Changes in chemiluminescence intensity for C-DNA, 5mC-DNA and 5mC-DNA; Figure 3C shows the correlation of measured values with actual input 5hmC levels in a mixture of 5hmC-DNA and 5mC-DNA.

Figure 4A is the chemiluminescence intensity of 5hmC in the genomic DNA of U-118 MG cells, A549 cells and HeLa cells quantified in the embodiment of the present invention; Figure 4B is the relationship between the chemiluminescence intensity and the concentration of U-118 MG cells in the embodiment of the present invention The number is linear in the range of 414 fg/μL - 4.14 ng/μL.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, 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 should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, rather than for limiting the protection scope of the present invention.

As mentioned above, the existing detection of 5-hydroxymethylcytosine generally has disadvantages such as complicated operation, high cost, long time and poor sensitivity.

In view of this, in a specific embodiment of the present invention, a chemiluminescence biosensor for detecting 5-hydroxymethylcytosine is provided, and the chemiluminescence biosensor includes at least:

T4 phage β-glucosyltransferase (T4β-GT), sodium periodate, aldehyde-based reactive probe ARP (Aldehydereactive probe), terminal transferase (TDT), heme and luminol solution.

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

Wherein, the glycosylation module includes T4 phage β-glucosyltransferase, NEBuffer 4, UDP-Glc;

The periodate oxidation module includes sodium periodate, sodium phosphate and sodium sulfite;

the biotinylation module includes ARP;

The TDT-assisted isothermal amplification module includes TDT, dGTP and dATP;

The chemiluminescence detection module includes hemin, luminol solution and hydrogen peroxide.

In yet another specific embodiment of the present invention, the application of the above chemiluminescence biosensor in detecting 5-hydroxymethylcytosine is provided;

In yet another specific embodiment of the present invention, a chemiluminescence detection method for 5-hydroxymethylcytosine is provided, the method comprising sequentially performing glycosylation, periodate oxidation, and biotinylation on a sample to be tested. , adding streptavidin-coated magnetic beads (MB) for enrichment, and after magnetic separation, the enriched biotinylated DNA produces a large amount of long-chain G-rich DNA under terminal transferase (TDT)-assisted isothermal amplification strategy sequence, followed by the addition of hemin to form a hemin _- G four-chain nanostructure, which catalyzes H2O2 _- mediated oxidation of luminol to generate a chemiluminescent signal, thereby completing the detection.

In another specific embodiment of the present invention, the glycosylation treatment is specifically as follows: incubating the sample to be tested, T4β-GT and UDP-Glc in NEBuffer 4;

In another specific embodiment of the present invention, the specific conditions of the incubation reaction are: react at 35-40°C for 1-3 hours, preferably at 37°C for 2 hours; further, use a QIAquick nucleotide excision purification column to remove unreacted nucleotides and ions;

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

In yet another specific embodiment of the present invention, the specific conditions of the reaction in the sodium periodate and sodium phosphate system are: 20-25°C for 10-20 h, preferably 22°C for 16 h; and the specific conditions for the reaction with sodium sulfite are: React at room temperature for 5-20 minutes, preferably 10 minutes; further, use QIAquick nucleotide excision purification column to remove salt ions;

In another specific embodiment of the present invention, the biotinylation treatment is specifically: mixing and incubating the above-mentioned periodate-oxidized sample with ARP; preferably, the specific conditions of the mixed incubation reaction are: at 35-40 ℃ reaction 0.5-2h, preferably 37 ℃ reaction 1h; further, use QIAquick nucleotide removal purification column to remove excess ARP;

In another specific embodiment of the present invention, the specific method for adding streptavidin-coated magnetic beads (MB) for enrichment and magnetic separation is as follows: the biotinylated sample is mixed with streptavidin-coated magnetic beads (MB). The coated magnetic beads (MBs) solution was mixed and incubated at room temperature in the dark for 10-20 minutes, preferably 15 minutes; then magnetic separation was performed; biotinylated 5hmC-DNA was obtained;

In yet another specific embodiment of the present invention, the specific method of TDT-mediated isothermal amplification is: mixing the above-mentioned biotinylated 5hmC-DNA, TDT, dATP and dGTP for amplification reaction;

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

In another specific embodiment of the present invention, the specific method of chemiluminescence detection is as follows: incubating the mixture of the luminol solution, the hemin solution and the above-mentioned amplification reaction product, so that the G-rich polymer product is folded into a G-quadruplex structure; Then add H ₂ O ₂ to the mixture and measure the chemiluminescence signal;

In another specific embodiment of the present invention, the specific conditions of the above incubation reaction are: incubation at room temperature for 10-60 minutes, preferably 30 minutes.

In summary, the present invention proposes a label-free, template-free chemiluminescence biosensor, which is based on glycosylation, periodate oxidation, biotinylation and terminal transferase (TDT)-assisted isothermal amplification strategies for genomic DNA samples. A method for the sensitive detection of 5hmC in . In the presence of 5hmC, T4 phage β-glucosyltransferase (T4 β-GT) transfers the glucosyl group from uridine diphosphate glucose (UDP-Glc) to the hydroxymethyl group of 5hmC for glycosylation. On the contrary, 5mC and C cannot react with T4 β-GT due to the absence of hydroxymethyl groups. Only 5hmC in genomic DNA can be covalently modified by glucose groups to form 5ghmC, which can effectively distinguish 5hmC, 5mC and C. Subsequently, 5ghmC is oxidized by sodium periodate to convert the ortho-hydroxy to the aldehyde. 5ghmC is further modified by ARP, so that biotin (biotin) is added to 5hmC to form biotin-5ghmC. After magnetic separation, biotinylated DNA was enriched and then incubated with 60% dGTP + 40% dATP + TDT, resulting in a large number 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 four-chain nanostructure, which catalyzes H2O2 _- mediated oxidation of luminol, resulting in pronounced chemiluminescence Signal. Notably, this method is based on selective chemoenzymatic reactions and does not involve isotopic labels or specific antibodies, eliminating potential radiological hazards and misinterpretation of genome-wide profiling data in antibody-based assays. This strategy is PCR-free and does not require changes in reaction temperature, nor specifically labeled nucleic acid probes or specific templates for signal amplification. In addition, this strategy does not involve bisulfate, it uses a combination of enzymatic and chemical reactions without any DNA degradation, and is able to isolate DNA with only one 5hmC from 5mC and C, making it possible to isolate DNA with only one 5hmC from a limited number of biological and clinical samples. Genome-wide analysis of 5hmC becomes possible.

The present invention is further explained and illustrated by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only intended to illustrate the present invention and not to limit the scope of the present invention.

Example

Preparation of biotinylated 5hmC-DNA products: The preparation of biotinylated 5hmC-DNA products involved three sequential steps, including glucosylation, iodic acid oxidation, and biotinylation. Perform the glucosylation reaction in 30 μl of reaction solution containing double-stranded DNA (dsDNAs), NEBuffer 4 (50 mM potassium acetate, 20 mM Tris-acetic acid, 10 mM magnesium acetate, 1 mM DTT, pH 7.9), 2 mM UDP-Glc and 4 U T4β-GT were reacted in a 37 °C water bath for 2 h, followed by a QIAquick nucleotide excision purification column to remove unreacted nucleotides and ions. The purified DNA was subjected to periodate oxidation in a 50 μl reaction system containing 20 mmol of sodium periodate and 100 mmol of sodium phosphate (pH 7.0) at 22°C for 16 h, and then reacted with 40 mmol of sodium periodate. Molar sodium sulfite was reacted at room temperature for 10 minutes, and then salt ions were removed using a QIAquick nucleotide excision cleanup column. The biotinylation reaction was performed by placing DNA in 30 μl of a solution containing 2 mM ARP, incubating at 37°C for 1 h, and then using a QIAquick nucleotide removal column to remove excess ARP.

Enrichment of biotinylated 5hmC-DNA with streptavidin-coated magnetic beads: Combine the synthetic biotinylated 5hmC-DNA product with 40 µl of 5 µl streptavidin-coated magnetic beads (MBs per µl) ) solution was mixed and incubated on a rotary mixer for 15 minutes at room temperature in the dark. Magnetic separation was then performed, and the mixture was washed 3 times with 1x binding and wash buffer (5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 mol NaCl), and the Biotinylated 5hmC-DNA conjugates with magnetic beads (5hmC-DNA-MB) were resuspended in ultrapure water.

TDT-mediated isothermal amplification: biotinylated 5hmC-DNA in reaction buffer containing 8 U TDT, 0.6 μl dATP (10 mmol), 0.9 μl dGTP (10 mmol), and 3 μl 10×TDT The amplification reaction was performed by reacting 30 μl of the reaction solution at 37° C. for 1 h.

Chemiluminescence detection: A total of 30 µL of the mixture of newly prepared luminol solution (0.1 mmol), hemin solution (2 µmol) and reaction product was added to 20 µL of HEPES buffer solution (40 mM HEPES, 20 mM KCl, pH 8.0) in 70 μl of the reaction solution at room temperature for 30 minutes to fold the G-rich polymer product into a G-quadruplex structure. After adding 80 microliters of H2O2 ₍ 100 mmol ₎ to the mixture, 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-118 MG cells (human glioblastoma cells), A549 cells (human lung adenocarcinoma cells) and HeLa cells (human cervical cancer cells) were put into cells containing Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was cultured at 37 °C in a humidified atmosphere with 5% _CO . Genomic DNA was extracted using the QIAamp DNA mini kit according to the instructions. The concentration of genomic DNA was determined using a NanoDrop 2000 spectrophotometer. Genomic DNA was digested to 50-200 bp using dsDNA fragmentase for 5hmC detection in whole genome DNA.

The experimental principle of this method is shown in Figure 1. designed a 5hmC-DNA sequence (TCG ATG TAG TGC GTCAC ^hm C GGA TGA TAG CTG TAG TCA) with a 5'-hydroxymethylcytosine modified at the 18th base at the 5' end ( ^hm C, Figure 1), T4 β-GTase can glycosylate 5hmC to 5ghmC ( ^ghmC , Figure 1), which is then treated with sodium sulfite to open the ring, and then the ARP aldehyde reaction probe can be added to 5hmC. Biotin (biotin-5ghmC, Figure 1) ), the streptavidin and biotin of the magnetic beads are combined and magnetically separated to separate 5hmC, which not only achieves the purpose of distinguishing 5hmC, 5mC and 5C, but also enriches 5hmC. TDT enzyme can amplify from the 3' end of 5hmC-DNA to generate a large number of long-chain G-rich sequences (Figure 1). After adding hemin, the G-rich products can combine with the cofactor hemin to form hemin-G quadruplex nanostructures , the structure can catalyze H2O2 _- mediated oxidation of _luminol , resulting in a distinct chemiluminescence signal.

1. Feasibility experiment

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

2. Sensitivity and specificity detection

Under optimal experimental conditions, the chemiluminescence intensities of hydroxymethylated DNA at different concentrations were measured. As shown in Figure 3A, the chemiluminescence signal increased significantly with increasing 5hmC-DNA concentration. Furthermore, the logarithm of chemiluminescence intensity (I) and 5hmC-DNA concentration (C) exhibited a good linear relationship over a large dynamic range of 5 orders of magnitude from 238 fM to 23.8 nM. The regression equation is I = 9.04×10 ⁵ + 6.30×10 ⁴ log ₁₀ C with a correlation coefficient of 0.991. The limit of detection was calculated to be 2.07×10 ⁻¹³ M by evaluating the mean response of the blank area, plus three times the standard deviation. Compared with fluorescence method (0.167 nM), capillary electrophoresis fluorescence method (90 pM), electrochemical immunosensor method (0.032 nM), and electrochemiluminescence method (16.3 pM), the sensitivity of 5hmC-GLIB-IAS increased by 806%, respectively times, 434 times, 154 times and 78.7 times. The specificity of this strategy was further assessed using three oligonucleotide sequences containing 5hmC-, 5mC-, C (ie 5hmC-DNA, 5mC-DNA and C-DNA). As shown in Figure 3B, the chemiluminescence intensity of 5mC-DNA and C-DNA remained at the background level, and the 5hmC-DNA signal was 50.0-fold and 43.9-fold higher than that of 5mC-DNA and C-DNA, respectively, indicating the high specificity and high specificity of this method for 5hmC. Single base resolution.

To investigate the feasibility of this method to accurately analyze hydroxymethylation levels in mixtures, a series of artificial mixtures were prepared by mixing different ratios of 5hmC-DNA and 5mC-DNA. The measured 5hmC-DNA level (Y) showed a good linear relationship with the input 5hmC-DNA level (X) (Fig. 3C). The regression equation was Y = 0.0479 + 0.988 X, and the correlation coefficient was 0.993, and the method was effective even at the 0.1% 5hmC level.

3. Detection of actual samples

To verify the feasibility of this strategy for genome-wide analysis of 5hmC, we measured 3 cancer cell lines, including human glioblastoma cell line (U-118 MG cells), human lung adenocarcinoma cell line (A549 cells) and human cervical cancer cell line 5hmC of cancer cell lines (HeLa cells). According to previous studies, the content of 5hmC varies greatly in different tissues, with the highest content in the brain, while the content of 5hmC is significantly reduced in various human tumors. In particular, there is no evidence for the presence of 5hmC in A549 and HeLa cells due to the limited sensitivity of the detection method. In this example, 300 ng of genomic DNA extracted from the above cancer cells was analyzed using the hmC-GLIB-IAS method, and the accurate 5hmC content in different types of cells was successfully quantified (Fig. 4A). The level of 5hmC in 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, and its abundance was much lower than that in U-118 MG cells (0.0240% for A549 cells and 0.0111% for HeLa cells).

Furthermore, U-118 MG cells were used as a model to study the relationship between chemiluminescence intensity and genomic DNA concentration. As shown in Figure 4B, the chemiluminescence intensity (I) was enhanced with increasing genomic DNA concentration (C), and in the range of 414 fg/μL – 4.14 ng/μL, the chemiluminescence intensity was logarithmically related to the genomic DNA concentration Linear correlation. The regression equation is I=6.33×10 ⁵ + 1.04×10 ⁵ log ₁₀ C (R ² = 0.992). The calculated detection limit was 39.2 fg/μL. These results indicate that this method has good accuracy in the whole genome analysis of 5hmC and has broad application prospects.

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

SEQUENCE LISTING

<110> Shandong Normal University

<120> A chemiluminescent biosensor for detecting 5-hydroxymethylcytosine and its detection method and application

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 36

<212> DNA

<213> Artificial sequences

<400> 1

tcgatgtagt gcgtcachmcgg atgatagctg tagtca 36

Claims (24)

1. A chemiluminescence biosensor for detecting 5-hydroxymethylcytosine, wherein the chemiluminescence biosensor comprises a glycosylation module, a periodate oxidation module, a biotinylation module, a terminal transferase assisted Isothermal amplification module and chemiluminescence detection module; The glycosylation module includes T4 phage β-glucosyltransferase, uridine diphosphate glucose and NEBuffer 4; The periodate oxidation module includes sodium periodate, sodium phosphate and sodium sulfite; The biotinylation module includes an aldehyde-based reaction probe; The terminal transferase-assisted isothermal amplification module includes terminal transferase, deoxyguanosine triphosphate and deoxyadenosine triphosphate; The chemiluminescence detection module includes hemin, luminol solution and hydrogen peroxide. 2. The application of the chemiluminescence biosensor of claim 1 in detecting 5-hydroxymethylcytosine. 3. A chemiluminescence detection method for 5-hydroxymethylcytosine, characterized in that the method comprises sequentially performing glycosylation, periodate oxidation, and biotinylation on the genomic DNA sample, adding streptomycin Avidin-coated magnetic beads were used for enrichment. After magnetic separation, the enriched biotinylated DNA produced a large number of long-chain G-rich sequences under the terminal transferase-assisted isothermal amplification strategy, and then hemin was added to form hemin. -G four _- stranded nanostructure, which catalyzes H2O2 _- mediated oxidation of luminol, resulting in a chemiluminescent signal. 4. The chemiluminescence detection method according to claim 3, wherein the glycosylation treatment is specifically: adding genomic DNA sample, uridine diphosphate glucose and T4 bacteriophage β-glucosyltransferase in NEBuffer 4 incubation reaction. 5 . The chemiluminescence detection method according to claim 4 , wherein the specific conditions of the incubation reaction are: reaction at 35-40° C. for 1-3 h. 6 . 6 . The chemiluminescence detection method according to claim 4 , wherein the specific conditions of the incubation reaction are: reaction at 37° C. for 2 h. 7 . 7. The chemiluminescence detection method of claim 4, wherein unreacted nucleotides and ions are removed by using a QIAquick nucleotide excision purification column. 8. The chemiluminescence detection method according to claim 4, wherein the periodate oxidation treatment is specifically: reacting the glycosylated sample in a sodium periodate and sodium phosphate system, Then react with sodium sulfite. 9. chemiluminescence detection method as claimed in claim 8, is characterized in that, in sodium periodate and sodium phosphate system, the concrete condition of reaction is: 20-25 ℃ of reaction 10-20 h; The concrete condition of reaction with sodium sulfite is: React at room temperature for 5-20 minutes. 10 . The chemiluminescence detection method according to claim 8 , wherein the specific reaction conditions in the sodium periodate and sodium phosphate system are: 22° C. for 16 hours. 11 . 11. The chemiluminescence detection method of claim 8, wherein the specific conditions for the reaction with sodium sulfite are: react at room temperature for 10 minutes. 12 . The chemiluminescence detection method according to claim 8 , wherein salt ions are removed by using a QIAquick nucleotide excision purification column. 13 . 13 . The chemiluminescence detection method according to claim 4 , wherein the biotinylation treatment is specifically: mixing and incubating the sample after the periodate oxidation treatment with the aldehyde group reaction probe. 14 . 14 . The chemiluminescence detection method according to claim 13 , wherein the specific conditions of the mixed incubation reaction are: reaction at 35-40° C. for 0.5-2 h. 15 . 15 . The chemiluminescence detection method according to claim 13 , wherein the specific conditions of the mixed incubation reaction are: reaction at 37° C. for 1 h. 16 . 16 . The chemiluminescence detection method of claim 13 , wherein a QIAquick nucleotide removal purification column is used to remove excess aldehyde group reaction probes. 17 . 17. The chemiluminescence detection method according to claim 4, wherein the specific method of adding streptavidin-coated magnetic beads for enrichment and magnetic separation is: combining the biotinylated sample with the chain The magnetic bead solution coated with mycovidin was mixed and incubated at room temperature in the dark for 10-20 minutes; then magnetic separation was performed; biotinylated 5hmC-DNA was obtained. 18. The chemiluminescence detection method of claim 17, wherein the incubation time in the dark is 15 minutes. 19. The chemiluminescence detection method as claimed in claim 4, wherein the specific method for isothermal amplification mediated by terminal transferase is: biotinylated 5hmC-DNA, terminal transferase, deoxyadenosine triphosphate and deoxygenation The guanosine triphosphate was mixed for the amplification reaction. 20 . The chemiluminescence detection method according to claim 19 , wherein the specific conditions of the amplification reaction are: reaction at 35-40° C. for 0.5-2 h. 21 . 21 . The chemiluminescence detection method according to claim 19 , wherein the specific conditions of the amplification reaction are: reaction at 37° C. for 1 h. 22 . 22. The chemiluminescence detection method according to claim 4, wherein the specific method of chemiluminescence detection is: incubating the mixture of luminol solution, hemin solution and the above-mentioned amplification reaction product, so that the G-rich polymer product is folded A G-quadruplex structure was formed; H ₂ O ₂ was then added to the mixture and the chemiluminescence signal was measured. 23. The chemiluminescence detection method according to claim 22, wherein the specific conditions of the incubation reaction are: incubation at room temperature for 10-60 minutes. 24. The chemiluminescence detection method according to claim 22, wherein the specific conditions of the incubation reaction are: incubation at room temperature for 30 minutes.

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