CN110196269B

CN110196269B - DNA methyltransferase detection paper, detection method and equipment

Info

Publication number: CN110196269B
Application number: CN201910476104.2A
Authority: CN
Inventors: 高培阔; 程昱川; 华乐天; 牛益民; 李志祥; 许高杰
Original assignee: Ningbo Institute of Material Technology and Engineering of CAS
Current assignee: Ningbo Institute of Material Technology and Engineering of CAS
Priority date: 2019-06-03
Filing date: 2019-06-03
Publication date: 2021-04-02
Anticipated expiration: 2039-06-03
Also published as: CN110196269A

Abstract

The application provides a DNA methyltransferase detecting test paper, a detecting method and a device, wherein the test paper comprises a detecting area, the detecting area is overlapped with a relatively separated saccharifying enzyme encapsulating area and a starch area, wherein saccharifying enzyme in the saccharifying enzyme encapsulating area can be released for the DNA methyltransferase, and starch is deposited in the starch area; when the saccharifying enzyme contacts starch in the starch zone, the starch is hydrolyzed to produce glucose, and a detection signal is generated in the detection zone. The test paper overcomes the defects that the traditional detection method needs to be operated in a professional laboratory by professional experimenters, cannot be used for portable and on-site timely detection and the like, and has wide application prospect.

Description

DNA methyltransferase detection paper, detection method and equipment

Technical Field

The application relates to the technical field of electrochemical biosensing, relates to nanotechnology and biomedical technology, and particularly relates to DNA methyltransferase detection paper, a detection method thereof and field detection equipment.

Background

DNA methyltransferases (e.g., Dam MTase, Dcm MTase, etc.) are enzymes that specifically catalyze methylation modification of DNA bases. DNA methylation can cause changes in DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby affecting cell function and gene expression. Therefore, the DNA methyltransferase can be used as a disease marker of various cancers (such as gastric cancer, lung cancer and colon cancer), and the portable detection of the activity of the DNA methyltransferase is helpful for early diagnosis and treatment of various diseases such as cancer. Currently, researchers have developed a variety of highly sensitive methods for detecting DNA methyltransferases, such as: fluorescence, colorimetry, electrochemical methods, and the like. However, these methods generally require specialized laboratories, specialized laboratory equipment, or specialized operators to perform the methods, which greatly limits their practical applications. Therefore, the development of an effective and reliable portable, point-of-care (POC) DNA methyltransferase activity assay tool is imminent.

As is well known, a blood glucose meter and a test strip matched with the blood glucose meter are the most common POC diagnostic tools which are widely applied in the market. The commercial blood glucose test paper is modified to prepare a POC diagnostic tool which is specifically responsive to disease markers, so that the method is an effective method. The invention is achieved accordingly.

Disclosure of Invention

The application aims to provide a DNA methyltransferase detection test paper to solve the problems in the prior art.

In order to accomplish the above objects, according to one aspect of the present application, there is provided a DNA methyltransferase assay strip comprising a detection region, wherein the detection region is overlapped with a relatively separated saccharifying enzyme-encapsulating region and starch region,

wherein the saccharifying enzyme can be released by DNA methyltransferase in the saccharifying enzyme encapsulating zone and starch is deposited in the starch zone; when the saccharifying enzyme contacts starch in the starch zone, the starch is hydrolyzed to produce glucose, and a detection signal is generated in the detection zone.

In a preferred embodiment, the glucoamylase in the glucoamylase encapsulating region is encapsulated by a DNA hydrogel, and the DNA hydrogel is a dendritic-like or tetrahedral network structure composed of deoxyribonucleic acid (DNA).

In a preferred embodiment, the DNA hydrogel is of a tetrahedral type, and the long-chain DNA tetrahedron is formed by amplifying four DNA single strands capable of hybridizing with each other, then cross-linking the DNA single strands by hybridization, and finally extending by dATP and dTTP, respectively.

In a preferred embodiment, the step of encapsulating a glucoamylase with the DNA hydrogel includes a step of hybridizing a long-chain DNA tetrahedron extended by dATP with a long-chain DNA tetrahedron extended by dATP in the presence of a glucoamylase, and encapsulating the glucoamylase.

In a preferred technical scheme, the starch area is formed by depositing starch on a test paper sheet and drying.

In a preferred technical scheme, the saccharifying enzyme encapsulating area and the starch area are respectively and independently arranged test paper sheets, and the saccharifying enzyme encapsulating area and the starch area form a foldout which is used as a detection area of the detection test paper.

Another object of the present invention is to provide a method for preparing the DNA methyltransferase detection test paper, which comprises:

(a) forming a saccharifying enzyme encapsulating zone and a starch zone which are independent from each other, wherein saccharifying enzyme in the saccharifying enzyme encapsulating zone can be released by DNA methyltransferase, and starch is deposited in the starch zone;

(b) the glucoamylase encapsulating region and the starch region are superposed on the detection region of the test strip.

Still another object of the present invention is to provide a method for detecting DNA methyltransferase, which comprises the steps of:

(1) the DNA methyltransferase releases the encapsulated saccharifying enzyme and contacts saccharifying enzyme with starch;

(2) saccharifying enzyme and starch produce hydrolysis reaction to generate glucose;

(3) the activity of DNA methyltransferase was determined by measuring a detection signal generated from glucose using a glucometer.

In a preferred technical scheme, the method specifically comprises the following steps:

I) preparing a DNA tetrahedron, and then extending the DNA tetrahedron to respectively obtain long-chain DNA tetrahedrons of terminal DNA tetrahedron growth A and T, which are respectively named as T-DNA-An and T-DNA-Tn;

II) modifying DNA hydrogel and starch on the test paper sheet: enabling the long-chain T-DNA-An and the long-chain T-DNA-Tn to be hybridized on the test paper sheet to form DNA hydrogel, and meanwhile, encapsulating saccharifying enzyme; dripping 0.1g/mL of starch on a test paper sheet, and drying to prepare starch modified filter paper;

III) combining two pieces of filter paper with a commercialized blood glucose test strip to prepare a page type test strip, and combining the page type test strip with a glucometer to form POC (particle oxidation catalyst) detection equipment;

IV) mixing Dam MTase to be detected, DpnI nickase and S-adenosylmethionine, then dropwise adding the mixture to a DNA hydrogel modified glucoamylase encapsulation area test strip, overlapping the test strip in the starch area to the test strip in the glucoamylase encapsulation area, reacting for 0.5-1.5 h, overlapping the two pieces of filter paper to a detection area of the blood glucose test strip, and detecting by a glucometer.

The specific detection steps may be as follows:

preparing a DNA tetrahedron: four DNA single-strands T1, T2, T3 and T4 hybridized with each other are respectively dissolved in 1 XTM buffer solution to prepare stock solution of 100 mu M, and then the four solutions and Tris buffer solution are mixed in equal volume to obtain DNA tetrahedron solution of 20 mu M. The solution was annealed at 95 ℃ for 10min, rapidly cooled at 4 ℃ and then stabilized overnight in a refrigerator at 4 ℃ overnight to give a DNA tetrahedral solution.

Extension of DNA tetrahedron: dividing the DNA tetrahedron solution obtained in the step I into two equal parts, and adding a mixed solution of TdT enzyme and dATP dissolved in a TdT buffer solution into one part; adding the other part into a mixed solution of TdT enzyme and dTTP dissolved in a TdT buffer solution, and then incubating at 37 ℃ for 18-30 h to finally obtain 4-8 mu M long-chain DNA tetrahedrons with terminal growth A and T, which are named as T-DNA-An and T-DNA-Tn respectively.

③ modifying DNA hydrogel and starch on paper: and (4) mixing the two solutions obtained in the step two with 2-4 g/L of saccharifying enzyme, dripping the mixture on a filter paper sheet with the diameter of 3-5 mm, heating to 50 ℃, and slowly cooling to ensure that the long-chain T-DNA-An and the long-chain T-DNA-Tn are hybridized on the paper to form DNA hydrogel and encapsulate the saccharifying enzyme.

Another piece of filter paper is taken, 0.1g/mL of starch is dripped on the filter paper, and the filter paper modified by starch is prepared after drying.

Preparing page type detection test paper and detection application: combining the two pieces of filter paper prepared in the third step with a commercial blood sugar test strip to prepare a page-type test paper, mixing 20 mu L of Dam MTase to be tested, 150-250U/mL of DpnI nickase and 150-250 mu M S-adenosylmethionine, dripping the mixture on the filter paper modified by DNA hydrogel, superposing the starch filter paper on the filter paper of the hydrogel, reacting for 0.5-1.5 h, superposing the two pieces of filter paper on the detection area of the blood sugar test paper, and detecting by using a glucometer.

The 1 × TM buffer comprises the following components: 20mM Tris, 50mM MgCl₂，pH＝8.0。

The Tris buffer solution comprises the following components: 20mM Tris, 100mM NaCl, pH 8.0.

The TdT buffer solution comprises the following components: 100mM potassium cacodylate, 1mM CoCl₂,，0.2mM DTT，pH＝7.2。

In the second step, the final concentration of the DNA tetrahedron is 4-8 μ M, preferably 6 μ M, and the final concentration of dATP and dTTP is 50-70 mM, preferably 60 mM.

In the third step, the two solutions obtained in the second step, namely T-DNA-An and T-DNA-Tn, are mixed according to the molar ratio of 1: 1.

another object of the present invention is to provide a POC detection apparatus for detecting DNA methyltransferase, the POC detection apparatus comprising:

1) the DNA methyltransferase detecting paper;

2) a blood glucose meter for detecting the level of glucose in a test strip of DNA methyltransferase upon release of the DNA methyltransferase.

The invention also aims to provide application of the DNA methyltransferase detection test paper in detecting DNA methyltransferases in situ.

The invention is based on the original blood sugar detector, and is improved on the blood sugar detection test paper, and the key of the improvement lies in how to fix the specific recognition sequence of DNA methyltransferase on the surface of the test paper. The invention adopts DNA hydrogel to wrap saccharifying enzyme. DNA hydrogels have unique advantages. The DNA hydrogel is a network structure material formed by mutually crosslinking DNA sequences, has very good biocompatibility, has natural advantages in enzyme immobilization, enzyme activity maintenance and the like, and is easy to combine with a substrate material (such as filter paper). Therefore, the material has wide application prospect in the field of preparing POC diagnostic tools.

The invention provides a preparation process of portable DNA methyltransferase-Dam MTase test paper. The process provided by the invention encapsulates the glucoamylase into the DNA hydrogel, fixes the glucoamylase on the filter paper, and then combines the immobilized glucoamylase with the filter paper and the blood glucose test paper for fixing the substrate, so that the defects that the traditional detection method needs to be operated in a professional laboratory and by professional experimenters, cannot be used for portable and on-site timely detection and the like are overcome, and the process has a wide application prospect. The invention provides a test paper for detecting the activity of DNA methyltransferase and a preparation method thereof. DNA methyltransferase is a marker of many diseases, and the development of a high-sensitivity and high-selectivity field detection method for the activity of the DNA methyltransferase has important practical application value. The invention utilizes the advantages that the DNA hydrogel can encapsulate enzyme and is easy to fix on paper, prepares the DNA hydrogel and starch modified test paper, and reforms commercial blood sugar test paper to prepare the page type DNA methyltransferase detection paper, and realizes the high-sensitivity and high-selectivity detection of the activity of the DNA methyltransferase by a glucometer. The detection method based on the test paper has the advantages of convenient use, no need of large-scale experimental instruments, no need of professional technicians for operation and the like, and can realize portable on-site instant detection, so the invention has wide application prospect in the field of early diagnosis and treatment of diseases.

The invention has the beneficial effects that:

(1) the DNA hydrogel is composed of pure DNA, has good biocompatibility, is easy to modify on the surface of filter paper, solves the problems of fixation and stability of enzyme on the paper and the like, does not need modification and has lower cost.

(2) The book-page type test paper prepared by the invention is convenient to use, can be detected by a commercialized glucometer, does not need to be operated by professional technicians, and does not need to be detected in professional laboratories and detection units.

(3) The DNA methyltransferase detection method can be used for detecting the activity of Dam MTase on site in the public.

(4) The DNA methyltransferase detection method has the advantages of simplicity, high sensitivity, good selectivity and the like, and has better application prospect in the field of early diagnosis of related diseases.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. In the drawings:

FIG. 1 is a schematic view of the Dam MTase activity test strip prepared in example 1.

FIG. 2 is a graph showing the signal response of different test strips prepared in example 1 and example 2 to Dam MTase activity.

FIG. 3 is a graph showing the signal response of the test strip prepared in example 3 to different concentrations of Dam MTase activity.

FIG. 4 is a linear plot of the log values of the test strips prepared in example 3 and the Dam concentration of MTase.

FIG. 5 is a graph showing the signal response of the Dam MTase activity assay strip prepared in example 4 to various interferents.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application 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 example embodiments according to the present application. 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 should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the application described herein are, for example, capable of operation in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

Spatially relative terms, such as "above … …", "above … …", "above … …", "above", and the like, may be used herein for ease of description to describe the spatial relationship of one component or module or feature to another component or module or feature as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the component or module in use or operation in addition to the orientation depicted in the figures. For example, if a component or module in the figures is turned over, components or modules described as "above" or "above" other components or modules or configurations would then be oriented "below" or "beneath" the other components or modules or configurations. Thus, the exemplary term "above … …" can include both an orientation of "above … …" and "below … …". The components or modules may also be oriented in other different ways (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

The invention provides a DNA methyltransferase detection test paper, which comprises a detection area and is characterized in that the detection area is overlapped with a relatively isolated saccharifying enzyme encapsulating area and a starch area,

The invention belongs to the technical field of electrochemical biosensing, and particularly relates to a preparation process of portable DNA methyltransferase activity electrochemical detection test paper based on a glucometer and DNA hydrogel. The DNA hydrogel encapsulating the glucoamylase and the enzyme substrate are respectively fixed on paper and then combined with the blood glucose test paper to prepare the portable Dam MTase activity test paper.

The process principle of the invention is as follows: the Dam MTase can recognize a 5 '-GATC-3' sequence in a DNA tetrahedron and catalyze N6 site of an A base to be methylated, so that the methyl site is specifically recognized by Dpn I nickase, double-stranded DNA is sheared, the structure of the DNA tetrahedron is damaged, and because the DNA tetrahedron is a crosslinking site of a plurality of pieces of double-stranded DNA in the DNA hydrogel, the damage of the structure of the DNA tetrahedron can cause the collapse of the structure of the DNA hydrogel, thereby releasing saccharifying enzyme encapsulated in the DNA hydrogel, enabling the saccharifying enzyme to be in contact with starch, hydrolyzing the starch to generate glucose, generating electrochemical signal response on a blood glucose test strip, further being detected by a glucometer, and realizing POC detection of DNA methyltransferase-Dam MTase activity.

Example 1: preparation of Dam MTase activity detection test paper and detection experiment thereof

Preparing Dam MTase activity test paper:

Extension of DNA tetrahedron: dividing the DNA tetrahedron solution obtained in the step I into two equal parts, and adding a mixed solution of TdT enzyme and dATP dissolved in a TdT buffer solution into one part; the other part was added with a mixture of TdT enzyme and dTTP dissolved in TdT buffer solution, and incubated at 37 ℃ for 18h to obtain 8. mu.M long-chain DNA tetrahedrons, named T-DNA-An and T-DNA-Tn, respectively, with terminal growth of A and T.

③ modifying DNA hydrogel and starch on paper: and (4) mixing the two solutions obtained in the step (II) with 3g/L of glucoamylase, dripping the mixture on a filter paper sheet with the diameter of 4mm, heating to 50 ℃, and slowly cooling to ensure that the long-chain T-DNA-An and the long-chain T-DNA-Tn are hybridized on the paper to form DNA hydrogel and encapsulate the glucoamylase. Another piece of filter paper is taken, 0.1g/mL of starch is dripped on the filter paper, and the filter paper modified by starch is prepared after drying.

Preparing page type test paper: combining the two pieces of filter paper prepared in the third step with the commercialized blood sugar test paper to prepare the page type test paper.

FIG. 1 is a schematic view of a Dam MTase activity test strip prepared in example 1, which is composed of a glucoamylase encapsulation zone (a DNA hydrogel-modified filter paper encapsulating a glucoamylase is made into a test paper sheet) 1, a starch zone (a paper sheet made of a filter paper) 2, and a blood glucose test strip 3. Referring to the figure before the superposition of figure 1, in use, 20 μ L of Dam MTase to be tested, 200U/mL of DpnI nickase and 213 μ M S-adenosylmethionine were mixed, added dropwise to the diastase envelope 1, the starch region 2 was superposed on the diastase envelope, and after 1 hour of reaction, both filters were superposed on the test region of the blood glucose test strip, as shown in the figure after the superposition of figure 1, and the test was performed by a glucometer.

In this example, the Dam MTase concentration to be measured was 0 and 10U/mL, respectively, and the mean values of the glucometer readings were shown in column iii and column iv in FIG. 2, respectively, in 3 replicates.

As can be seen from the graph, the mean reading of the blood glucose meter is relatively small, about 2 (column iii in FIG. 2) without Dam MTase, and is relatively large, about 22.5 (column iv in FIG. 2) when the Dam MTase is at a concentration of 10U/mL, indicating that the test strip prepared according to the present invention is specifically responsive to Dam MTase.

Example 2: response study of starch alone and glucoamylase-encapsulating DNA hydrogel modified blood glucose dipsticks to Dam MTase

Extension of DNA tetrahedron: dividing the DNA tetrahedron solution obtained in the step I into two equal parts, and adding a mixed solution of TdT enzyme and dATP dissolved in a TdT buffer solution into one part; the other part was added with a mixture of TdT enzyme and dTTP dissolved in TdT buffer solution, and incubated at 37 ℃ for 22h to obtain 6. mu.M long-chain DNA tetrahedrons, named T-DNA-An and T-DNA-Tn, respectively, with terminal growth of A and T.

③ modifying DNA hydrogel and starch on paper: and (4) mixing the two solutions obtained in the step (II) with 4g/L of glucoamylase, dripping the mixture on a filter paper sheet with the diameter of 3mm, heating to 50 ℃, and slowly cooling to ensure that the long-chain T-DNA-An and the long-chain T-DNA-Tn are hybridized on the paper to form DNA hydrogel and encapsulate the glucoamylase. Another piece of filter paper is taken, 0.1g/mL of starch is dripped on the filter paper, and the filter paper modified by starch is prepared after drying.

Preparing page type detection test paper and detection application: combining the two pieces of filter paper prepared in the third step with a commercialized blood sugar test paper respectively to prepare two types of page-type test paper, which are respectively: starch modified filter paper and blood glucose test paper, DNA hydrogel modified filter paper and blood glucose test paper encapsulating saccharifying enzyme. Then 20. mu.L of 10U/mL Dam MTase, 150U/mL DpnI nickase and 150. mu. M S-adenosylmethionine were mixed and dropped on the starch or DNA hydrogel modified filter paper, respectively, and after 1.5 hours of reaction, the filter paper was superimposed on the detection area of the blood glucose test paper, respectively, and the detection was performed by a glucometer, and the results of the three replicates were shown in column i and column ii of FIG. 2.

As can be seen from columns i and ii in fig. 2, the reading of the glucometer was close to 0, indicating that only starch-modified filter paper or hydrogel-modified filter paper encapsulating saccharifying enzyme could not generate a signal response to Dam MTase. Therefore, starch and DNA hydrogel encapsulating saccharifying enzyme are all indispensable parts of the test paper for testing the activity of Dam MTase.

Example 3: detection experiment of different concentrations of Dam MTase

Preparing Dam MTase activity test paper:

Extension of DNA tetrahedron: dividing the DNA tetrahedron solution obtained in the step I into two equal parts, and adding a mixed solution of TdT enzyme and dATP dissolved in a TdT buffer solution into one part; the other part was added with a mixture of TdT enzyme and dTTP dissolved in TdT buffer solution, and incubated at 37 ℃ for 24h to obtain 6. mu.M long-chain DNA tetrahedrons, named T-DNA-An and T-DNA-Tn, respectively, with terminal growth of A and T.

Preparing page type detection test paper and detection application: combining the two filter papers prepared in the third step with a commercialized blood sugar test strip to prepare a page-type test paper, wherein the concentration of 20 mu L is as follows: 0. 0.003, 0.01, 0.05, 0.2, 1, 5, 10U/mL Dam MTase was mixed with 200U/mL DpnI nickase and 213. mu. M S-adenosylmethionine, respectively, and dropped on the DNA hydrogel-modified filter paper, and then the starch filter paper was laminated on the hydrogel filter paper, and after 1 hour of reaction, both the filter papers were laminated on the detection area of the blood glucose test paper and detected by a glucometer, and the detection results are shown in FIGS. 3 and 4.

As can be seen from FIG. 3, the prepared Dam MTase activity test strip showed a gradually increasing signal on the blood glucose meter as the Dam MTase concentration increased, and although the signal was not linear to the Dam MTase concentration, it was found to be linear to the blood glucose meter signal by taking the logarithm of the Dam MTase concentration, as shown in FIG. 4.

Example 4: selective assay for Dam MTase Activity

Preparing Dam MTase activity test paper:

Extension of DNA tetrahedron: dividing the DNA tetrahedron solution obtained in the step I into two equal parts, and adding a mixed solution of TdT enzyme and dATP dissolved in a TdT buffer solution into one part; the other part was added with a mixture of TdT enzyme and dTTP dissolved in TdT buffer solution, and incubated at 37 ℃ for 30h to obtain 4. mu.M long-chain DNA tetrahedrons, named T-DNA-An and T-DNA-Tn, respectively, with terminal growth of A and T.

③ modifying DNA hydrogel and starch on paper: and (4) mixing the two solutions obtained in the step (II) with 2g/L of glucoamylase, dripping the mixture on a filter paper sheet with the diameter of 5mm, heating to 50 ℃, and slowly cooling to ensure that the long-chain T-DNA-An and the long-chain T-DNA-Tn are hybridized on the paper to form DNA hydrogel and encapsulate the glucoamylase. Another piece of filter paper is taken, 0.1g/mL of starch is dripped on the filter paper, and the filter paper modified by starch is prepared after drying.

Preparing page type detection test paper and detection application: combining the two filter papers prepared in the third step with a commercial blood sugar test strip to prepare a page-type test strip, mixing 20 mu L of each of 10U/mL Dam MTase, 100U/mL alkaline phosphatase, lysozyme enzyme and bovine serum albumin solution with 250U/mL DpnI nickase and 250 mu M S-adenosylmethionine, dripping the mixture on the DNA hydrogel modified filter paper, superposing the starch filter paper on the hydrogel filter paper, reacting for 0.5h, superposing the two filter papers on the detection area of the blood sugar test strip, and detecting by using a glucometer, wherein the detection result is shown in figure 5. Wherein: the columns i to iv are the results of detection of Dam MTase, alkaline phosphatase, lysozyme enzyme, and bovine serum albumin, respectively.

As can be seen from FIG. 5, only Dam MTase can produce a larger reading on a blood glucose meter, and even if the concentrations of alkaline phosphatase, lysozyme, bovine serum albumin and other substances are ten times that of Dam MTase, no signal response can be produced, which indicates that the Dam MTase activity test paper prepared by the invention has good selectivity.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A DNA methyltransferase assay strip for assaying the activity of a DNA methyltransferase, comprising a test strip comprising a test region, wherein the test region is overlaid with a separate saccharifying enzyme encapsulating region in which saccharifying enzyme is releasable by DNA methyltransferase and a starch region in which starch is deposited; when the saccharifying enzyme contacts starch in the starch area, hydrolyzing the starch to generate glucose, and generating a detection signal in the detection area;

the saccharifying enzyme in the saccharifying enzyme encapsulating area is encapsulated by DNA hydrogel which is a dendritic-like and tetrahedral-like reticular structure formed by DNA, and the DNA tetrahedron has a 5 '-GATC-3' sequence.

2. The DNA methyltransferase detection paper of claim 1 wherein the DNA hydrogel is of a tetrahedral type, and a long-chain DNA tetrahedron is formed by amplifying four DNA single strands that can hybridize with each other, then cross-linking the DNA single strands by hybridization, and finally extending by dATP and dTTP, respectively.

3. The DNA methyltransferase assay paper according to claim 2, wherein the step of encapsulating a saccharifying enzyme with the DNA hydrogel comprises a step of hybridizing a long-chain DNA tetrahedron extended by dATP with a long-chain DNA tetrahedron extended by dATP in the presence of a saccharifying enzyme, and encapsulating the saccharifying enzyme.

4. The DNA methyltransferase detecting paper of claim 1 wherein the starch area is formed by depositing starch on a strip and drying.

5. The DNA methyltransferase detection test paper according to claim 1, wherein the saccharifying enzyme-encapsulating region and the starch region are separately provided as test paper sheets, and they constitute a hinge as a detection region of the test paper.

6. A method for producing the DNA methyltransferase detection test paper according to any one of claims 1 to 5, characterized by comprising:

7. A method for detecting DNA methyltransferase, comprising the steps of:

(1) providing the DNA methyltransferase assay strip of any of claims 1-5 and releasing the encapsulated saccharifying enzyme with the DNA methyltransferase and contacting the saccharifying enzyme with starch;

8. A POC detection apparatus for DNA methyltransferase, comprising:

1) the DNA methyltransferase detection test paper according to any one of claims 1 to 5;

9. Use of the DNA methyltransferase detection test paper according to any one of claims 1 to 5 for the in-situ detection of DNA methyltransferase activity.