阅读说明：本技术 主客体探针结合纳米孔技术联用的酶活性浓度检测方法 (Enzyme activity concentration detection method combining host-guest probe and nanopore technology ) 是由 吴海臣 郭秉元 于 2019-08-27 设计创作，主要内容包括：本发明涉及一种主客体探针结合纳米孔技术联用的酶活性浓度检测方法,本发明首先提供了一种用于检测酶活性浓度的分子组合,包括DNA-肽分子和葫芦脲,所述DNA-肽分子由双链DNA分子和肽分子组成,肽分子的羧基端共价连接于双链DNA分子的5’端或3’端；所述DNA-肽分子经待测酶作用后,改变氨基端芳香族氨基酸的暴露状态。当待测酶通过酶促反应将部分肽链切除后,暴露出(或丢失掉)的芳香族氨基酸能够(不能)与葫芦脲发生主客体作用而形成(无法形成)DNA主客体探针。这种DNA主客体探针穿越纳米孔时将产生特征电流信号,通过检测电流信号即可实现对酶活性浓度的高灵敏度、特异性检测。(The invention relates to a method for detecting enzyme activity concentration by combining a subject probe and an object probe with a nanopore technology, and firstly provides a molecular combination for detecting the enzyme activity concentration, which comprises a DNA-peptide molecule and cucurbituril, wherein the DNA-peptide molecule consists of a double-stranded DNA molecule and a peptide molecule, and the carboxyl end of the peptide molecule is covalently connected with the 5 'end or the 3' end of the double-stranded DNA molecule; after the DNA-peptide molecule is acted by the enzyme to be detected, the exposure state of the amino-terminal aromatic amino acid is changed. When the enzyme to be detected cuts off partial peptide chains through enzymatic reaction, the exposed (or lost) aromatic amino acid can (cannot) generate the host-object action with cucurbituril to form (cannot) a DNA host-object probe. When the DNA subject probe passes through the nano-pore, a characteristic current signal is generated, and high sensitivity and specificity detection on the enzyme activity concentration can be realized by detecting the current signal.)

1. A combination of molecules for detecting the concentration of enzymatic activity, wherein said combination of molecules comprises a DNA-peptide molecule and cucurbituril;

the DNA-peptide molecule consists of a double-stranded DNA molecule and a peptide molecule, wherein the carboxyl end of the peptide molecule is covalently linked to the 5 'end or the 3' end of the double-stranded DNA molecule;

after the DNA-peptide molecules are acted by enzyme to be detected, the exposure state of amino-terminal aromatic amino acid is changed;

the exposure state of the amino-terminal aromatic amino acid is changed as follows: (i) the amino terminal of the DNA-peptide molecule does not have exposed aromatic amino acid, and the amino terminal of the DNA-peptide molecule acted by the enzyme to be detected has exposed aromatic amino acid; or, (ii) the amino terminus of the DNA-peptide molecule has an exposed aromatic amino acid, and the amino terminus of the DNA-peptide molecule after the action of the enzyme to be detected does not have an exposed aromatic amino acid;

the molar ratio of the DNA-peptide molecules to the cucurbituril is 1: 10-250.

2. The molecular combination of claim 1, wherein the double stranded DNA molecule is 20-60bp in length.

3. The molecular combination of claim 1, wherein the aromatic amino acid is phenylalanine, tryptophan, or tyrosine.

4. The molecular combination of claim 1, wherein the number of amino acids in the DNA-peptide molecule after the action of the enzyme to be detected is 20 or less.

5. The molecular combination according to claim 1, wherein the cucurbituril is selected from the group consisting of: cucurbit [6] urils, cucurbit [7] urils, cucurbit [8] urils.

6. A host-guest probe for detecting the activity concentration of enzyme is characterized by being formed by co-incubating DNA-peptide molecules and cucurbituril;

the DNA-peptide molecule consists of a double-stranded DNA molecule and a peptide molecule, wherein the carboxyl end of the peptide molecule is covalently linked to the 5 'end or the 3' end of the double-stranded DNA molecule;

after the DNA-peptide molecules are acted by enzyme to be detected, the exposure state of amino-terminal aromatic amino acid is changed;

the exposure state of the amino-terminal aromatic amino acid is changed as follows: (i) the amino terminal of the DNA-peptide molecule does not have exposed aromatic amino acid, and the amino terminal of the DNA-peptide molecule acted by the enzyme to be detected has exposed aromatic amino acid; or, (ii) the amino terminus of the DNA-peptide molecule has an exposed aromatic amino acid, and the amino terminus of the DNA-peptide molecule after the action of the enzyme to be detected does not have an exposed aromatic amino acid;

when the exposure state of amino-terminal aromatic amino acid is changed to be (i) after the DNA-peptide molecules are acted by enzyme to be detected, the host-guest probe is formed by co-incubation of the DNA-peptide molecules acted by the enzyme to be detected and the cucurbituril, and the cucurbituril is connected with the amino-terminal aromatic amino acid in the DNA-peptide molecules acted by the enzyme to be detected through host-guest supramolecule action;

when the exposure state of amino-terminal aromatic amino acid is changed to be (ii) after the DNA-peptide molecule is acted by enzyme to be detected, the host-guest probe is formed by co-incubation of the DNA-peptide molecule without the action of the enzyme to be detected and the cucurbituril, and the cucurbituril is connected with the amino-terminal aromatic amino acid in the DNA-peptide molecule through the action of host-guest supramolecules;

the molar ratio of the DNA-peptide molecules to the cucurbituril is 1: 10-250.

7. The host-guest probe of claim 6, wherein the double-stranded DNA molecule has a length of 20-60 bp;

preferably, the aromatic amino acid is phenylalanine, tryptophan, or tyrosine;

preferably, the number of amino acids in the DNA-peptide molecule subjected to the action of the enzyme to be detected is less than or equal to 20;

preferably, the cucurbiturils are selected from the group consisting of: cucurbit [6] urils, cucurbit [7] urils, cucurbit [8] urils;

preferably, the reaction condition of co-incubation of the DNA-peptide molecule and the cucurbituril is 4 ℃, and the reaction time is 1-3 h.

8. A system comprising the molecular combination of any one of claims 1-5 and a sample cell, or the host-guest probe of any one of claims 6-7 and a sample cell;

the sample cell comprises two compartments separated by an insulating membrane; the insulating film is provided with a through hole with the diameter of 100-; an Ag/AgCl electrode is applied in the sample cell to form a closed loop.

9. Use of the molecular composition according to any one of claims 1-5, the host-guest probe according to any one of claims 6-7, or the system according to claim 8 for detecting the concentration of an enzyme, which is a protease;

preferably, the enzyme is selected from the group consisting of: leucine aminopeptidase, cathepsin B, matrix metalloproteinase, carboxypeptidase M, methionine aminopeptidase.

10. A method for detecting the activity concentration of enzyme, comprising the steps of mixing enzyme to be detected with the DNA-peptide molecule of claim 1, allowing the enzyme to be detected to perform enzymatic reaction on the DNA-peptide molecule, and then adding cucurbituril for co-incubation to obtain a solution to be detected; and adding the solution to be detected into the sample cell, and detecting the enzyme activity concentration by analyzing the characteristic electric signal.

Technical Field

The invention relates to the technical field of supramolecular chemistry, in particular to an enzyme activity concentration detection method combining a host-guest probe with a nanopore technology.

Background

The protease is used as an important biomarker, and the rapid and sensitive detection of the activity of the protease is not only significant for early diagnosis of tumors, but also an important means for observation of curative effect, judgment of tumor stages and prognosis. Fluorescence spectroscopy is currently the most common technique for detecting enzyme activity concentration. Although fluorescence methods have high sensitivity and good spatial resolution, fluorescent probes usually require complex design and background interference caused by auto-fluorescence phenomenon will cause decrease of signal-to-noise ratio and thus affect the sensitivity and imaging quality of the method. With the rapid development of nanotechnology in recent years, various methods based on nanomaterials to achieve highly sensitive, highly selective detection of the concentration of enzyme activity have been developed in succession. But still require the assistance of large instruments and are cumbersome in process. Therefore, the development of simple and efficient enzyme activity concentration detection methods is becoming more urgent.

DNA has abundant and diversified 'tool box' properties, and can be designed into various nanometer machines with different functions due to the advantages of various modifications on bases or chain ends and the like. In 2019, Ricci et al used a DNA nanomachine modified with cyanine dyes to achieve high-sensitivity rapid detection of urease. However, the method relies on optical signals, so the method has the problems of photobleaching, short fluorescence lifetime, high background interference and the like, and particularly, false positive conclusion can be easily obtained by the background interference. Therefore, the research and development of the enzyme activity concentration detection method based on the DNA molecular probe with high sensitivity, high selectivity and low background interference has important significance for the early diagnosis, treatment and prognosis of the tumor.

Nanopore single channel technology is an emerging detection tool that has been developed on the basis of electrophysiological studies since the mid-nineties of the twentieth century. The nano-pores are the channels with the pore size of nanometer scale, and are usually 1-100 nanometers. When two mutually insulated compartments filled with electrolyte are communicated through a nano-scale pore channel, under the action of an external electric field, electrolyte ions in the solution directionally migrate and pass through the nano-pores to generate current, and the current is measured, amplified and converted by a patch clamp system and then recorded. When a substance to be detected exists in a solution, the substance passes through the nanopore under the drive of diffusion action or voltage, the number of ions passing through the pore channel changes due to the occupation of the substance to be detected, so that the recorded current changes, and a current signal comprises two characteristic quantities: the current retardation amplitude (amplitude) and the current retardation time (dwell) from which rich physical property information can be analyzed: species, structure, conformational changes, and molecular composition of the substance, among others.

In general, the sensitivity and signal-to-noise ratio of biological nanopores are much better than those of artificial nanopores, so that biological nanopores are more favorable for high-sensitivity detection. Biological nanopores are expressed from plasmids, however, whose geometric dimensions are fixed and therefore can only be traversed by molecules of a size that matches them. The protease is usually about ten nanometers in size, so that the protease cannot penetrate through the alpha HL nanopore. JACS published work in 2011 to detect ***e based on aptamer binding to α HL nanopores. When the aptamer is combined with ***e, a Y-shaped composite structure is formed. The structure can not penetrate through the nano-pores to generate long-time current blockage, and the ***e rapid detection is realized by utilizing the blockage current signal. However, the occlusion signal is very unfavorable for high sensitivity detection because of low signal characteristics and strong background interference.

Disclosure of Invention

The invention aims to provide an enzyme activity concentration detection method combining a host-guest probe and a nanopore technology.

To this end, in a first aspect of the invention, there is provided a combination of molecules for detecting the concentration of enzymatic activity, said combination of molecules comprising a DNA-peptide molecule and cucurbituril;

the DNA-peptide molecule consists of a double-stranded DNA molecule and a peptide molecule, wherein the carboxyl end of the peptide molecule is covalently linked to the 5 'end or the 3' end of the double-stranded DNA molecule;

after the DNA-peptide molecules are acted by enzyme to be detected, the exposure state of amino-terminal aromatic amino acid is changed;

the exposure state of the amino-terminal aromatic amino acid is changed as follows: (i) the amino terminal of the DNA-peptide molecule does not have exposed aromatic amino acid, and the amino terminal of the DNA-peptide molecule acted by the enzyme to be detected has exposed aromatic amino acid; or, (ii) the amino terminus of the DNA-peptide molecule has an exposed aromatic amino acid, and the amino terminus of the DNA-peptide molecule after the action of the enzyme to be detected does not have an exposed aromatic amino acid;

the molar ratio of the DNA-peptide molecules to the cucurbituril is 1:10-250, preferably 1: 10-150.

Further, the length of the double-stranded DNA molecule is 20-60 bp.

Further, the aromatic amino acid is phenylalanine, tryptophan, or tyrosine; phenylalanine is preferred.

Furthermore, the number of amino acids in the DNA-peptide molecule after the action of the enzyme to be detected is less than or equal to 20, preferably less than or equal to 15.

Further, the cucurbiturils are selected from the group consisting of: cucurbit [6] urils (CB [6]), cucurbit [7] urils (CB [7]), cucurbit [8] urils (CB [8 ]); cucurbit [7] urils are preferred.

In a second aspect of the invention, a host-guest probe for detecting the concentration of enzyme activity is provided, which is formed by co-incubating a DNA-peptide molecule and cucurbituril;

the DNA-peptide molecule consists of a double-stranded DNA molecule and a peptide molecule, wherein the carboxyl end of the peptide molecule is covalently linked to the 5 'end or the 3' end of the double-stranded DNA molecule;

after the DNA-peptide molecules are acted by enzyme to be detected, the exposure state of amino-terminal aromatic amino acid is changed;

the exposure state of the amino-terminal aromatic amino acid is changed as follows: (i) the amino terminal of the DNA-peptide molecule does not have exposed aromatic amino acid, and the amino terminal of the DNA-peptide molecule acted by the enzyme to be detected has exposed aromatic amino acid; or, (ii) the amino terminus of the DNA-peptide molecule has an exposed aromatic amino acid, and the amino terminus of the DNA-peptide molecule after the action of the enzyme to be detected does not have an exposed aromatic amino acid;

when the exposure state of amino-terminal aromatic amino acid is changed to be (i) after the DNA-peptide molecules are acted by enzyme to be detected, the host-guest probe is formed by co-incubation of the DNA-peptide molecules acted by the enzyme to be detected and the cucurbituril, and the cucurbituril is connected with the amino-terminal aromatic amino acid in the DNA-peptide molecules acted by the enzyme to be detected through host-guest supramolecule action;

when the exposure state of amino-terminal aromatic amino acid is changed to be (ii) after the DNA-peptide molecule is acted by enzyme to be detected, the host-guest probe is formed by co-incubation of the DNA-peptide molecule without the action of the enzyme to be detected and the cucurbituril, and the cucurbituril is connected with the amino-terminal aromatic amino acid in the DNA-peptide molecule through the action of host-guest supramolecules;

the molar ratio of the DNA-peptide molecules to the cucurbituril is 1:10-250, preferably 1: 10-150.

Further, the length of the double-stranded DNA molecule is 20-60 bp.

Further, the aromatic amino acid is phenylalanine, tryptophan, or tyrosine; phenylalanine is preferred.

Furthermore, the number of amino acids in the DNA-peptide molecule after the action of the enzyme to be detected is less than or equal to 20, preferably less than or equal to 15.

Further, the cucurbiturils are selected from the group consisting of: cucurbit [6] urils (CB [6]), cucurbit [7] urils (CB [7]), cucurbit [8] urils (CB [8 ]); cucurbit [7] urils are preferred.

Further, the reaction condition of co-incubation of the DNA-peptide molecules and the cucurbituril is 4 ℃, and the reaction time is 1-3h, preferably 2 h.

In a third aspect of the present invention, there is provided a method for preparing the subject-guest probe, comprising,

when the exposure state of amino-terminal aromatic amino acid is changed to be (i) after the DNA-peptide molecule is acted by enzyme to be detected, the preparation method of the subject-guest probe comprises the following steps:

mixing the DNA-peptide molecules with enzyme to be detected to enable the enzyme to be detected to perform enzymatic reaction on the DNA-peptide molecules, and then adding the cucurbituril for co-incubation to obtain the subject-guest probe;

when the exposure state of amino-terminal aromatic amino acid of the DNA-peptide molecule is changed to be (ii) after the DNA-peptide molecule is acted by enzyme to be detected, the preparation method of the subject-guest probe comprises the following steps:

and co-incubating the DNA-peptide molecules and the cucurbituril to obtain the subject-object probe.

Further, the reaction condition of the co-incubation is 4 ℃, and the reaction time is 1-3h, preferably 2 h.

In a fourth aspect of the invention, there is provided a system comprising the molecular combination and the sample cell of the invention, or the host-guest probe and the sample cell of the invention;

the sample cell comprises two compartments separated by an insulating membrane; the insulating film is provided with a through hole with the diameter of 100-; an Ag/AgCl electrode is applied in the sample cell to form a closed loop.

Further, the insulating film is a polytetrafluoroethylene film. Still further, the thickness of the polytetrafluoroethylene film was 20 μm.

In a fifth aspect of the invention, there is provided the use of the molecular composition, the host-guest probe or the system for detecting the concentration of an enzyme activity, the enzyme being a protease.

Further, the enzyme is selected from the group consisting of: leucine aminopeptidase, cathepsin B, matrix metalloproteinase, carboxypeptidase M, methionine aminopeptidase.

The sixth aspect of the invention provides a method for detecting the activity concentration of enzyme, which comprises the following steps of mixing enzyme to be detected with the DNA-peptide molecule, enabling the enzyme to be detected to perform enzymatic reaction on the DNA-peptide molecule, and then adding cucurbituril for co-incubation to obtain solution to be detected; and adding the solution to be detected into the sample cell, and detecting the enzyme activity concentration by analyzing the characteristic electric signal.

Further, the reaction condition of the co-incubation is 4 ℃, and the reaction time is 1-3h, preferably 2 h.

Further, the DNA-peptide molecule is in excess relative to the maximum catalytic amount of the enzyme to be detected. In a specific embodiment, for example, for a sample from a tumor patient, the concentration of the DNA-peptide molecule in the mixed reaction solution of the enzyme to be tested and the DNA-peptide molecule may be 200 nM.

Further, an electrolyte solution is pre-added into the compartment of the sample cell, wherein the electrolyte solution is a KCl electrolyte solution or a NaCl electrolyte solution, K⁺Or Na⁺The concentration of (A) is 1-3M, preferably 3M; what is needed isThe pH of the electrolyte solution is 4 to 6. In a specific embodiment, the electrolyte solution is prepared with 10mM Mes-Tris buffer.

Further, the sampling frequency of the electric signal is 100kHz, and the Bessel low-pass filtering cutoff frequency is 10 kHz.

The inventor designs a DNA subject probe and combines the DNA subject probe with a nanopore technology, modifies small peptides with different sequences at the tail end of a DNA molecule, and uses the modified small peptides as substrates corresponding to enzymes to be detected, so as to construct a DNA-peptide molecule required for detecting the activity concentration of the enzymes. And then incubating the DNA-peptide molecules with the enzyme to be detected, and after the enzyme to be detected cuts off partial peptide chains through enzymatic reaction, exposing (or losing) aromatic amino acids which can (cannot) generate subject-object action with cucurbituril to form (cannot) DNA subject-object probes. The DNA subject-object probe generates a characteristic current signal when passing through the nanopore, and the signal has extremely strong specificity, so that the molecular identification can be accurately realized, the problem of background interference is avoided, and the sensitivity is extremely high. In addition, the method for detecting the enzyme activity concentration provided by the invention has the advantages of simple operation, little actual consumption, no need of large instruments and the like.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1 is a schematic diagram of the detection of leucine aminopeptidase;

FIG. 2 is a schematic diagram showing the detection of cathepsin B;

FIG. 3 is a working curve of the determination of the concentration of the enzyme activity of leucine aminopeptidase;

FIG. 4 shows the results of a selective detection assay for leucine aminopeptidase;

FIG. 5 is a working curve of the detection of the enzyme activity concentration of cathepsin B;

FIG. 6 shows the results of selective detection of cathepsin B.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. The quantitative tests in the following examples, each set up three replicates.

DNA-peptide molecules

The DNA-peptide molecule consists of a double-stranded DNA molecule and a peptide molecule, wherein the carboxyl end of the peptide molecule is covalently connected to the 5 'end or the 3' end of the double-stranded DNA molecule. In a specific embodiment, the peptide molecule can be modified at the end of the DNA molecule by a "click reaction" (click reaction), for example, common click reactions include thiol-maleimide addition reaction, amino-carboxyl condensation reaction, alkynyl-azide addition reaction, aldehyde-amino condensation reaction, and the like.

The specific sequence of the double-stranded DNA molecule does not affect its function of detecting the concentration of the enzyme activity, and in a specific embodiment, a DNA sequence of 20-60bp in length should be designed to form a fully base-paired double helix structure without forming other types of secondary structures (e.g., stem loop, hairpin, endocyclic loop, bulge loop, polycyclic loop, etc.).

The peptide chain part in the DNA-peptide molecule can be specifically identified by the enzyme to be detected and subjected to enzyme digestion, so that the exposure state of amino-terminal aromatic amino acid is changed. And the DNA-peptide molecules can not be identified or subjected to enzyme digestion by other molecules except the enzyme to be detected in the detection system.

Concentration of enzyme Activity

The international unit of enzymatic activity is IU (often abbreviated as U), defined as 1IU of enzyme catalyzing 1. mu. mol of substrate per minute under specified conditions. Enzyme activity per volume is commonly used in the art to characterize enzyme concentration, i.e., "enzyme activity concentration", in U/L, which is also the unit used in clinical enzymology assays.

Principle of detection

The following description will take leucine aminopeptidase and cathepsin B as examples to illustrate the principle of enzyme activity concentration detection according to the present invention:

leucine Aminopeptidase (LAP) can hydrolyze the N end of a peptide chain, peptide bonds formed by leucine and other amino acids are widely distributed in tissues such as liver, kidney, pancreas and the like, when the tissues are diseased, the serum LAP level can be increased, and the clinical detection of the enzyme activity concentration of the LAP has important significance for the diagnosis of related diseases. The invention designs a DNA-peptide molecular probe for detecting LAP, wherein the sequence of a peptide molecule is LFGK, as shown in figure 1, when phenylalanine is exposed after the LAP reacts with the peptide molecule, the DNA-peptide molecule acted by the LAP and cucurbit [7] urea form a host-guest probe, the host-guest probe passes through a nanopore to generate a characteristic current signal, and the enzyme activity concentration can be measured by using the frequency change of the characteristic current signal.

Cathepsin B (cat.B) is cysteine proteolytic enzyme existing in lysosomes, and the expression of cat.B is higher than that of adjacent normal tissues by times, even 3-9 times, in various malignant tumor tissues such as lung cancer, gastric cancer, prostatic cancer, breast cancer and the like. B can selectively recognize and degrade polypeptide fragments such as Val-Cit (guanadine), Phe-Lys, Gly-Phe-Leu-Gly, and the like. The invention designs a DNA-peptide molecular probe for detecting cat.B, wherein the sequence of a peptide molecule is FGVCitK, as shown in figure 2, when the cat.B reacts with the peptide molecule to remove phenylalanine, the DNA-peptide molecule acted by the cat.B cannot form a subject-object probe with cucurbit [7] urea, the DNA-peptide molecule can not generate a characteristic current signal after passing through a nanopore, and the enzyme activity concentration can be measured by using the frequency change of the characteristic current signal.