阅读说明：本技术 一种具有双荧光发射的小分子荧光探针的应用 (Application of small-molecule fluorescent probe with double fluorescence emission ) 是由 罗亮 孟凡玲 何珍艳 高玉婷 于 2019-10-09 设计创作，主要内容包括：本发明属于分子检测技术领域,更具体地,涉及一种具有双荧光发射的小分子荧光探针的应用。该小分子荧光探针与双链DNA相互作用时,在350-550nm的激发波长激发条件下,出现双荧光发射,其最强荧光发射峰分别为640±5nm和540±5nm。该荧光探针分子由于其独特的分子结构,使得其与双链DNA相互作用时表现出独有的双荧光发射现象,利用这一现象将其应用于制备双链DNA的特异性识别试剂,由此解决现有技术检测dsDNA过程繁琐复杂的技术问题。该荧光探针具有如式(一)所示的结构：<Image he="609" wi="700" file="DDA0002226744840000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>其中,R为：<Image he="221" wi="251" file="DDA0002226744840000012.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>或者<Image he="266" wi="347" file="DDA0002226744840000013.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>n为1-12的整数。(The invention belongs to the technical field of molecular detection, and particularly relates to application of a small molecular fluorescent probe with double fluorescent emission. When the small molecular fluorescent probe interacts with double-stranded DNA, double fluorescence emission occurs under the excitation condition of excitation wavelength of 350-550nm, and the strongest fluorescence emission peaks are 640 +/-5 nm and 540 +/-5 nm respectively. Due to the unique molecular structure of the fluorescent probe molecule, the fluorescent probe molecule shows unique double-fluorescence emission phenomenon when interacting with double-stranded DNA, and is applied to the preparation of a specific recognition reagent of the double-stranded DNA by utilizing the phenomenon, so that the technical problem that the dsDNA detection process in the prior art is complicated is solved. The fluorescent probe has a structure as shown in formula (I): wherein R is: or n is an integer of 1 to 12.)

1. The application of a fluorescent probe with double fluorescence emission in preparing a detection reagent for specifically recognizing double-stranded DNA (deoxyribonucleic acid), wherein the fluorescent probe has a structure as shown in a formula (I):

wherein R is:

when the fluorescent probe interacts with double-stranded DNA, double fluorescence emission occurs under the excitation condition of excitation wavelength of 350-550nm, and the strongest fluorescence emission peaks are 640 +/-5 nm and 540 +/-5 nm respectively.

2. The use of claim 1, wherein the fluorescent probe molecule is dissolved in water to prepare a detection reagent for specifically recognizing double-stranded DNA.

3. The use of claim 2, wherein the concentration of fluorescent probe in the detection reagent is not less than 1 μ M.

4. The application of a fluorescent probe with double fluorescence emission in preparing a detection reagent for detecting double-stranded DNA of single nucleotide base mutation comprises the following components in percentage by weight:

wherein R is:

when the fluorescent probe interacts with double-stranded DNA, double fluorescence emission occurs under the excitation condition of excitation wavelength of 350-550nm, and the strongest fluorescence emission peaks are 640 +/-5 nm and 540 +/-5 nm respectively;

when the fluorescent probe acts with mismatched double-stranded DNA, the mismatched double-stranded DNA is the single nucleotide mutation dsDNA, and compared with the fully paired double-stranded DNA, the increase rate of the fluorescence intensity of the strongest fluorescence emission peak within the range of 540nm +/-5 nm is weakened along with the increase of the concentration of the mismatched double-stranded DNA; and the greater the degree of attenuation, the greater the degree of mismatch of the double-stranded DNA with respect to the perfectly matched double-stranded DNA.

5. The use according to claim 4, wherein the fluorescent probe molecule is dissolved in water to prepare a detection reagent for detecting double-stranded DNA having a single nucleotide base mutation.

6. The use of claim 5, wherein the concentration of fluorescent probe in the detection reagent is not less than 1 μ M.

7. The application of a fluorescent probe with double fluorescence emission in preparing a detection reagent for detecting damaged double-stranded DNA, wherein the fluorescent probe has a structure as shown in the formula (I):

wherein R is:

when the fluorescent probe interacts with double-stranded DNA, double fluorescence emission occurs under the excitation condition of excitation wavelength of 350-550nm, and the strongest fluorescence emission peaks are 640 +/-5 nm and 540 +/-5 nm respectively;

when the fluorescent probe acts with damaged double-stranded DNA, the increase rate of the fluorescence intensity of the strongest fluorescence emission peak in the range of 540nm +/-5 nm is weakened along with the increase of the concentration of the double-stranded DNA compared with the undamaged double-stranded DNA, and the larger the weakening degree is, the larger the damage degree of the double-stranded DNA relative to the undamaged double-stranded DNA is.

8. The use of claim 7, wherein the fluorescent probe molecule is dissolved in water to prepare a detection reagent for detecting damaged double-stranded DNA.

9. The use of claim 8, wherein the concentration of fluorescent probe in the detection reagent is not less than 1 μ M.

Technical Field

The invention belongs to the technical field of molecular detection, and particularly relates to application of a small molecular fluorescent probe with double fluorescent emission. The fluorescent probe is suitable for specifically recognizing double-stranded DNA (dsDNA) due to the unique double fluorescence emission phenomenon, and has the application of detecting Single Nucleotide Polymorphisms (SNP) and dsDNA damage on the premise.

Background

The DNA strand is a double helix structure constructed by two nucleotides with complementary paired sequences, and can carry all genetic information of organisms. In recent years, a great deal of research and analysis has been conducted on DNA because of its importance in genetic diseases, clinical diagnosis and molecular biology. SNPs are nucleic acid sequence polymorphisms induced by single nucleotide variations in the genome, the most common form of sequence variation in an organism. Accurate detection of SNPs helps to distinguish the genomes of biological individuals and provides a clinical diagnosis for a particular genetic predisposition to a disease.

Most current diagnostic methods for SNPs are based on DNA single base pair mutation hybridization sensors, which consist of a detection method and a signal transducer. Detection methods for specifically recognizing DNA generally utilize single-stranded DNA (ssDNA) and complementary molecular probes to form base pairs. For the detection of DNA, fluorescent or electroactive labels are often coupled to probes or analytes as signal transducers to report hybridization events, with molecular beacons being the most popular example. However, detection by such hybridization requires not only complicated modification and Polymerase Chain Reaction (PCR) amplification of ssDNA, but also denaturation and hybridization of the analyte, which is cumbersome and complicated.

Recently, cationic polythiophene derivatives can rapidly detect SNPs without any chemical reaction of a probe or an analyte. However, precise characterization of these cationic polythiophene derivatives is a difficult problem and requires optimization of detection sensitivity. Furthermore, hybridization is necessary for detection, since the single-stranded DNA probe must first be mixed with the polythiophene before the complementary DNA strand is added.

Commercial DNA intercalating dyes (ethidium bromide, propidium iodide, etc.) or trench binding dyes (Hoechst33258, etc.) show little difference in spectral position or amplitude when compared to ssDNA and dsDNA binding. Therefore, a convenient detection method is necessary, and direct detection of dsDNA can make DNA diagnosis more rapid, sensitive and convenient.

Disclosure of Invention

In view of the above defects or improvement needs of the prior art, the present invention provides an application of a fluorescent probe with dual fluorescence emission, wherein due to its unique molecular structure, the fluorescent probe molecule shows a unique dual fluorescence emission phenomenon when interacting with double-stranded DNA, and the phenomenon is utilized to apply the fluorescent probe molecule to the preparation of a specific recognition detection reagent for double-stranded DNA, thereby solving the technical problem of complex and complicated dsDNA detection process in the prior art.

To achieve the above objects, according to one aspect of the present invention, there is provided a use of a fluorescent probe having dual fluorescence emission for preparing a detection reagent for specifically recognizing a double-stranded DNA, the fluorescent probe having a structure represented by formula (one):

wherein R is:

orn is an integer of 1 to 12;

when the fluorescent probe interacts with double-stranded DNA, double fluorescence emission occurs under the excitation condition of excitation wavelength of 350-550nm, and the strongest fluorescence emission peaks are 640 +/-5 nm and 540 +/-5 nm respectively.

Preferably, the fluorescent probe molecule is dissolved in water to prepare a detection reagent for specifically recognizing double-stranded DNA.

Preferably, the concentration of the fluorescent probe in the detection reagent is not less than 1. mu.M.

According to another aspect of the present invention, there is provided a use of a fluorescent probe having dual fluorescence emission in the preparation of a detection reagent for detecting double-stranded DNA having a single nucleotide base mutation, the fluorescent probe having a structure represented by formula (one):

wherein R is:

or

n is an integer of 1 to 12;

when the fluorescent probe interacts with double-stranded DNA, double fluorescence emission occurs under the excitation condition of excitation wavelength of 350-550nm, and the strongest fluorescence emission peaks are 640 +/-5 nm and 540 +/-5 nm respectively;

when the fluorescent probe acts with mismatched double-stranded DNA, the mismatched double-stranded DNA is the single nucleotide mutation dsDNA, and compared with the fully paired double-stranded DNA, the increase rate of the fluorescence intensity of the strongest fluorescence emission peak within the range of 540nm +/-5 nm is weakened along with the increase of the concentration of the mismatched double-stranded DNA; and the greater the degree of attenuation, the greater the degree of mismatch of the double-stranded DNA with respect to the perfectly matched double-stranded DNA.

Preferably, the fluorescent probe molecule is dissolved in water to prepare a detection reagent for detecting a single nucleotide base mutation of double-stranded DNA.

Preferably, the concentration of the fluorescent probe in the detection reagent is not less than 1. mu.M.

According to another aspect of the present invention, there is provided a use of a fluorescent probe having dual fluorescence emission in preparing a detection reagent for detecting damaged double-stranded DNA, the fluorescent probe having a structure represented by formula (one):

wherein R is:

or

n is an integer of 1 to 12;

when the fluorescent probe interacts with double-stranded DNA, double fluorescence emission occurs under the excitation condition of excitation wavelength of 350-550nm, and the strongest fluorescence emission peaks are 640 +/-5 nm and 540 +/-5 nm respectively;

when the fluorescent probe acts with damaged double-stranded DNA, the increase rate of the fluorescence intensity of the strongest fluorescence emission peak in the range of 540nm +/-5 nm is weakened along with the increase of the concentration of the double-stranded DNA compared with the undamaged double-stranded DNA, and the larger the weakening degree is, the larger the damage degree of the double-stranded DNA relative to the undamaged double-stranded DNA is.

Preferably, the fluorescent probe molecule is dissolved in water to prepare a detection reagent for detecting damaged double-stranded DNA.

Preferably, the concentration of the fluorescent probe in the detection reagent is not less than 1. mu.M.

In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:

(1) the probe of the invention does not need complex modification, PCR amplification, denaturation, analyte hybridization and other complex operations on the ssDNA of the probe, and can conveniently, rapidly, accurately and specifically detect the dsDNA, thereby being used for preparing a detection reagent for specifically recognizing the double-stranded DNA.

(2) The probe of the invention can not only specifically recognize dsDNA, but also detect dsDNA with single nucleotide base mutation. By weakening the action of the probe and the mononucleotide mutation dsDNA, the fluorescence intensity of the probe and the mononucleotide mutation DNA after action can be sensitively detected by a fluorometer to be obviously reduced, the nucleotide mutation of the DNA can be conveniently judged, and the method can be used for preparing a detection reagent for detecting the nucleotide mutation of the DNA.

(3) The probe of the invention can not only specifically identify dsDNA and detect single nucleotide mutant DNA, but also detect the damage condition of dsDNA, and can be used for preparing a detection reagent for detecting damaged double-stranded DNA. The binding of UV damaged dsDNA to the probe TPBT is reduced, which can be detected rapidly by a fluorometer, and different dsDNA damage degrees have different fluorescence intensity reductions at 540 nm. Therefore, the probe can rapidly judge the damage degree of dsDNA. The probe disclosed by the invention has the advantages of convenience, sensitivity and universality in SNP detection.

Drawings

FIG. 1 is a schematic representation of the specific recognition of dsDNA by the fluorescent probe TPBT provided in the present invention.

FIG. 2 is the AIE performance of 10. mu.M of the fluorescent probe TPBT in example 1. Fig. 2(a) is a fluorescence spectrum of TPBT in different volume ratios of a good solvent (water) and a poor solvent (ethanol), and the volume ratios of ethanol are 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, respectively. FIG. 2(b) is a fluorescence spectrum of TPBT after adding polyanionic Heparin sodium (an anticoagulant), the concentration of Heparin is 0, 10, 20, 30, 40, 50 μ g/mL in sequence^-1. FIG. 2(c) is a fluorescence spectrum of TPBT after gradual addition of polyanionic polyacrylic acid (PAA, Mw: 450000), wherein the concentration of PAA is 0, 10, 20, 40, 60 μ g/mL^-1. FIG. 2(d) is a fluorescence spectrum of TPBT after the gradual addition of negatively charged Bovine Serum Albumin (BSA), wherein the BSA concentrations are 0, 50, 100, 150, 200, 250. mu.g.mL^-1. The excitation wavelength is 450 nm; wherein the abscissa is the wavelength and the ordinate is the fluorescence intensity value.

FIG. 3 is a graph showing the fluorescence spectrum of the reaction of 10. mu.M probe TPBT with dsDNA in example 2. FIG. 3 is a fluorescence spectrum of the effect of TPBT on ctDNA at a concentration of 0-20. mu.g/mL^-1. Insert pictures are 10 μ MTPBTRespectively 10 ug/mL^-1And 20. mu.g.mL^-1Fluorescent photograph of ctDNA irradiated under UV lamp.

FIG. 4 shows the specificity of the probe TPBT to dsDNA in example 3 to generate 540nm fluorescence emission. FIG. 4(a) is a fluorescence spectrum of TPBT and ctDNA at different environmental temperatures. The ambient temperature was room temperature, heated to 95 ℃ and cooled from 95 ℃ to room temperature, respectively. FIG. 4(b) shows TPBT and ssDNA (50 adenine residues, A)₅₀) Fluorescence spectrum of action, A₅₀The concentration is 0-1. mu.M. FIG. 4(c) shows TPBT mixed with 1 μ M A₅₀After the action, gradually adding T dropwise₅₀(consisting of 50 thymines) fluorescence spectrum, T₅₀The concentration is 0-2. mu.M. FIG. 4(d) shows TPBT and dsDNA (A)₅₀And T₅₀Mixed) fluorescence spectrum after the action.

FIG. 5 shows the limit of detection of ctDNA detectable by the probe TPBT in example 4. FIG. 5(a) shows that the fluorescence peak at 640nm is saturated to form a mixed system after the probe TPBT reacts with the Heparin. The ctDNA was titrated in the mixed system and the resulting fluorescence spectrum was obtained. FIG. 5(b) is a graph of fluorescence intensity at 540nm versus ctDNA concentration for TPBT versus ctDNA fluorescence titration. Wherein the abscissa is ctDNA concentration and the ordinate is fluorescence intensity at 540 nm.

FIG. 6 is the experiment for detecting HIV mononucleotide mutation by TPBT in example 5. FIG. 6(a) is a nucleotide sequence of HIV DNA, single nucleotide mutant DNA (mis-HIV-A indicates that the base T is substituted by the base A, mis-HIV-G indicates that the base T is substituted by the base G, and mis-HIV-C indicates that the base T is substituted by the base C), and perfect mismatch DNA (comp. mis-HIV). FIG. 6(b) is a fluorescence spectrum of titrated HIV DNA in a mixed system of TPBT and Heparin, HIV DNA concentration: 0-100 nM. FIG. 6(c) is a graph showing the fluorescence spectrum of a titration of mis-HIV-A in a mixed system of TPBT and Heparin, at a concentration of mis-HIV-A: 0-100 nM. Fig. 6(d) is the fluorescence spectrum of the titration of comp.mis-HIV in the TPBT and Heparin mixed system, comp.mis-HIV concentration: 0-100 nM. FIG. 6(e) is a graph showing the relationship between the concentrations of HIV DNA, 3 single nucleotide mutant DNAs and comp.mis-HIV added dropwise to a mixed system of TPBT and Heparin, respectively, and the fluorescence intensities at 540nm corresponding thereto, wherein the abscissa is the concentration of the added DNA and the ordinate is the fluorescence intensity at 540 nm.

FIG. 7 is the experiment for detecting tau DNA single nucleotide mutation by TPBT in example 6. FIG. 7(a) is a nucleotide sequence of tau DNA, single-nucleotide mutant DNA (R406W tau DNA, which shows a substitution of nucleotide C with nucleotide A), and perfect mismatch DNA (Non-match DNA). FIG. 7(b) is a fluorescence spectrum of tau DNA titrated in a mixed system of TPBT and Heparin, and the concentration of tau DNA: 0-96 nM. FIG. 7(c) is a fluorescence spectrum of titrated R406W tau DNA in a TPBT and Heparin mixed system, R406W tau DNA concentration: 0-96 nM. FIG. 7(d) is a fluorescence spectrum obtained by titrating Non-match DNA in a mixed system of TPBT and Heparin, the concentration of Non-match DNA: 0-96 nM. FIG. 7(e) is a graph showing the relationship among tau DNA, R406W tau DNA and Non-match DNA at different concentrations and their corresponding fluorescence intensities at 540nm, which were added dropwise to TPBT and heparin sodium systems, respectively, and the abscissa is the concentration of DNA added and the ordinate is the fluorescence intensity at 540 nm.

FIG. 8 is the result of the TPBT assay on dsDNA UV damage and damage level test in example 7. FIG. 8(a) is a schematic diagram of the experimental procedure of TPBT, which shows that the same concentration of ctDNA is exposed to UV light for different time periods. FIG. 8(b) is a graph of the fluorescence intensity at 540nm versus ctDNA concentration for TPBT versus ctDNA exposed to UV light at different times. FIG. 8(c) is a circular dichroism plot of ctDNA subjected to UV light at various times.

FIG. 9 is a graph of the 540nm and 640nm dual fluorescence emission generated by the fluorescent probe and dsDNA in example 8.

FIG. 10 is a graph showing the single fluorescence emission at 640nm from the dsDNA of the fluorescent probe in comparative example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The invention provides an application of a small molecular fluorescent probe in preparing a detection reagent for specifically recognizing double-stranded DNA, wherein the small molecular fluorescent probe has a structure as shown in a formula (I):

wherein R is:

orn is an integer of 1 to 12; preferably, n is an integer from 1 to 6.

When the small molecular fluorescent probe interacts with double-stranded DNA, double fluorescence emission occurs under the excitation condition of the excitation wavelength of 350-550nm (the maximum absorption wavelength is 450nm), and the strongest fluorescence emission peaks are 640 +/-5 nm and 540 +/-5 nm respectively.

Dissolving the small molecular fluorescent probe in water to prepare the detection reagent for specifically recognizing the double-stranded DNA, wherein in some embodiments, the concentration of the fluorescent probe molecules in the detection reagent is not higher than 10 mu M.

The fluorescent probe and the detection reagent prepared correspondingly can identify double-stranded DNA from a set of solvent, protein, polyanion and double-stranded DNA; the fluorescent probe interacts with a solvent, protein or polyanion and single-stranded DNA (ssDNA), only single fluorescence emission occurs under the condition of an excitation wavelength of 350-550nm, and the peak of the strongest fluorescence emission is 640 +/-5 nm.

The method is applied to identifying double-stranded DNA from a solvent, protein, polyanion and double-stranded DNA, and in some embodiments, the method specifically comprises the following steps: and respectively dripping solution samples containing a solvent, protein, polyanion and double-stranded DNA into the aqueous solution of the fluorescent probe to obtain mixed solution samples of the fluorescent probe, the solvent, the protein, the polyanion and the double-stranded DNA.

And testing the fluorescence spectrogram of each mixed solution sample under the excitation condition of the excitation wavelength of 350-550nm, comparing the fluorescence spectrogram with the initial fluorescence spectrum of the aqueous solution of the fluorescence probe, and obtaining the mixed solution sample with double fluorescence emission and the strongest fluorescence emission peaks of 640 +/-5 nm and 540 +/-5 nm respectively, namely the solution sample containing the double-stranded DNA.

In some embodiments, the initial concentration of the aqueous solution of the fluorescent probe is not less than 1 μ M, and the solution samples containing the solvent, the protein, the polyanion, and the double-stranded DNA are respectively added dropwise to the aqueous solution of the fluorescent probe until each solution sample reaches an equilibrium state in which the rate of increase in fluorescence intensity of each solution sample at the strongest fluorescence emission peak in the range of 540 ± 5nm does not change any more as the concentration thereof increases.

The fluorescent probe of the present invention is a fluorescent probe having AIE properties, which is abbreviated as TPBT in the present invention. In a mixed system of a good solvent and a poor solvent with different volume ratios, the unique fluorescence emission intensity of 640nm is obviously increased along with the increase of the content of the poor solvent.

The fluorescent probe interacts with protein or polyanion with negative charge, and aggregation state can be formed due to electrostatic interaction, so that the unique 640nm fluorescence emission is obviously enhanced. Increasing the ssDNA content in the fluorescent probe solution also resulted in a unique 640nm fluorescence emission enhancement.

When the fluorescent probe molecule provided by the invention interacts with double-stranded DNA, double fluorescence emission occurs under the condition of an excitation wavelength of 450nm, the fluorescence emission peaks are 640 +/-5 nm and 540 +/-5 nm respectively, and the possible mechanism is as follows: the groove combination effect is generated between the planar structure and the large groove of the double-stranded DNA with the double-helix structure, and the molecule rotation and vibration are limited, so that the fluorescent probe molecule generates double-fluorescence emission, and the fluorescent probe molecule can specifically distinguish the double-stranded DNA from other substances, such as solvent, protein molecules and polyanion (macromolecule with negative charge).

The fluorescent probe of the invention acts with dsDNA, and double fluorescence emission is caused, and the fluorescence emission peaks are 640nm and 540nm respectively. With increasing dsDNA content, the 540nm fluorescence emission was significantly enhanced. The mixed system of the action of the fluorescent probe and the dsDNA is heated to 95 ℃ to uncoil the dsDNA and eliminate the 540nm fluorescence emission. Then, the temperature is returned to the room temperature from 95 ℃, and the 540nm fluorescence emission appears again.

In some embodiments of the invention, the use of the fluorescent probe TPBT for specific recognition of dsDNA is verified by the following method. The method comprises the following steps:

(1) preparation of dsDNA: dissolving the ssDNA purified by HPLC with ultrapure water, mixing two ssDNAs with the same amount, diluting, and uniformly oscillating for 5-10min at 37 ℃ and 225rad/min for later use. According to the experimental content, the two ssDNA sequences include the following cases: complementary pairing, single nucleotide mutation, complete mismatch.

(2) Preparation of TPBT aqueous solution: dissolving the purified TPBT in dimethyl sulfoxide to prepare 10 mmol.L^-1The mother liquor is ready for use. Adding 1 μ L of TPBT mother liquor into 999 μ L of ultrapure water to prepare 110 μmol/L^-1TPBT aqueous solution.

(3) And (3) measuring the fluorescence intensity of the step (2) by using a fluorometer, gradually dripping the dsDNA, and measuring the fluorescence spectrum after each dripping of the dsDNA.

In some embodiments, calf thymus DNA (ctDNA) is selected as dsDNA. A similar experimental phenomenon was observed when the dsDNA was exchanged for another.

Further, experiments show that the fluorescent probe can be used for preparing a detection reagent for detecting dsDNA with single nucleotide base mutation, namely the detection reagent for detecting SNP.

Specifically, when the fluorescent probe molecule of the present invention is used with a single nucleotide-mutated dsDNA, the fluorescent probe emits fluorescence at 540nm with a reduced intensity compared to a dsDNA without single nucleotide base mutation.

When the fluorescent probe acts with mismatched double-stranded DNA (namely single nucleotide mutation dsDNA, SNP) compared with the fully paired double-stranded DNA, the increase rate of the fluorescence intensity of the strongest fluorescence emission peak in the range of 540nm +/-5 nm is weakened along with the increase of the concentration of the mismatched double-stranded DNA; and the greater the degree of attenuation, the greater the degree of mismatch of the double-stranded DNA with respect to the perfectly matched double-stranded DNA.

In some examples, the fluorescent probe molecule is dissolved in water to prepare a detection reagent for detecting a single nucleotide base mutation in a double-stranded DNA.

In some embodiments, the concentration of the fluorescent probe in the detection reagent is no less than 1 μ M.

In some embodiments, when the fluorescent probe is used for preparing a detection reagent for detecting dsDNA (i.e., SNP) having a single nucleotide base mutation, i.e., preparing a detection reagent for detecting SNP, and then the detection reagent is used for SNP detection, the method specifically comprises the following steps:

(1) taking a plurality of double-stranded DNAs as samples to be detected, wherein two single-stranded oligonucleotides of each double-stranded DNA have mismatching with different degrees;

(2) respectively dripping the double-stranded DNA samples to be detected with different mismatching degrees into the aqueous solution of the fluorescent probe to obtain a plurality of mixed solution samples of the fluorescent probe and the mismatching double-stranded DNA; dripping the completely paired double-stranded DNA into the fluorescent probe aqueous solution to obtain a mixed solution sample of the fluorescent probe and the completely paired double-stranded DNA;

(3) taking the increase rate of the fluorescence intensity of the strongest fluorescence emission peak of the fully paired double-stranded DNA within the range of 540nm +/-5 nm along with the increase of the concentration of the double-stranded DNA in the dripping process as a reference, and testing the increase rate of the fluorescence intensity of the strongest fluorescence emission peak of the fully paired double-stranded DNA within the range of 540nm +/-5 nm along with the increase of the concentration of the double-stranded DNA in each mixed solution sample in the dripping process; the faster the fluorescence intensity increases, the smaller the degree of mismatch corresponding to the double-stranded DNA; when the fluorescent probe acts with mismatched double-stranded DNA (of single nucleotide mutant dsDNA) compared with fully paired double-stranded DNA, the increase rate of the fluorescence intensity of the fluorescence emission peak at 540nm is weakened with the increase of the concentration of the double-stranded DNA, and the larger the weakening degree, the larger the mismatch degree of the double-stranded DNA relative to the fully paired double-stranded DNA is.

In some embodiments, the double-stranded DNAs with different degree of mismatch in step (1) include double-stranded DNAs with only one base mismatch and double-stranded DNAs with complete base mismatches.

In some embodiments, the concentration of the fluorescent probe in the mixed sample is not less than 1. mu.M, and the concentration of the double-stranded DNA is not more than 100nM, in which the fluorescence intensity of the strongest fluorescence emission peak in the range of 540 nM. + -. 5nM increases linearly with increasing concentration.

In some embodiments, SNP detection of HIV DNA and tau DNA using the fluorescent probes of the invention is universal. After the fluorescent probe is acted with the mononucleotide mutant HIV DNA, the fluorescence intensity of the strongest fluorescence emission peak within the range of 540nm +/-5 nm is obviously reduced and is 0.3-0.5 times of the fluorescence intensity of non-mutant dsDNA. After the fluorescent probe and the single nucleotide mutant tau DNA are reacted, the fluorescence intensity of the strongest fluorescence emission peak within the range of 540nm +/-5 nm is obviously reduced, and is about 0.5 times of the fluorescence intensity of the non-mutant dsDNA.

In a specific embodiment of the invention, single nucleotide mutations in dsDNA that are associated with disease, such as HIVDNA and tau DNA, are selected. Specifically, two ssDNAs with complete mismatch are mixed, wherein one ssDNA is a nucleotide sequence related to diseases, so as to verify the application of the fluorescent probe in the detection of SNP.

In addition, the fluorescent probe of the invention can also be used for preparing a detection reagent for detecting damaged dsDNA.

Specifically, the fluorescent probe of the present invention, when acted upon by damaged dsDNA, exhibits a reduced fluorescence emission at 540 nm.

When the fluorescent probe acts with damaged double-stranded DNA, the increase rate of the fluorescence intensity of the fluorescence emission peak at 540nm is weakened with the increase of the concentration of the double-stranded DNA compared with undamaged double-stranded DNA, and the larger the weakening degree, the larger the damage degree of the double-stranded DNA relative to undamaged double-stranded DNA is.

In some embodiments, the fluorescent probe molecule is dissolved in water to prepare a detection reagent for detecting damaged double-stranded DNA.

In some embodiments, the concentration of the fluorescent probe in the detection reagent is no less than 1 μ M.

When the fluorescent probe of the invention is acted with damaged dsDNA, the fluorescence intensity emitted by fluorescence at 540nm is 0.24-0.69 times of the fluorescence intensity after the action with undamaged DNA.

When the fluorescent probe is used for preparing a detection reagent for identifying damage of dsDNA and/or detecting damage degree of dsDNA, and the reagent is used for detecting damaged double-stranded DNA, some embodiments specifically comprise the following steps:

and taking the double-stranded DNA with different damage degrees as a sample to be detected, and respectively dripping the double-stranded DNA with different damage degrees into the fluorescent probe solution to obtain a mixed solution sample of the double-stranded DNA with different damage degrees and the fluorescent probe solution.

Taking the increase rate of the fluorescence intensity of the strongest fluorescence emission peak of undamaged double-stranded DNA in the range of 540nm +/-5 nm along with the increase of the concentration of the double-stranded DNA in the dripping process as a reference, and testing the increase rate of the fluorescence intensity of the strongest fluorescence emission peak of the double-stranded DNA in the range of 540nm +/-5 nm along with the increase of the concentration of the double-stranded DNA in each mixed solution sample in the dripping process, wherein the faster the increase rate of the fluorescence intensity is, the smaller the damage degree of the double-stranded DNA is.

When the fluorescent probe acts with damaged double-stranded DNA, the increase rate of the fluorescence intensity of the strongest fluorescence emission peak in the range of 540nm +/-5 nm is weakened along with the increase of the concentration of the double-stranded DNA, and the larger the weakening degree is, the larger the damage degree of the double-stranded DNA relative to the undamaged double-stranded DNA is.

In some embodiments, the concentration of the fluorescent probe in the mixed sample is not less than 1. mu.M, and the concentration of the double-stranded DNA is not more than 4. mu.g/mL, in which the fluorescence intensity of the strongest fluorescence emission peak in the range of 540 nm. + -. 5nm is linearly increased as the concentration thereof is increased.

The excitation wavelength of the fluorescent probe applied in the invention is 450nm, the fluorescence intensity is directly measured under a fluorometer, and the emission spectrum is 460nm-800 nm.

The damage to dsDNA described herein is damage caused by ultraviolet light irradiation, x-ray irradiation, or chemical contamination. Such as damage after uv irradiation for different times, such as 0, 30, 120 min.

In view of the characteristics of the fluorescent probe of the present invention, the fluorescent probe can detect DNA structures having double helix-like structure, such as G-quadruplex and i-motif structure, by fluorescence emission at 540 nm.

FIG. 1 is a schematic representation of the specific recognition of dsDNA by the fluorescent probe TPBT provided in the present invention. The figure shows a fluorescent probe of the structure of formula (I) according to the invention, wherein R is

n-1 has good planarity, and may generate a groove effect with DNA having a double helix structure to affect molecular vibration, thereby causing a double fluorescence phenomenon.

The synthesis method of the fluorescent probe molecule TPBT can be found in patent CN 108727256A.

The invention discloses a fluorescent probe with aggregation-induced emission (AIE) performance, which can be used for preparing a detection reagent for specifically identifying dsDNA and Single Nucleotide Polymorphism (SNP) detection by using a unique double-fluorescence emission phenomenon of double-stranded DNA (dsDNA), belonging to the technical field of molecular detection. The invention provides a small molecular probe with positive charge called TPBT, which has weak luminescence in a solution, but acts with polyanion, protein and single-stranded DNA (ssDNA), and the fluorescence emission near 640nm is enhanced. However, when TPBT binds to the dsDNA trench, TPBT shows a new fluorescence emission peak at 540nm, showing highly specific recognition of dsDNA. More importantly, the method can detect SNP and dsDNA damage by a convenient fluorescence detection mode without complex processes such as denaturation, renaturation or hybridization and the like. The invention researches the fluorescent probe which can specifically identify dsDNA and can quickly detect SNP and dsDNA damage, provides a sensitive, convenient and quick method for detecting unmarked SNP and dsDNA damage, and has the monitoring application potential of diseases related to gene mutation.

The fluorescence emission peak of 640nm or 540nm mentioned in the present invention may be shifted in position depending on the species mixed in the solution, and is generally in the range of 640. + -. 5nm and 540. + -.5 nm.

The following are examples: