CN104316705B - Preparation method and application of hybridization indicator 5, 7-dinitro-2-sulfo-acridone - Google Patents

Abstract

The invention provides a preparation method and application of a hybridization indicator 5, 7-dinitro-2-sulfo-acridone, and provides a preparation method of an electrochemical biosensor based on triple signal amplification technology of 'membrane modified electrode', 'exonuclease III auxiliary target sequence circulation' and 'DNA long distance self-assembly', and the electrochemical biosensor is used for ultra-sensitive and high-specificity detection of a lung cancer related target sequence c-erbB-2 related gene. The linear range of the sensor is 2? aM ~ 50? fM, detection limit up to 0.5? aM, and can better identify complete complementary and mismatched sequences, and can realize the detection of the ultra-low content target sequence in clinical practical lung cancer serum samples.

Description

Preparation method and application of a hybridization indicator 5,7-dinitro-2-sulfo-acridone

technical field

The invention relates to a preparation method and application of a hybridization indicator 5,7-dinitro-2-sulfo-acridone, belonging to the field of organic synthesis.

Background technique

Lung cancer is one of the major malignant tumors that threaten human life in the world. In recent years, its morbidity and mortality have increased year by year, ranking first among malignant tumors. Among them, non-small cell lung cancer (NSCLC) accounts for 80% of all lung cancers. . At present, research on the treatment of lung cancer has made great progress, but the overall 5-year survival rate of lung cancer is still only 15%. Even for patients with stage IA NSCL, the 5-year survival rate after surgery is only 80%. Therefore, early and rapid diagnosis of lung cancer is very important for the selection of treatment options and prognosis evaluation.

In recent years, with the development of technologies such as biochemistry, molecular biology and immunology, the research on lung cancer has continued to deepen, and medical workers have made breakthroughs in the screening of tumor markers for lung cancer. Related protein, gene and other markers, especially tumor marker c-erbB-2 and so on. Some researchers have found that c-erbB-2 is abnormally expressed in the serum of lung cancer patients and is stable in nature. It is gradually becoming one of the new tumor markers for reliable early diagnosis of lung cancer. Many scholars have studied the gene markers closely related to NSCLC from the gene level by PCR method. Although PCR can realize the quantitative detection of gene expression, its application in the clinical diagnosis of lung cancer is limited due to the disadvantages of cumbersome operation process, high cost, and the need for specialized technicians. Therefore, the development of a simple, rapid, sensitive and economical c-erbB-2 detection technology has extremely broad application prospects, and it is expected to solve the urgent clinical problem of early diagnosis of NSCLC, which is of great significance.

Biosensors are sensory detectors that use biologically specific recognition processes to detect proteins, genes, antibodies, toxins, etc., and are a new generation of biological detection technology. Among them, electrochemical biosensing is a sensor that uses biological materials (DNA, enzymes, antigens, cells, tissues, etc.) as sensitive elements, electrodes as conversion elements, and detects signals with potential or current characteristics. Determined that this sensor is highly selective. Therefore, scholars have designed many electrochemical biosensing strategies for detecting DNA. Although these sensing strategies can achieve sensitive and specific detection of DNA, there are still the following defects: 1) It is complex, time-consuming and lacks economy. 2) The existence of traditional indicators is expensive, and it will interact with both single- and double-stranded DNA, and the specificity of hybridization detection is not high. 3) Poor stability. 4) Certain functional group labeling methods have large background signals and low sensitivity. 5) False positives and false negatives are still inevitable. These electrical biosensing strategies still face great difficulties in replacing traditional clinical techniques in actual sample detection. Therefore, it is necessary to continue to study a new method for electrochemical biosensing.

The following design and synthesis of a new type of acridone derivatives - "5,7-dinitro-2-sulfo-acridone" with high water solubility and electrochemical activity for the inventors , and using this as a hybridization indicator, developed an electrochemical sensor for the determination of c-erbB-2 related genes - based on "membrane modified electrode", "exonuclease III assisted target sequence recycling" and "DNA long Electrochemical biosensor based on distance self-assembly" triple signal amplification technology: L-lysine is modified on the surface of glassy carbon electrodes, and the hairpin probe DNA is fixed to the terminal carboxyl group of polylysine by covalent modification. Through the hybridization of probe DNA and target DNA, and after exonuclease III cleavage cycle, the surface of the modified electrode leaves a flexible short oligonucleotide sequence, which can hybridize with auxiliary probe genes AP1 and AP2 to form a super sandwich structure. 5,7-Dinitro-2-sulfo-acridone was selected as the hybridization indicator, which can be embedded in the double helix structure of DNA, and the electrochemical biosensor with triple signal amplification technology was successfully developed. Since polylysine contains carboxyl groups, it is beneficial for the immobilization of DNA on the electrode surface. In addition, "polylysine has good conductivity", "exonuclease III assists target sequence circulation" and "DNA long-distance self-assembly" are all The detection sensitivity of the sensor is greatly enhanced. Thus, a highly sensitive and specific c-erbB-2 related gene detection method was established. In the future, on the basis of the successful application of this new technology in the early diagnosis of lung cancer, we will extend it to the early diagnosis of other types of tumors and the screening of new anti-tumor drugs. Therefore, the present invention has huge potential application value and far-reaching significance.

Contents of the invention

The object of the present invention is to provide a preparation method and application of a hybridization indicator 5,7-dinitro-2-sulfo-acridone, to provide a method based on "membrane modified electrode", "exonuclease III assisted The preparation method of electrochemical biosensors based on the triple signal amplification technology of "target sequence recycling" and "DNA long-distance self-assembly", and it is used for ultra-high sensitivity and high specificity of lung cancer-related target sequence c-erbB-2 related genes detection.

To achieve the above object, the present invention adopts the following technical solutions:

A hybridization indicator 5,7-dinitro-2-sulfo-acridone, the structural formula of the 5,7-dinitro-2-sulfo-acridone is .

A method for preparing a hybridization indicator 5,7-dinitro-2-sulfo-acridone, the steps are: weigh 0.6 g of acridone, add 5 mL of concentrated H ₂ SO ₄ , stir to dissolve, Heat to 100°C and react for 30 minutes, pour the solution into ice water (0~4°C) while it is still hot, the solid precipitates, filter and dry to obtain light green solid 2-sulfo-acridone; weigh 0.6g 2-sulfo-acridone Pyridone, add 3mL of 36% acetic acid, then add 0.36mL of concentrated nitric acid and 0.8mL of glacial acetic acid in turn, stir and react at 56~60°C for 2h, pour the solution into ice water while it is hot, the solid precipitates, filter, and use 10~ Rinse with 20 mL of water, and further recrystallize and purify with absolute ethanol to obtain 5,7-dinitro-2-sulfo-acridone as a yellow needle-like powder.

Application of a hybridization indicator 5,7-dinitro-2-sulfo-acridone in an electrochemical sensor.

The preparation method of the electrochemical sensor includes: 1) pretreatment of the electrode

The glassy carbon electrode was ground on 0.05 μm Al ₂ O ₃ powder, and ultrasonically oscillated with absolute ethanol and double distilled water for 5 minutes to remove impurities attached to the electrode surface, and the electrode surface was characterized with 10 mM potassium ferricyanide. Until the peak voltage difference between the front and back is less than 80V, wash with distilled water, dry it, take it out, and blow it dry with nitrogen;

2) Preparation of probe-modified electrodes

Place the treated glassy carbon electrode in pH 8.0, 0.2MPBS buffer solution containing 0.01M/L lysine, use cyclic voltammetry to electrically modify the polylysine membrane, and then rinse the electrode thoroughly with double distilled water and dried at room temperature to obtain a polylysine modified electrode. Invert the polylysine modified electrode, drop 20 μL of coupling activator on the surface, evaporate to dryness at room temperature, and then wash with ultrapure water for 10 seconds to remove unbound Modify the coupling activator on the surface of the electrode, dry it with nitrogen gas for use; then drop 10 μL of 1 μM probe gene P solution with a hairpin structure on the surface of the electrode, evaporate to dryness at room temperature, and wash with ultrapure water to remove the probe that is not bound to the surface of the electrode. Needle gene P, wash with 10mM pH 7.0 PBS buffer solution, and dry at room temperature to obtain a hairpin probe modified electrode;

3) Enzyme-assisted target sequence recycling

Place the hairpin probe modified electrode in a series of concentrations of the target sequence c-erbB-2 related gene (1aM~100fM) solution, hybridize at room temperature for 1 hour, wash and dry, and place the hybridized electrode in 10U exonuclease In the buffer solution of III, react at 37°C for 30 minutes, wash and dry for use;

4) DNA long-distance self-assembly and electrochemical detection

Take 10 μL of 1 μM auxiliary probes AP1 and AP2 solutions (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.) and drop-coat them on the surface of the above electrodes, carry out DNA long-distance self-assembly at room temperature, wash and dry after 2 hours; 5mM K ₃ [Fe(CN) ₆ ]/K ₄ [Fe(CN) ₆ ](1:1) 100mMpH7.0PBS+0.1MKCl buffer solution to measure the CV curves of different modified electrodes, the scan rate is 0.05V/s, sampling The interval is 0.001V, and the resting time is 2s; the electrode after DNA long-distance self-assembly is placed in a pH7.0 PBS buffer solution containing 100μM 5,7-dinitro-2-sulfo-acridone DSA for 20min After washing and drying; then place the electrode in pH5.0 phosphate PB buffer solution to scan CV and square wave voltammetry (SWVs) curves.

The conditions of the cyclic voltammetry are: voltage -1.0~+2.4V, scan rate 100mVs-1, and number of characterization cycles is 14 cycles.

The coupling activator is pH 7.4 phosphate buffer containing 5 mM EDC and 8 mM NHS.

The scan rate of the CV method is 0.50V/s, the sampling interval is 0.001V, and the rest time is 2s; the scan potential range of the square wave voltammetry is-0.2V～+0.15V, and the potential increment is 0.005V, The amplitude is 0.025V.

The sequence of the probe P is 5'-NH2-AAAAATTTATTTGATAGGCGAACTATTTGTTTTTAATATCAAATAATGGTT-3'; the sequence of the probe AP1 is 5'-CAAAATATATGATAGGCGAA-3'; the sequence of the probe AP2 is 5'-ATATATTTTGTTCGCCTATC-3';

The advantages of the present invention are:

1. Synthesized a new type of electrochemical hybridization indicator acridone derivative - "5,7-dinitro-2-sulfo-acridone" (DSA), which has high water solubility and Electrochemical activity, and strong selectivity for single and double strands.

2. Using DSA as a hybridization indicator, an electrochemical biosensor based on the triple signal amplification technology of "membrane modified electrode", "exonuclease III assisted target sequence recycling" and "DNA long-distance self-assembly" was developed. It can be used for ultra-high sensitivity and high specificity detection of lung cancer-related target sequence c-erbB-2 related genes.

3. The linear range of the sensor is 2aM~50fM, the detection limit reaches 0.5aM, and it can better identify fully complementary and mismatched sequences, which can realize the detection of ultra-low content target sequences in actual clinical lung cancer serum samples.

Description of drawings

Figure 1 is the mass spectrum of the electrochemical hybridization indicator 5,7-dinitro-2-sulfo-acridone.

Fig. 2 is a [Fe(CN) ₆ ] ^3-/4- characterization diagram of different modified electrodes of the present invention. Characterization of [Fe(CN) ₆ ] ^3-/4- on different modified electrodes: membrane modified electrode (a), exonuclease III-assisted target sequence cycling before (c) and after (b), DNA long distance self-assembly (d)

Fig. 3 is an electrochemical signal detection diagram of the present invention. Comparison of electrochemical signals A, B, C, D, E, F, and G of different concentrations of target DNA.

detailed description

The present invention is described in detail below in conjunction with accompanying drawing and embodiment:

Example 1

The characterization data of DSA are as follows:

Elemental analysis of DSA: C, 42.83%; H, 1.99%; N, 11.52%. (calculated value: C, 42.75%; H, 1.93%; N, 11.50%); DSA infrared analysis IR (KBr) ν: 3410(υNH), 1592(υC-C), 1642(υC=C), 1389(υC-NO2), 1190(υSO2OH).

Mass spectrometry of DSA FAB-MS: m/z366([M+1]+).

DSA hydrogen spectrum analysis 1HNMR (CDCl3, δ): 9.04 (s, ArH), 8.76 (s, ArH), 8.13 (s, ArH), 7.85 (d, ArH), 6.87 (d, ArH), 4.21 (s , NH).

Example 2

The electrochemical sensor of the present invention comprises a glassy carbon electrode GCE, the surface is coated with a sensitive film, ExoIII enzyme, and auxiliary sequences AP1 and AP2. The sensitive membrane is composed of poly-lysine PLLy and immobilized hairpin probe gene P (5'-NH2-AAAAATTTATTTGATAGGCGAACTATTTGTTTTTAATATCAAATAATGGTT-3'). The glassy carbon electrode immobilized with probe DNA is immersed in a glassy carbon electrode containing a certain concentration of complementary DNA (T1). The hybridization reaction was carried out in PBS (phosphate pH 7.0) buffer solution. Place the hybridized electrode in 10 U of ExoIII enzyme buffer, and eliminate it by shearing at 37°C. Since ExoIII can gradually degrade the probe gene P from the blunt end of the 3′ end until the part that hybridizes with T1 to form a double strand is completely degraded, the remaining The part of the gene not hybridized with T1 remains on the surface of the electrode. At the same time, T1 is released intact and can continue to re-hybrid with another P. In this way, a cycle of hybridization, degradation, and re-hybridization occurs, and one T1 can cause multiple Ps to be digested and degraded. After the above cycle is completed, the probe gene P can transform from a hairpin structure to a flexible short linear structure. Auxiliary probe gene AP1 (5'-CAAAATATATGATAGGCGAA-3') and AP2 (5'-ATATATTTTGTTCGCCTATC-3') solutions were added dropwise on the electrode surface, and DNA long-distance self-assembly was performed at room temperature. The long-distance self-assembled electrode was placed in a pH 7.0 PBS buffer solution containing 5,7-dinitro-2-sulfo-acridone to make it embedded in the double helix structure of DNA. The electrodes can be used for electrochemical detection after cleaning and drying.

The preparation method of above-mentioned sensor

1) Preparation of electrochemical hybridization indicator 5,7-dinitro-2-sulfo-acridone: Weigh 0.6g of acridone, add 5mL of concentrated H ₂ SO ₄ , stir to dissolve, and heat to 100°C After reacting for 30 minutes, pour the solution into ice water while it was hot, the solid precipitated out, filtered and dried to obtain light green solid 2-sulfo-acridone; weighed 0.6g of 2-sulfo-acridone, added 3mL of 36% acetic acid, and then added 0.36mL of concentrated nitric acid and 0.8mL of glacial acetic acid were stirred and reacted at 56~60°C for 2h. Pour the solution into ice water (0-4°C) while hot, and the solid precipitates out. Filter, rinse with 10-20 mL of water, and further recrystallize and purify with absolute ethanol to obtain 5,7-dinitro-2-sulfo-acridone as a yellow needle-like powder.

2) Preparation of polylysine-modified glassy carbon electrode: place the treated glassy carbon electrode in 0.2MPBS (pH8.0) buffer solution containing 0.01ML-lysine, and use cyclic voltammetry to electrically modify poly Lysine film, the conditions are: voltage -1.0~+2.4V, scan rate 100mVs-1, characterization cycle is 14 cycles, then thoroughly rinse the electrode with double distilled water and dry at room temperature to obtain polylysine modified electrode.

3) Design probes for hairpin probe P, c-erbB-2 related genes, AP1 and AP2: hairpin probe P: 5'-NH2-AAAAATTTATTTGATAGGCGAACTATTTGTTTTTAATATCAAATAATGGTT-3' (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.) ; c-erbB-2 related gene: 5'-AACCATTATTTGATATTAAAACAAATAGGCTTG-3' is a specific sequence DNA (also c-erbB-2 related gene synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.); AP1: 5'-CAAAATATATGATAGGCGAA- 3' (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.); AP2: 5'-ATATATTTTGTTCGCCTATC-3' (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.); Probe P was dissolved in TE buffer ( Prepared from 10mM Tris, 1.0mM EDTA and 0.10mM NaCl, and adjusted to pH7.4 with 10mmol/L HCl) to make a 100μM probe solution; dissolve the target DNA in pH7.0PBS buffer to make a 100μM target DNA solution.

4) Immobilization of the hairpin probe gene on the modified electrode: the polylysine modified glassy carbon electrode is fully washed in ethanol and secondary water to remove excess lysine. Add 20 μL of coupling activator (phosphate buffer solution at pH 7.4 containing 5 mM EDC and 8 mM NHS) dropwise on the surface of the electrode, evaporate to dryness naturally at room temperature, and then wash with ultrapure water for 10 seconds to remove unbound on the modified electrode. The coupling activator on the surface was blown dry with nitrogen. Then, 10 μL of 1 μM probe gene P solution with hairpin structure was added dropwise on the electrode surface, and the probe gene P with terminal modified amino group was covalently immobilized on the carboxyl group of polylysine. The P bound on the surface of the electrode was washed with pH 7.0 PBS buffer solution and dried at room temperature to obtain a hairpin probe modified electrode.

5) Enzyme-assisted target sequence cycling: place the hairpin probe modified electrode in a series of concentrations of the target sequence c-erbB-2 related gene solution, hybridize at room temperature for 1 hour, wash and dry. The hybridized electrode was placed in 10U exonuclease III buffer, reacted at 37°C for 30min, washed and dried for use.

6) DNA long-distance self-assembly: Take 10 μL of 1 μM auxiliary probe AP1 and AP2 solutions and drop-coat them on the surface of the above electrode, perform DNA long-distance self-assembly at room temperature, wash and dry after 2 hours. The cyclic voltammetry curves of different modified electrodes were measured in 100mMpH7.0PBS+0.1MKCl buffer solution containing 5mMK3[Fe(CN)6]/K4[Fe(CN)6](1:1), and the scan rate was 0.05V/ s, the sampling interval is 0.001V, and the rest time is 2s. From the experimental results (see Figure 2), the more DNA content on the electrode surface, the smaller the peak current value (Ip); the less the DNA content on the electrode surface, the larger the peak current value (Ip). This is because the DNA phosphate backbone is negatively charged, which prevents [Fe(CN) ₆ ] ^3-/4- with the same charge from going to the electrode surface for redox reaction, resulting in blocked electron transfer and reduced peak current.

The required sensor can be obtained through the above method.

Example 3

The above-mentioned electrochemical sensor is used to detect c-erbB-2 related genes, and the specific detection method is as follows: the glassy carbon electrode immobilized with the hairpin probe DNA is immersed in a solution containing a certain concentration of complementary DNA to carry out hybridization reaction. The hybridized electrode was placed in 10 U of exonuclease III buffer, and the shearing was eliminated at 37°C, and the probe P was transformed from a hairpin structure into a flexible short linear structure. Auxiliary probes AP1 and AP2 solutions were added dropwise on the electrode surface, and DNA long-distance self-assembly was carried out at room temperature. The long-distance self-assembled electrode was placed in a pH 7.0 PBS buffer solution containing 5,7-dinitro-2-sulfo-acridone to allow it to be embedded in the double helix structure of DNA. After the above-mentioned electrodes are washed and dried, they can be placed in PB buffer solution and detected by square wave voltammetry.

The invention uses the square wave voltammetry electrochemical detection technology in the prior art to observe the redox signal change of the indicator. The experimental results are shown in Figure 3. It can be seen from the figure that within a certain range, as the target DNA concentration increases, the amount of double-stranded deoxyribonucleic acid (dsDNA) formed by hybridization increases, and the more flexible short sequences produced by enzyme digestion, thus forming long DNA. The more the amount of distance self-assembly, the more the amount of indicator embedded, and the peak current signal will increase. The peak potential of indicator 5,7-dinitro-2-sulfo-acridone is -0.028V. Determination conditions: the determination medium is PB buffer solution, pH=5.0. Measurement parameters of square wave voltammetry: the potential increment is 0.005V, and the amplitude is 0.025V. Its linear range is: 2aM~50fM. The regression equation is I(μA)=0.5955Log[C/(aM)]+0.5814, and the linear correlation coefficient r is 0.9947. The lowest detection limit of this method for specific sequence DNA is 0.5aM.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCELISTING

<110> Fujian Medical University

<120> Preparation method and application of a hybridization indicator 5,7-dinitro-2-sulfo-acridone

<130>4

<160>4

<170>PatentInversion3.3

<210>1

<211>51

<212>DNA

<213> Probe P

<400>1

aaaaatttatttgataggcgaactatttgtttttaatatcaaataatggtt51

<210>2

<211>20

<212>DNA

<213> probe AP1

<400>2

caaaatatatgataggcgaa20

<210>3

<211>20

<212>DNA

<213> Probe AP2

<400>3

atatattttgttcgcctatc20

<210>4

<211>33

<212>DNA

<213> c-erbB-2 related gene

<400>4

aaccattatttgatattaaaacaaataggcttg33

Claims (8)

1. a hybridization indicator 5,7-dinitro-2-sulfo-acridone, characterized in that: the 5,7-dinitro-2-sulfo-acridone structural formula is . 2. a kind of preparation method of a kind of hybridization indicator 5,7-dinitro-2-sulfo-acridone as claimed in claim 1, is characterized in that: described preparation method step is: weigh 0.6 g of acridone, add 5mL of concentrated H ₂ SO ₄ , stir to dissolve, heat to 100°C for 30min, pour the solution into ice water at 0~4°C while hot, solid precipitates, filter and dry to obtain light green solid 2-sulfo -Acridone: Weigh 0.6g of 2-sulfo-acridone, add 3mL of 36% acetic acid, then add 0.36mL of concentrated nitric acid and 0.8mL of glacial acetic acid in turn, stir and react at 56~60°C for 2h, and dissolve while hot The solution was poured into ice water, the solid precipitated out, filtered, rinsed with 10-20 mL of water, and further recrystallized and purified with absolute ethanol to obtain 5,7-dinitro-2-sulfo-acridone as a yellow needle-like powder. 3. The application of a hybridization indicator 5,7-dinitro-2-sulfo-acridone as claimed in claim 1 on an electrochemical sensor. 4. the application of a kind of hybridization indicator 5,7-dinitro-2-sulfo-acridone on electrochemical sensor according to claim 3, is characterized in that: described electrochemical sensor preparation method Including: 1) Electrode pretreatment The glassy carbon electrode was ground on 0.05 μm Al ₂ O ₃ powder, and ultrasonically oscillated with absolute ethanol and double distilled water for 5 minutes to remove impurities attached to the electrode surface, and the electrode surface was characterized with 10 mM potassium ferricyanide. Until the peak voltage difference between the front and back is less than 80V, wash with distilled water, dry it, take it out, and blow it dry with nitrogen; 2) Preparation of probe-modified electrodes Place the treated glassy carbon electrode in pH 8.0, 0.2MPBS buffer solution containing 0.01M/L lysine, use cyclic voltammetry to electrically modify the polylysine membrane, and then rinse the electrode thoroughly with double distilled water and dried at room temperature to obtain a polylysine modified electrode. Invert the polylysine modified electrode, drop 20 μL of coupling activator on the surface, evaporate to dryness at room temperature, and then wash with ultrapure water for 10 seconds to remove unbound Modify the coupling activator on the surface of the electrode, dry it with nitrogen gas for use; then drop 10 μL of 1 μM probe gene P solution with a hairpin structure on the surface of the electrode, evaporate to dryness at room temperature, and wash with ultrapure water to remove the probe that is not bound to the surface of the electrode. Needle gene P, wash with 10mM pH 7.0 PBS buffer solution, and dry at room temperature to obtain a hairpin probe modified electrode; 3) Enzyme-assisted target sequence recycling Place the hairpin probe modified electrode in a series of concentrations of the target sequence c-erbB-2 related gene solution, wash and dry after hybridization at room temperature for 1 hour, and place the hybridized electrode in 10U exonuclease III buffer , after reacting at 37°C for 30 minutes, wash and dry for later use; 4) DNA long-distance self-assembly and electrochemical detection 10 μL of 1 μM auxiliary probe gene AP1 and AP2 solutions were drip-coated on the surface of the above electrodes, and DNA long-distance self-assembly was carried out at room temperature, washed and dried after 2 hours; respectively, in 5 mM K ₃ [Fe( CN) ₆ ]/K ₄ [Fe(CN) ₆ ] 100mMpH7.0PBS+0.1MKCl buffer solution to measure the CV curves of different modified electrodes, the scan rate is 0.05V/s, the sampling interval is 0.001V, and the resting time is 2s ; Place the electrode after DNA long-distance self-assembly in 10mM pH7.0PBS buffer solution containing 100μM 5,7-dinitro-2-sulfo-acridone, wash and dry after 20min; then place the electrode at 100mMpH5 Scanning CV and square wave voltammetry curves in .0 phosphate PB buffer solution. 5. the application of a kind of hybridization indicator 5,7-dinitro-2-sulfo-acridone on electrochemical sensor according to claim 4, is characterized in that: the condition of described cyclic voltammetry It is: voltage -1.0~+2.4V, scanning rate is 100mVs-1, and the number of characterizing circles is 14 circles. 6. the application of a kind of hybridization indicator 5,7-dinitro-2-sulfo-acridone on the electrochemical sensor according to claim 4, is characterized in that: the coupling activator is containing 5 mM EDC and 8 mM NHS in pH 7.4 phosphate buffer. 7. the application of a kind of hybridization indicator 5,7-dinitro-2-sulfo-acridone on the electrochemical sensor according to claim 4, is characterized in that: the scanning rate of described CV method is 0.50V/s, the sampling interval is 0.001V, and the resting time is 2s; the scanning potential range of square wave voltammetry is -0.2V~+0.15V, the potential increment is 0.005V, and the amplitude is 0.025V. 8. The application of a hybridization indicator 5,7-dinitro-2-sulfo-acridone on an electrochemical sensor according to claim 4, characterized in that: the probe P sequence is 5 '-NH2-AAAAATTTATTTGATAGGCGAACTATTTGTTTTTAATATCAAATAATGGTT-3'; the sequence of the probe AP1 is 5'-CAAAATATATGATAGGCGAA-3'; the sequence of the probe AP2 is 5'-ATATATTTTGTTCGCCTATC-3'.