CN102590494A - Molecular probe for detecting single-stranded and/or double-stranded DNA and application thereof - Google Patents

Abstract

The invention belongs to the technical field of material preparation, and relates to a molecular probe for detecting single-strand and/or double-strand DNA and its application. The present invention optimizes the synthesis method in the prior art, and optimizes the synthesis of [Ru(bpy)2(dppz)] ²⁺ , and finds that it has the functions of single-strand and double-strand probes. The inventor preliminarily believes that the optimization of the synthesis method has caused the change in the spatial configuration of the molecule, that is, there are differences in the bonding modes between the isomers obtained by the synthesis method of the present invention and DNA, which makes the present invention The molecular probe has the function of a single-stranded probe, and the inventors will further explore and study the mechanism of this action.

Description

Molecular probe for detecting single-stranded and/or double-stranded DNA and application thereof

technical field

The invention belongs to the technical field of material preparation, and relates to a molecular probe for detecting single-strand and/or double-strand DNA and its application; specifically, it is a fluorescent molecular probe based on a Ru complex, and its application lies in its ability to combine with single-strand DNA (ssDNA) or double-stranded DNA (dsDNA) interact to generate a fluorescent signal.

Background technique

Nucleic acid is an important carrier of genetic information. In the research and application of biosensing systems, the detection and analysis of nucleic acid has always been a very important research content. Among them, the specific recognition of DNA is very important in genomics, disease diagnosis, virology, and molecular biology. Science and other fields are getting more and more attention and research. In the prior art, research on double-stranded DNA (dsDNA) has been relatively common and mature. However, there are relatively few fluorescent molecular probes capable of detecting single-stranded DNA molecules, which can simultaneously detect single-stranded DNA (ssDNA) and double-stranded DNA. Simple, fast, low-cost, and highly sensitive identification has great challenges, and these technologies are an extremely important part of DNA detection and analysis.

In recent years, absorbance method, fluorescence method, Rayleigh light scattering method, chemiluminescence method and electrochemical method have been widely used in nucleic acid analysis. Fluorescence method has been widely studied because of its easy operation, high sensitivity and visibility. Since Barton et al. found that a series of Ru(II) polypyridine complexes interacted with nucleic acids, they had no fluorescence in aqueous solution, but had strong fluorescence when dsDNA existed in the solution, so they were called Nucleic acid molecular "optical switch". In recent years, nucleic acid molecular "optical switch" _probes have been extensively and deeply studied, and the synthesis and development of specific fluorescent probes has become the ^key to DNA fluorescence sensing strategies. The most classic one, most of the currently known literatures are studies on [Ru(bpy) ₂ dppz] ²⁺ as a dsDNA probe, however, there is no use of [Ru(bpy) ₂ dppz] ²⁺ to detect Reporting of ssDNA molecules.

Although the research on [Ru(bpy)2(dppz)] ²⁺ has been carried out for nearly 20 years, its binding mechanism with DNA has been controversial. At present, it is more recognized that the dppz ligand of this probe is inserted into Layer action inserts into double-stranded DNA. However, in 2009, Barton’s research group found that the various isomers of the probe have different bonding methods with DNA, such as Δ (right-handed isomer) complexes are easy to bind to B-DNA, while ∧ (left-handed isomers) Heterogeneous) type complexes are easy to bond with Z-DNA ^[1] . The main reason for this result is that the bonding mode changes due to the different spatial structures of the probes.

References involved in the present invention:

[1] Lim, M.H., et al., Sensitivity of Ru(bpy)(2)dppz(2+)Luminescence to DNA Defects. Inorganic Chemistry, 2009.48(12): p.5392-5397.

[2] Friedman, A.E., et al., A molecular light switch for DNA: Ru(bpy)2(dppz)2+. Journal of the American Chemical Society, 1990.112(12): p.4960-4962.

[3]HolmLin, R.E., E.D.A.Stemp, and J.K.Barton, Ru(phen)2dppz2+Luminescence: Dependence on DNA Sequences and Groove-Binding Agents. Inorganic Chemistry, 1998.37(1): p.29-34.

[4] Wang, J., et al., Aptamer-Based ATP Assay Using a Luminescent Light Switching Complex. Analytical Chemistry, 2005.77(11): p.3542-3546.

[5]Barton, J.K., E.D.Olmon, and P.A.Sontz, Metal complexes for DNA-mediated chargetransport. Coordination Chemistry Reviews, 2011.255(7-8): p.619-634.

Contents of the invention

The technical problem to be solved by the present invention is to provide a molecular probe capable of detecting ssDNA and/or dsDNA by optimizing the synthesis method, so that the synthesis process of the molecular probe of the present invention is simple and controllable, and the obtained molecular probe can be simply and quickly reflected Whether there are DNA molecules in the solution, and the biggest advantage of the present invention is that it can directly and effectively detect ssDNA and/or dsDNA in the solution, avoiding the cumbersome operation steps and high detection cost caused by the use of expensive fluorescently labeled DNA probes The problem.

The technical solution adopted by the present invention to solve the problems of the technologies described above is as follows:

One, a molecular probe of chemical structural formula I (name: 2,2'-bipyridyl dipyrido[3,2-a:2',3'-c] ruthenium phenazine, referred to as [Ru(bpy) ₂ dppz] ²⁺ ); It is characterized in that it is prepared by the method described in synthetic route II:

1) Synthesis of Compound 1: Dried o-phenanthroline and potassium bromide, added them to fully cooled concentrated sulfuric acid, added dropwise HNO ₃ after fully cooled; heated the reaction system to 40-50° C. for 2 hours, and then Raise the temperature to 80-90°C for 2 hours, then to 120°C for 1 hour, then cool down to 80°C overnight; after cooling to room temperature, neutralize the reaction system to pH=6-7, filter under reduced pressure and use CH ₂ Cl ₂ extraction to CH ₂ Cl ₂ was nearly colorless to obtain the crude product of compound 1.

Further, the crude product was recrystallized in methanol to obtain a pure yellow solid;

Further, when adjusting and neutralizing the product pH, use concentrated NaOH solution first, and then use Na ₂ CO ₃ solution until no bubbles are generated.

2) Synthesis of Compound 2: Dissolve Compound 1 in ethanol, then add dropwise the ethanol solution of o-phenylenediamine; reflux for two hours to obtain a light yellow flocculent precipitate, vacuum filter under reduced pressure, recrystallize and vacuum dry to obtain a spongy yellow solid that is Compound 2.

3) Synthesis of Compound 3: Add N,N'-dimethylformamide to the mixed system of ruthenium trichloride and 2,2'-bipyridyl, heat to reflux for 4 hours, evaporate most of the resulting solution Solvent, after the remaining solution was cooled to room temperature, acetone was added and the temperature was kept at 0°C for 24 hours, and a brown-black solid insoluble matter, namely the crude product of compound 3, was precipitated.

Further, the crude product of compound 3 was filtered under reduced pressure and washed with distilled water twice; the obtained crude product was placed in a mixed solution of water and ethanol with a volume ratio of 1:1, heated and refluxed for one hour, filtered, and then added with chlorine Lithium chloride was fully stirred; finally, the ethanol in the solution was evaporated, and the remaining aqueous solution was cooled and then continued to be cooled in an ice bath; after standing for 24 hours, black crystals were precipitated in the solution; filtered and vacuum-dried to obtain the pure compound 3.

4) Synthesis of the molecular probe of chemical structural formula I: adding compound 3 and compound 2 into ethanol at a molar ratio of 1:1.1, heating and refluxing under nitrogen protection until the reaction solution turns into a red transparent solution, and cooling to room temperature After steaming most of the solvent, adding water to boil, then cooling in an ice-water bath to separate out insoluble matter; after filtering, washing, rotary steaming, and recrystallization, the probe molecule of chemical structural formula I is obtained;

Two, the preparation method of the molecular probe of chemical structural formula I of the present invention:

1) Synthesis of Compound 1: Dried o-phenanthroline and potassium bromide, added them to fully cooled concentrated sulfuric acid, added dropwise HNO ₃ after fully cooled; heated the reaction system to 40-50° C. for 2 hours, and then Raise the temperature to 80-90°C for 2 hours, then to 120°C for 1 hour, then cool down to 80°C overnight; after cooling to room temperature, neutralize the reaction system to pH=6-7, filter under reduced pressure and use CH ₂ Cl ₂ extraction to CH ₂ Cl ₂ was nearly colorless to obtain the crude product of compound 1.

Further, the crude product was recrystallized in methanol to obtain a pure yellow solid.

Further, when adjusting and neutralizing the product pH, use concentrated NaOH solution first, and then use Na ₂ CO ₃ solution until no bubbles are generated.

2) Synthesis of Compound 2: Dissolve Compound 1 in ethanol, then add dropwise the ethanol solution of o-phenylenediamine; reflux for two hours to obtain a light yellow flocculent precipitate, vacuum filter under reduced pressure, recrystallize and vacuum dry to obtain a spongy yellow solid that is Compound 2.

3) Synthesis of Compound 3: Add N,N'-dimethylformamide to the mixed system of ruthenium trichloride and 2,2'-bipyridyl, heat to reflux for 4 hours, evaporate most of the resulting solution Solvent, after the remaining solution was cooled to room temperature, acetone was added and the temperature was kept at 0°C for 24 hours, and a brown-black solid insoluble matter, namely the crude product of compound 3, was precipitated.

Further, the crude product of compound 3 was filtered under reduced pressure and washed with distilled water twice; the obtained crude product was placed in a mixed solution of water and ethanol with a volume ratio of 1:1, heated and refluxed for one hour, filtered, and then added with chlorine Lithium chloride was fully stirred; finally, the ethanol in the solution was evaporated, and the remaining aqueous solution was cooled and then continued to be cooled in an ice bath; after standing for 24 hours, black crystals were precipitated in the solution; filtered and vacuum-dried to obtain the pure compound 3.

4) Synthesis of the molecular probe of chemical structural formula I: adding compound 3 and compound 2 into ethanol at a molar ratio of 1:1.1, heating and refluxing under nitrogen protection until the reaction solution turns into a red transparent solution, and cooling to room temperature After steaming most of the solvent, adding water to boil, then cooling in an ice-water bath to separate out insoluble matter; after filtering, washing, rotary steaming, and recrystallization, the probe molecule of chemical structural formula I is obtained;

3. Application of the molecular probe of the chemical structural formula I of the present invention.

Specifically, the molecular probe with chemical structural formula I and single-stranded and/or double-stranded DNA are dissolved in HCl-Tris buffer to prepare an aqueous solution, and the fluorescent probe obtained by compounding is used for single-stranded and/or double-stranded DNA fluorescence analysis.

Further, the Tris-HCl is weakly acidic or weakly basic.

Compared with prior art, the beneficial effect of the present invention is:

1. The present invention has synthesized a water-soluble fluorescent probe molecule that can interact with single-stranded DNA; through the measurement of the fluorescence phenomenon of the detection system, the probe molecule can directly and effectively detect ssDNA and dsDNA in the water medium; the present invention does not need Labeled fluorescent probes have low detection cost, easy operation, and high feasibility of the preparation process, which avoids the problems of cumbersome operation and high detection cost caused by the use of expensive fluorescently-labeled DNA probes;

2. The present invention optimizes the synthesis method in the prior art. The specific difference is: in the step 1) of the present invention, when the product pH is adjusted and neutralized, a concentrated NaOH solution is used first, and then Na ₂ CO ₃ solution Until no bubbles are generated; and after neutralization, vacuum suction filtration is added before extraction. And the method of prior art is directly neutralized with NaOH solution, and does not have suction filtration operation. In addition, in the step 4) of the present invention, the precipitation product is purified by evaporation concentration and mixed solvent method recrystallization; while the traditional method adds BF4-type anion precipitation product for purification.

In the prior art, it has been widely used to produce fluorescence when the probe with the same chemical structure described in the present invention is bonded to double-stranded DNA, but there is no report on the phenomenon of fluorescence when it interacts with single-stranded DNA ^[2-5] . In the present invention, [Ru(bpy)2(dppz)] ²⁺ is optimized and synthesized by the above-mentioned method, and it is found that it has the functions of single-strand and double-strand probes. The inventor preliminarily believes that the optimization of the synthesis method has caused the change in the spatial configuration of the molecule, that is, there are differences in the bonding modes between the isomers obtained by the synthesis method of the present invention and DNA, which makes the present invention The molecular probe has the function of a single-stranded probe, and the inventors will further explore and study the mechanism of this action.

Description of drawings

Fig. 1 is the NMR spectrum of [Ru(bpy) ₂ (dppz)] ²⁺ in CD ₃ CN (deuterated acetonitrile) of the present invention;

δH(CD3CN)9.66(2H, d, J=8.4Hz), 8.57(4H, t, J=9.3Hz), 8.48(2H, dd, J=3.3, 6.6Hz), 8.20-8.10(6H, overlapping multiplets ), 8.03 (2H, td, J=1.0, 7.9Hz), 7.90 (2H, dd, J=5.4, 8.1Hz), 7.87 (2H, dd, J=0.9, 5.1Hz), 7.74 (2H, d, J = 5.5Hz), 7.48 (2H, ddd, J = 0.6, 6.4Hz), and 7.27 (2H, ddd, J = 1.2, 6.3Hz).

Fig. 2 is the ultraviolet-visible absorption spectrum of [Ru(bpy) ₂ (dppz)] ²⁺ in aqueous solution of the present invention.

Fig. 3 is the fluorescence emission spectrogram of [Ru(bpy) ₂ dppz] ²⁺ solution of the present invention and certain concentration dsDNA solution of different concentrations;

Among them, the main figure: different concentrations of [Ru(bpy) ₂ (dppz)] ²⁺ (0, 2, 20, 40, 100, 200, 400 μmol L ^-1 ) and 10 μmol L ^-1 dsDNA fluorescence curves, The intensity of fluorescence signal increases with the concentration of [Ru(bpy) ₂ (dppz)] ²⁺ ; inset: the graph of the change of fluorescence intensity with the concentration of [Ru(bpy) ₂ (dppz)] ²⁺ .

Fig. 4 is different concentration ssDNA solution and certain concentration [Ru(bpy) ₂ dppz] ²⁺ solution fluorescence emission spectrogram of the present invention;

Among them, the main figure: 10 μL and 40 μmol ^{L -1 [} Ru(bpy) ₂ (dppz )] ²⁺ fluorescence curve; the inset is the curve of fluorescence intensity changing with DNA3 concentration.

Fig. 5 is the fluorescence curves of [Ru(bpy)2(dppz)] ²⁺ with 10 μmol·L ^-1 and 40 μmol·L ^-1 of 6 kinds of ssDNA.

Among them, the DNA1-6 sequence is placed at the bottom.

Fig. 6 is a cyclic voltammetry (CV) diagram of using the sulfhydryl group on DNA4 to immobilize the gold electrode, and placing the electrode in [Ru(bpy) ₂ (dppz)] ²⁺ probe solution and buffer blank solution.

Fig. 7 is a chronoelectric (CC) diagram of using DNA4 immobilized on a gold electrode and placing the electrode in [Ru(bpy) ₂ (dppz)] ²⁺ probe solution and buffer blank solution.

Detailed ways

Example 1

This example illustrates the synthesis method of the molecular probe [Ru(bpy) ₂ dppz] ²⁺ with chemical structural formula I described in the present invention, as shown in Route II.

1) Synthesis of Compound 1: Weigh 2.0 g of o-phenanthroline and 3.5 g of potassium bromide and put them in a vacuum drying oven to dry overnight, add them to 20 mL of concentrated sulfuric acid fully cooled by an ice-water bath, and drop them after fully cooling Add 10mL HNO ₃ , remove the ice-water bath, heat to 40-50°C with an oil bath, keep the temperature constant for 2 hours, raise the temperature to 80-90°C and keep the temperature for 2 hours, then keep the temperature at 120°C for 1 hour, then cool down to 80°C and keep the temperature overnight. After cooling to room temperature, neutralize to pH=6-7 (not greater than 7), filter under reduced pressure, and then extract the filtrate with CH ₂ Cl ₂ until CH ₂ Cl ₂ is nearly colorless. Rotary evaporation and recrystallization in methanol gave 3.8 g of yellow solid.

2) Synthesis of compound 2: 1.2 g of o-phenanthrolinedione was dissolved in 40 mL of ethanol, and then an ethanol solution of 0.64 g of o-phenylenediamine was added dropwise. Reflux for two hours to obtain a pale yellow flocculent precipitate, which was filtered under reduced pressure, recrystallized and dried in vacuo to obtain a spongy yellow solid.

3) Synthesis of compound 3: Weigh 1.04g of ruthenium trichloride and 1.29g of 2,2'-bipyridine, put the two drugs into a 100mL dry and clean flask, and then add N,N'-dimethyl Formamide 50mL, after heating to reflux for 4 hours, evaporate most of the solvent of the obtained solution, after cooling the remaining solution to room temperature, add 50mL acetone, put it in the refrigerator and keep the temperature at 0°C, and let it stand for 24 months hour, at this time, a brownish-black solid insoluble matter was precipitated, and then the product was filtered out by vacuum filtration, and the crude product was washed twice with an appropriate amount of distilled water; In the mixed solution, continue to heat and reflux for one hour, filter, then add 15g of lithium chloride, and stir fully; evaporate the ethanol in the solution, and continue to cool the remaining aqueous solution in an ice bath after cooling; after standing for 24 hours, Black crystals precipitated out of the solution. Filter and dry in vacuo.

4) Synthesis of compound 4: Add 0.30g Ru(bpy) ₂ Cl ₂ ·2H ₂ O and 0.18g dppz (molar ratio 1:1.1) into a 50mL round bottom flask, use 25mL ethanol as solvent, and protect Heated to reflux until the reaction solution turned into a red transparent solution, and cooled to room temperature. Most of the solvent was evaporated, then a certain amount of water was added and boiled at the same time, and then cooled in an ice-water bath. At this time, a large amount of insoluble matter was precipitated (dppz is insoluble in water). After filtration, washing, rotary evaporation and recrystallization by mixed solvent method, the target product can be obtained.

5) Dissolve the [Ru(bpy) ₂ dppz] ²⁺ complex and single-stranded (double-stranded) DNA in HCl-Tris buffer to prepare an aqueous solution of a certain concentration, then mix, and the resulting fluorescent probe can be used For the fluorescence analysis of single-stranded (double-stranded) DNA.

In the step 3), when adjusting and neutralizing the pH of the product, the concentrated NaOH solution is first used, and then the Na ₂ CO ₃ solution is used until no bubbles are generated.

In the step 5), the Tris-HCl used can be weakly acidic or weakly alkaline.

The finally obtained chemical structure formula is [Ru(bpy) ₂ (dppz)] ²⁺ in CD ₃ CN (deuterated acetonitrile) in the nuclear magnetic resonance spectrogram of I as shown in Figure 1; Its ultraviolet-visible absorption spectrogram in aqueous solution as shown in picture 2.

Example 2

This example illustrates the application of the molecular probe with the chemical structural formula I, that is, dissolving it with single-stranded and/or double-stranded DNA in HCl-Tris buffer to prepare an aqueous solution, and compounding the obtained fluorescent probe for single-stranded and/or fluorescence analysis of double-stranded DNA.

Wherein, the ssDNA involved in the six groups of ssDNA sample solutions are:

DNA1: CTCACTATAGGAAGAGATGGATGTCTGT;

DNA2: ACAGACATCATCTCTGAAGTAGCGCCGCCGTATAGTGAG;

DNA3: ACTCACTATAGGAAGAGATTCTGT;

DNA4: HS-(CH2)6-AAAGCGGTTGTGTTCAGTTGC;

DNA5: GCACGCCTCACTATAGGAAGAGATGATTGCGTGC;

DNA6: AATCATCTCTGAAGTAGCGCCGCCGTATAGTGAG.

The above primers were synthesized and purified by Shanghai Sangon Inc.

The dsDNA involved in the dsDNA solution is formed by the complementarity of DNA5 and DNA6, and the above primers were synthesized and purified by Shanghai Sangon Inc.

(1) dsDNA interacts with [Ru(bpy) ₂ (dppz) of the present invention] ²⁺ probe molecules of different concentrations

Weigh an appropriate amount of the Ru complex probe synthesized in Example 1, dissolve it in 5mmol·L ^-1 Tris, 50mmol·L ^-1 NaCl, pH7.4 buffer solution, and prepare a series of probe solutions with different concentrations. Pipette 100 μL of 7 probe solutions with different concentrations and drop them into 100 μL of 10 μmol L ^-1 dsDNA solution, so that the final concentration of the probe solution is 0, 1, 10, 20, 50, 100, 200 μmol ^L-1 . At room temperature, use a Molecular Devicws Model Spectramax M5e microplate reader to excite at a wavelength of 440nm, and detect the wavelength range of 500nm to 800nm. The experimental results are shown in Figure 3: the dsDNA can cause the probe molecules to generate fluorescence, and as the concentration of the probe molecules increases, the fluorescence intensity first increases and then becomes flat, indicating that the combination of the probe and dsDNA tends to be saturated, indicating that the present invention dsDNA in aqueous media can be efficiently detected without a large number of probes.

(2) Detection of ssDNA molecules based on [Ru(bpy) ₂ (dppz)] ²⁺ probe

An appropriate amount of the synthesized Ru complex probe was weighed and dissolved in 5mmol·L ^-1 Tris, 50mmol·L ^-1 NaCl, pH 7.4 buffer solution to prepare a probe solution with a concentration of 40μmol·L ^-1 . a. Pipette 100 μL of the probe solution and add dropwise to the DNA3 to be tested at concentrations of 0, 1×10 ^-3 , 1×10 ^-2 , 0.1, 1, 5, 10, 25, 50, 100 ^{μmol·L -1} in solution. At room temperature, use a Molecular Devicws Model Spectramax M5e microplate reader to excite at a wavelength of 440nm, and detect the wavelength range of 500nm to 800nm. The experimental results are shown in Figure 4: when there is no ssDNA target, no fluorescence occurs; as the concentration of ssDNA increases, the fluorescence intensity increases continuously, indicating that this probe can effectively detect ssDNA in aqueous media. b. Pipette 100 μL of the probe solution and add dropwise to 100 μL of 10 μmol L ^-1 DNA1-DNA6 sample solution respectively, and perform a fluorescence test under the same conditions. The experimental results are shown in Figure 5: 6 kinds of single-stranded DNA and probe molecules Fluorescence was generated after the action, indicating that this probe can be widely used in the detection of various single-stranded DNA, wherein DNA4 is DNA containing sulfhydryl modification.

(3) Electrochemical characterization of the interaction between [Ru(bpy) ₂ (dppz)] ²⁺ probe and ssDNA

In the electrochemically modified electrode, the gold electrode (2mm diameter, Shanghai Chenhua) is modified with DNA4 labeled at the 5-terminal with a mercapto group, and the exposed part of the electrode is protected with mercaptoethanol to keep the DNA4 in an upright structure, which can well avoid electrode contamination. Physisorption with [Ru(bpy) ₂ dppz] ²⁺ probe present. Immerse the cleaned gold electrode in a mixed solution containing 1 μmol L ^-1 DNA4, 100 mmol L ^-1 oxalate/100 mmol L ^-1 phosphate buffer solution (pH 5.5) and 1 mmol L ^-1 TCEP for 10 h , and then submerged in 1mmol·L ^-1 mercaptoethanol solution for 2h, and after washing, a gold electrode with assembled DNA4 was obtained. The prepared DNA4 assembly electrodes were placed in the blank solution and 50 μmol L ^-1 [Ru(bpy) ₂ dppz] ²⁺ solution for electrochemical (cyclic voltammetry CV, chronocoulometry CC) scanning (scan Both speeds are 50mV·s ^-1 ), and the corresponding CV/CC electrical signals are obtained (see Figure 6 for CV and Figure 7 for CC). It can be seen from Figure 6 that the potential peak of [Ru(bpy) ₂ dppz] ²⁺ combined with ssDNA was at -0.17V, and the current value increased significantly. It can be seen from Figure 7 that after adding the [Ru(bpy) ₂ dppz] ²⁺ probe solution, the charge density increases obviously, and the charge transfer accelerates, indicating that the probe has bound to ssDNA.

The invention proposes a method for detecting single-strand and double-strand DNA based on Ru complex fluorescent molecular probe. By synthesizing the [Ru(bpy) ₂ (dppz)] ²⁺ probe with good water solubility, it can be used to quench the fluorescence in aqueous solution and produce obvious fluorescence in the presence of ssDNA or dsDNA to detect ssDNA in aqueous medium and dsDNA, the detection system involved in the present invention does not contain expensive fluorescently labeled probes, and is simple to operate, which lays a certain foundation for the application of simultaneous detection of ssDNA and dsDNA. Therefore, in the preparation and detection of ssDNA and dsDNA probes It has important practical application value and potential application prospect.

Claims (10)

1. A molecular probe of chemical structural formula I is characterized in that it is prepared by the method described in synthetic route II: 1) Synthesis of Compound 1: Dried o-phenanthroline and potassium bromide and added them to fully cooled concentrated sulfuric acid, then added dropwise HNO ₃ after fully cooled; heated the reaction system to 40-50°C for 2 hours, and then Raise the temperature to 80-90°C for 2 hours, then to 120°C for 1 hour, then cool down to 80°C overnight; after cooling to room temperature, neutralize the reaction system to pH=6-7, filter under reduced pressure and use CH ₂ Cl ₂ extraction to CH ₂ Cl ₂ is nearly colorless to obtain the crude product of compound 1; 2) Synthesis of Compound 2: Dissolve Compound 1 in ethanol, then add o-phenylenediamine ethanol solution dropwise; reflux for two hours to obtain light yellow flocculent precipitate, filter under reduced pressure, recrystallize and vacuum dry to obtain a spongy yellow solid that is Compound 2; 3) Synthesis of compound 3: Add N,N'-dimethylformamide to the mixed system of ruthenium trichloride and 2,2'-bipyridyl, heat and reflux for 4 hours, evaporate most of the obtained solution Solvent, after cooling the remaining solution to room temperature, add acetone and keep the temperature at 0°C for 24 hours, and a brown-black solid insoluble matter is precipitated, which is the crude product of compound 3; 4) Synthesis of the molecular probe of chemical structural formula I: add compound 3 and compound 2 into ethanol at a molar ratio of 1:1.1, heat and reflux under nitrogen protection until the reaction solution turns into a red transparent solution, and cool to room temperature After steaming most of the solvent, adding water to boil, then cooling in an ice-water bath to separate out insoluble matter; after filtering, washing, rotary steaming, and recrystallization, the probe molecule of chemical structural formula I is obtained;

I;

II. 2. The molecular probe of chemical structural formula I according to claim 1, characterized in that the crude product in step 1) is recrystallized in methanol to obtain a pure yellow solid. 3. The molecular probe of chemical structural formula I according to claim 1, characterized in that in the step 1) when adjusting and neutralizing the pH of the product, first use concentrated NaOH solution, then use Na ₂ CO ₃ solution until there are no bubbles produce. 4. The molecular probe of chemical structural formula I according to claim 1, characterized in that in step 3) the crude product of compound 3 is filtered under reduced pressure and washed with distilled water twice; then the crude product obtained is placed in Water and ethanol are heated and refluxed for one hour in the mixed solution of water and ethanol with a volume ratio of 1:1, then filtered, and then lithium chloride is added to fully stir; finally, the ethanol in the solution is evaporated, and the remaining aqueous solution is cooled and then continued to be placed in an ice bath for cooling; After standing still for 24 hours, black crystals were precipitated in the solution; filtered and vacuum dried to obtain pure compound 3. 5. the preparation method of the molecular probe of chemical structural formula I described in claim 1 is characterized in that it obtains by the method described in synthetic route II: 1) Synthesis of Compound 1: Dried o-phenanthroline and potassium bromide and added them to fully cooled concentrated sulfuric acid, then added dropwise HNO ₃ after fully cooled; heated the reaction system to 40-50°C for 2 hours, and then Raise the temperature to 80-90°C for 2 hours, then to 120°C for 1 hour, then cool down to 80°C overnight; after cooling to room temperature, neutralize the reaction system to pH=6-7, filter under reduced pressure and use CH ₂ Cl ₂ extraction to CH ₂ Cl ₂ is nearly colorless to obtain the crude product of compound 1; 2) Synthesis of Compound 2: Dissolve Compound 1 in ethanol, then add o-phenylenediamine ethanol solution dropwise; reflux for two hours to obtain light yellow flocculent precipitate, filter under reduced pressure, recrystallize and vacuum dry to obtain a spongy yellow solid that is Compound 2; 3) Synthesis of compound 3: Add N,N'-dimethylformamide to the mixed system of ruthenium trichloride and 2,2'-bipyridyl, heat and reflux for 4 hours, evaporate most of the obtained solution Solvent, after cooling the remaining solution to room temperature, add acetone and keep the temperature at 0°C for 24 hours, and a brown-black solid insoluble matter is precipitated, which is the crude product of compound 3; 4) Synthesis of the molecular probe of chemical structural formula I: add compound 3 and compound 2 into ethanol at a molar ratio of 1:1.1, heat and reflux under nitrogen protection until the reaction solution turns into a red transparent solution, and cool to room temperature After steaming most of the solvent, adding water to boil, then cooling in an ice-water bath to separate out insoluble matter; after filtering, washing, rotary steaming, and recrystallization, the probe molecule of chemical structural formula I is obtained; I;

II. 6. The method for preparing the molecular probe of chemical structural formula I according to claim 5, characterized in that the crude product in step 1) is recrystallized in methanol to obtain a pure yellow solid. 7. The preparation method of the molecular probe of the chemical structural formula I according to claim 1, characterized in that in the step 1) when neutralizing the product pH, first use a concentrated NaOH solution, and then use a Na ₂ CO ₃ solution until no bubbles are produced. 8. The method for preparing the molecular probe of chemical structural formula I according to claim 1, characterized in that in step 3) the crude product of compound 3 is filtered under reduced pressure and the crude product is washed twice with distilled water; The product is placed in a mixed solution of water and ethanol with a volume ratio of 1:1, heated and refluxed for one hour, then filtered, and then lithium chloride is added to stir thoroughly; finally, the ethanol in the solution is evaporated, and the remaining aqueous solution is cooled and then placed in an ice bath Cooled in medium; after standing for 24 hours, black crystals were precipitated in the solution; filtered and vacuum-dried to obtain the pure product of compound 3. 9. the application of the molecular probe of chemical structural formula I described in claim 1 is characterized in that chemical structural formula is that the molecular probe of I and single-stranded and/or double-stranded DNA are dissolved in the HCl-Tris damping fluid and are mixed with aqueous solution , the resulting fluorescent probes are used for fluorescence analysis of single-stranded and/or double-stranded DNA. 10. The application according to claim 9, characterized in that said Tris-HCl is weakly acidic or weakly alkaline.

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