Preparation method of bisazopyridine borate and quantum dot fluorescent probe

Abstract

The invention discloses a preparation method of a bisazopyridine borate and quantum dot fluorescent probe, and belongs to the technical field of molecular switch type fluorescent sensors. Comprising the following steps: diluting water-soluble carboxyl quantum dots in proportion by taking a boric acid buffer solution as a solvent; the preparation method comprises the following steps: dissolving bisazopyridine borate in an ethanol solution, and carrying out ultrasonic dissolution; and then adding into a diluted quantum dot solution, and uniformly mixing to obtain the bisazopyridine borate and the quantum dot fluorescent probe. On the basis of the electron energy transfer principle (PET), pyridine groups of azo molecules have an electrostatic attraction effect on the periphery of charges around the quantum dots, so that the fluorescence of the quantum dots is quenched; glucose and boric acid groups on azo molecules form cyclic borate, and charges are redistributed, so that fluorescence of the quantum dots is recovered; the switch type fluorescence sensor for detecting glucose has the advantages of simplicity in manufacturing, sensitivity and stability.

Description

Technical Field

[0001] The invention relates to a method for preparing a disazopyridine borate@quantum dot fluorescent probe, and belongs to the technical field of molecular switch type fluorescent sensors. Background Art

[0002] As people's living standards improve, the prevalence of diabetes continues to increase, and the prevalence among adults has reached 11.9%. The International Diabetes Federation (IDF) predicts that the number of diabetes patients will increase to 629 million by 2045. Therefore, blood sugar concentration detection has become a focus of increasing attention.

[0003] At present, the development of blood glucose sensors has gone through four generations. The first three generations used glucose oxidase and electrodes to achieve glucose sensing. There were problems such as unstable enzyme-based systems and harsh storage environments. Therefore, non-enzyme blood glucose sensors have attracted more attention. Compared with enzyme sensors, non-enzyme glucose sensors have the advantages of low cost, stability and long service life, and have a large application market in the field of blood glucose testing.

[0004] Carboxyl water-soluble quantum dots are obtained by surface modification of oil-soluble quantum dots with polymers. They can be evenly dispersed in aqueous solutions (such as borate buffer, PBS buffer solution, etc.). The surface carboxyl groups are reactive, with a wide absorption spectrum and a narrow emission spectrum in the visible light range. Their optical properties can be controlled by adjusting the size and composition of the particles, making them attractive for use in the field of fluorescent probes.

[0005] In aqueous media, the best ligand for glucose is boric acid, which can reversibly bind to dihydroxy compounds on glucose with high affinity. Boric acid is widely available, inexpensive, and stable, and can work in harsh environments. Therefore, it is often used as a sugar molecule recognition unit to design synthetic sugar sensors.

[0006] Azopyridine compounds are a type of photosensitizing material. Their -N=N- double bond can transform into cis and trans isomers under the conditions of light of a specific wavelength or heating. During the transformation of these two isomers, the dipole moment, geometric size and energy of the azobenzene compound change, thus giving the material some special properties. It is widely used as an effective fluorescence quencher in fluorescence sensing probes.

[0007] Photoinduced electron transfer (PET) occurs between the photoexcited electron acceptor (expressed as a fluorophore) and the electron donor (expressed as an acceptor unit), thereby quenching fluorescence. In a PET fluorescent molecular probe, once the acceptor unit binds to a guest, PET will be inhibited or even completely blocked, while the fluorophore will emit fluorescence.

[0008] Therefore, a method for preparing a disazopyridine borate@quantum dot fluorescent probe (switch-type fluorescent sensor) is developed, and the electrostatic interference of the azopyridine group is used to disrupt the charge distribution of the quantum dots, so that the fluorescence of the quantum dots is quenched; and the synthesized pyridine group can be combined with boric acid to form a pyridinium salt. As an electron-withdrawing group, the pyridinium salt has an inductive effect. Its electron-withdrawing effect can reduce the electron density on the boron atom, thereby making the borate more acidic and the pKa value lower, close to the glucose sensing conditions under physiological conditions, thereby solving the problem that the pH value of the general glucose response is higher than the physiological pH value, and can also optimize the problem of instability of the enzyme-based system of the traditional glucose sensor; compared with general fluorescent sensors, it has the characteristics of fast response and easy use, which has become a technical problem that urgently needs to be solved in this technical field. Summary of the invention

[0009] One of the purposes of the present invention is to provide a method for preparing a disazopyridine borate, which utilizes the electrostatic interference effect of an azopyridine group to disrupt the charge distribution of quantum dots, so that the fluorescence of the quantum dots is quenched; and the synthesized pyridine group can be combined with boric acid to form a pyridinium salt, which, as an electron-withdrawing group, has an inductive effect, and its electron-withdrawing effect can reduce the electron density on the boron atom, thereby making the borate more acidic, reducing the pKa value, and approaching the glucose sensing conditions under physiological conditions, thereby solving the problem that the pH value of the general glucose response is higher than the physiological pH value, and can also optimize the problem of the instability of the enzyme-based system of the traditional glucose sensor; compared with a general fluorescent sensor, it has the characteristics of fast response, easy use, etc.

[0010] The above object of the present invention is achieved through the following technical solutions:

[0011] A method for preparing a disazopyridine borate comprises the following steps:

[0012] (1) 4-aminopyridine and phenol undergo a diazo coupling reaction in the presence of nitrite to obtain the intermediate 4-hydroxyphenylazopyridine;

[0013] (2) the intermediate 4-hydroxyphenylazopyridine in step (1) undergoes an etherification reaction with 1,8-dibromooctane to generate a disazopyridine derivative;

[0014] (3) The disazopyridine derivative prepared in step (2) undergoes a substitution reaction with 2-bromomethylphenylboronic acid to generate a disazopyridine salt compound.

[0015] Preferably, the reaction process of the intermediate 4-hydroxyphenylazopyridine in step (1) is as follows:

[0016]

[0018] Preferably, step (1) is as follows: sodium nitrite and phenol are placed in a sodium hydroxide aqueous solution, cooled to about 0°C, the mixture is added dropwise to a hydrochloric acid aqueous solution of 4-aminopyridine, stirred in an ice bath, and the pH is adjusted to 6-7 with a sodium hydroxide aqueous solution, filtered, the precipitate is washed with water, recrystallized, and dried to obtain the intermediate 4-hydroxyphenylazopyridine.

[0019] Preferably, in step (1), a magnetic stirrer is used with a rotation speed of 400 r / min for 4-5 h; and the filtration equipment is a SHZ-D (III) circulating water vacuum pump and a Buchner funnel.

[0020] Preferably, the structure of the disazopyridine compound generated in step (2) is as follows:

[0021]

[0022] Preferably, the reaction process of step (2) is as follows:

[0023]

[0024] Preferably, the specific steps of step (2) are as follows: dissolving 4-hydroxyphenylazopyridine, 1,8-diiodooctane or 1,8-dibromooctane, potassium iodide and potassium carbonate in N,N-dimethylformamide (DMF) solution; heating to 113-115° C., condensing and reflux for 4-5 hours, and introducing nitrogen; placing in water, filtering to obtain a pink-purple precipitate, washing with water, and drying to obtain a pink-purple solid; then, performing column chromatography with a petroleum ether-ethyl acetate mixed solvent, collecting the front fraction; vacuum drying the solvent in the front fraction to obtain an orange-yellow powder of a disazopyridine derivative.

[0025] Preferably, in step (2), the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixture is 1:2.5.

[0026] Preferably, the structure of the azopyridinium salt compound generated in step (3) is as follows:

[0027]

[0028] Preferably, the specific steps of step (3) are as follows: dissolving the bisazopyridine compound derivative and 2-(bromomethyl)phenylboronic acid in N,N-dimethylformamide (DMF) solution, heating to 70° C., condensing and refluxing for 48 hours, and introducing nitrogen; dripping deionized water, centrifuging, filtering, and vacuum drying to obtain a dark brown powder bisazopyridine borate.

[0029] Another object of the present invention is to provide a method for preparing a disazopyridinium borate@quantum dot fluorescent probe. The azopyridine group of the prepared disazopyridinium salt has an electrostatic attraction effect and can effectively quench the quantum dots.

[0030] The above object of the present invention is achieved through the following technical solutions:

[0031] A method for preparing a disazopyridine borate@quantum dot fluorescent probe comprises the following steps:

[0032] (1) Using boric acid buffer as solvent, diluting water-soluble carboxyl quantum dots in proportion;

[0033] (2) Dissolving bisazopyridinium borate in an ethanol solution and dissolving by ultrasonication; then adding the solution to the diluted quantum dot solution and mixing evenly to obtain bisazopyridinium borate and a quantum dot fluorescent probe.

[0034] Preferably, in step (1), the pH of the borate buffer is 8-9 (Shanghai MacLean Biochemical Technology Co., Ltd.).

[0035] Another object of the present invention is to provide an application of a disazopyridinium borate@quantum dot fluorescent probe in the field of blood glucose detection sensing.

[0036] The above object of the present invention is achieved through the following technical solutions:

[0037] Application of bis(azopyridinium) borate@quantum dot fluorescent probe in blood glucose detection sensing.

[0038] Preferably, the specific steps of the application are as follows:

[0039] (1) mixing at least 5 concentrations of glucose aqueous solutions with disazopyridinium borate and quantum dot fluorescent probe solutions, respectively, and then measuring the FL spectrum to obtain the fluorescence spectrum intensity;

[0040] (2) According to the fluorescence spectrum intensity and the concentration of the biochemical analyte obtained in step (1), a working curve is prepared to obtain a linear equation.

[0041] Beneficial effects:

[0042] The preparation method of the bisazopyridinium borate fluorescent probe@quantum dot fluorescent probe of the present invention utilizes the electrostatic interference effect of the azopyridine group to disturb the charge distribution of the quantum dots, so that the fluorescence of the quantum dots is quenched; and the synthesized pyridine group can be combined with boric acid to form a pyridinium salt. The pyridinium salt, as an electron-withdrawing group, has an inductive effect. Its electron-withdrawing effect can reduce the electron density on the boron atom, thereby making the borate more acidic and the pKa value lower, which is close to the glucose sensing conditions under physiological conditions, thereby solving the problem that the pH value of the general glucose response is higher than the physiological pH value, and can also optimize the problem of instability of the enzyme-based system of the traditional glucose sensor.

[0043] The detection limit of the bisazopyridinium borate fluorescent probe @ quantum dot fluorescent probe prepared by the present invention for glucose concentration is 0.0059mmol / L, and this sensor can be applied in the field of blood sugar detection.

[0044] The present invention is further described below through the accompanying drawings and specific implementation modes, but it is not intended to limit the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a schematic diagram of the mechanism of the switch type fluorescence sensor in the preparation method of the bisazopyridine borate @ quantum dot fluorescence probe of the present invention;

[0046] Figure 2 is a nuclear magnetic resonance spectrum of the bisazopyridine compound prepared in Example 1 of the present invention in a deuterated chloroform solution;

[0047] Figure 3 is a nuclear magnetic resonance spectrum of the disazopyridine borate prepared in Example 1 of the present invention in a deuterated chloroform solution;

[0048] Figure 4 is the infrared spectrum of the disazopyridine borate prepared in Example 1 of the present invention;

[0049] Figure 5-1 is the TG curve of the disazopyridine borate prepared in Example 1 of the present invention;

[0050] Figure 5-2 is the DSC curve of the disazopyridine borate prepared in Example 1 of the present invention;

[0051] Figure 6 UV determination of the disazopyridine compound and disazopyridine borate prepared in Example 1 of the present invention in an ethanol solution;

[0052] Figure 7 is a nuclear magnetic resonance spectrum of the disazopyridine borate prepared in Example 4 of the present invention in a deuterated chloroform solution;

[0053] Figure 8 is a comparison diagram of fluorescence quenching of quantum dots added with disazopyridinium salt in Example 5 of the present invention;

[0054] Fig. 9 is a graph showing the change of the FL intensity of quantum dots with concentration when different concentrations of 8-chain disazopyridinium borate are added in Example 5 of the present invention;

[0055] Fig.10 is a graph showing the response of FL intensity to glucose concentration when different concentrations of glucose are added in Example 6 of the present invention;

[0056] Fig.11is a linear variation diagram of FL intensity with glucose concentration and a variation diagram of (F-F0) with glucose concentration when different concentrations of glucose are added in Example 6 of the present invention;

[0057] Fig.12 This is a linear variation diagram of (F-F0) / F0 with the concentration of glucose when different concentrations of glucose are added in Example 6 of the present invention. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with the examples. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and reaction equipment available on the market in the technical field, and the reaction conditions are conventional conditions; and the units are all weight units.

[0059] Example 1 Synthesis of Bis-azopyridine Borate (1a)

[0060]

[0061] 1. Synthesis of intermediate 4-hydroxyphenylazopyridine

[0062] 4.00 g (58 mmol) of sodium nitrite and 5.00 g (53 mmol) of phenol were placed in a 10% (w / w) sodium hydroxide aqueous solution (20 mL), and the mixture was cooled to 0°C. The mixture was added dropwise into a 45 mL hydrochloric acid solution (25 mL of 11N hydrochloric acid and 20 mL of water) containing 6.00 g (64 mmol) of 4-aminopyridine, and stirred for 4-5 h under an ice bath. Then, the pH value of the reaction mixture was adjusted to 6-7 with a 10% (w / w) sodium hydroxide aqueous solution, and the mixture was filtered, the precipitate was washed with water, recrystallized, and dried to obtain yellow solid 4-hydroxyphenylazopyridine; yield: 35.74%; a magnetic stirrer was used with a rotation speed of 400 r / min for 4-5 h; the filtration equipment was a SHZ-D (III) circulating water vacuum pump and a Buchner funnel;

[0063] The reaction formula is as follows:

[0064]

[0065] 2. Synthesis of bis(azopyridine) compounds (8DAP)

[0066] 4-Hydroxyphenylazopyridine (0.9950 g, 5 mmol), 1,8-dibromooctane (0.6800 g, 2.5 mmol), potassium iodide (very small amount) and potassium carbonate (0.7700 g, 5.57 mmol) were dissolved in N,N dimethylformamide (DMF) (40 mL) solution, and the oil bath was heated to 114°C-115°C, and condensed and refluxed for 4-5 hours. Nitrogen was introduced during the reaction. After the reaction was completed, the temperature was cooled, filtered, and washed with water to obtain a purple-red precipitate. After drying, column chromatography was performed with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 1:2.5, and the front fraction was collected. The solvent in the front fraction was dried under vacuum to obtain orange-yellow powder of disazopyridine (DAP-8), with a yield of 4.2%.

[0067] The reaction process of step (2) is as follows:

[0068]

[0070] 3. Synthesis of 8DAPB

[0071] The orange-yellow powder of disazopyridine (8DAP) (0.8122 g, 1.6 mmol) and 2-(bromomethyl)phenylboronic acid (0.8809 g, 4.1 mmol) obtained in step 2 were weighed and dissolved in N,N-dimethylformamide (DMF) solution (20 mL). The mixture was heated to 70 ° C in an oil bath and refluxed for 48 h, during which nitrogen was introduced. After the reaction, deionized water was added dropwise, the mixture was centrifuged, filtered, and dried in vacuo to obtain dark brown powder of disazopyridine borate (8DAPB) with a yield of 30.5%.

[0072] Example 2

[0073] The rest is the same as Example 1, except that 1,8-dibromooctane (0.6800 g, 2.5 mmoL) is replaced by 1,8-diiodooctane (0.6800 g, 2.5 mmoL).

[0074] Example 3 Synthesis of Bis-azopyridine Borates (2a) with Different Alkyl Chains

[0075]

[0076] The synthesis steps and principles are the same as those of Example 1, except that 1,8-dibromooctane (0.6800 g, 2.5 mmoL) is replaced with 1,10-dibromodecane (0.7500 g, 2.5 mmoL).

[0077] The 8-chain disazopyridine compound was replaced with a 10-chain disazopyridine compound (10DAP; 0.8571 g, 1.6 mmol) and mixed with phenylboronic acid to undergo a substitution reaction to generate disazopyridine borate salts with different alkyl chains.

[0078] Example 4 Synthesis of Bis-azopyridine Borates (3a) with Different Alkyl Chains

[0079]

[0080] The synthesis steps and principles are the same as those of Example 1, except that 1,8-dibromooctane (0.6800 g, 2.5 mmoL) is replaced with 1,12-dibromododecane (0.8200 g, 2.5 mmoL).

[0081] The 8-chain disazopyridine compound was replaced with a 12-chain disazopyridine compound (12DAP; 0.9020 g, 1.6 mmol) and mixed with phenylboronic acid to undergo a substitution reaction to generate disazopyridine borate salts with different alkyl chains, such as Figure 7 .

[0082] Example 5 Preparation of Bis(azo)pyridinium borate@quantum dot fluorescent probe

[0083] By using the principle of photoinduced electron transfer (PET), after the disazopyridinium borate prepared in Example 1 of the present invention is added to the fluorescent quantum dots with high brightness, the carboxylate ions generated by the hydrolysis and ionization of the carboxylate are negatively charged, and electrostatic attraction occurs with the positively charged pyridine group, thereby disrupting the charge distribution on the luminescent quantum dots, resulting in a decrease in the luminescence efficiency of the fluorescent quantum dots and fluorescence quenching;

[0084] The specific process is as follows:

[0085] (1) 8 μmol / L water-soluble carboxyl quantum dots (Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd.) were dissolved in boric acid buffer (pH=8) (Shanghai MacLean Biochemical Technology Co., Ltd.) and diluted at a ratio of 1:200 to obtain a diluted quantum dot aqueous solution;

[0086] (2) preparing an aqueous solution of bis(azo)pyridinium borate with the optimal quenching quantum dot concentration: 100 μmoL / L;

[0087] (3) The prepared disazopyridinium borate aqueous solution in step (2) is added to the diluted quantum dot aqueous solution prepared in step (1), and the two solutions are mixed evenly at a volume ratio of 1:1. The quantum dot aqueous solution changes from pink to orange-brown, and no longer emits fluorescence under the irradiation of ultraviolet light, and the fluorescence is quenched (such as Figure 8 ), which is the "off" state of the fluorescent probe switch.

[0088] Example 6: Bis(azopyridinium) borate@quantum dot fluorescent probe for glucose detection

[0089] Based on the fluorescence quenching mechanism, after adding glucose, cyclic borate esters are formed with the boric acid groups on the azo molecules, and the charge is redistributed, so that the fluorescence of the quantum dots is restored. Glucose aqueous solutions of different concentrations are prepared (the concentrations are 0, 10, 50, 100, 300, 500, 1000, 1500, and 2000 μMol / L), and then the glucose aqueous solutions of different concentrations are mixed with the fluorescence quenched quantum dot aqueous solution in Example 5 at a volume ratio of 1:1. Subsequently, the corresponding fluorescence spectra (such as Fig.10 ), make a working curve based on the relationship between fluorescence spectrum intensity and concentration, calculate the linear range and detection limit, (such as Fig.11 and 12 ).

[0090] Test (Confirmation of Bisazopyridinium Borate (1a)):

[0091] Using a Bruker-400MHz nuclear magnetic resonance spectrometer, the disazopyridine (DAP-8) and disazopyridine borate (8DAPB) in Example 1 of the present invention were loaded into a nuclear magnetic resonance tube, and deuterated chloroform reagent was added. The test results were as follows: Figure 2 and Figure 3 At the same time, the infrared spectrum of the disazopyridine borate (8DAPB) in Example 1 of the present invention is measured, as shown in FIG. Figure 4 As shown; the bis-azo borate was observed at 1000, 1100 and 1200 cm -1 There are obvious bands on the left and right, which are due to BO stretching, BOH bending and BO- deformation vibration, indicating that the surface is successfully modified by boric acid groups. -1 There are aromatic ring peaks on the left and right, 1630cm -1 There are peaks of nitrogen-nitrogen double bonds on the left and right, indicating the presence of azo bonds, 1590cm -1 There is a nitrogen-oxygen double bond on the left and right, 1500cm -1 There is an alkyl peak at 1400cm -1 The presence of benzene ring peaks on the left and right indicates that the substance is the target product;

[0092] like Figure 5-1 As shown, it is the TG curve of the disazopyridine borate prepared in Example 1 of the present invention (temperature range: RT-600°C, heating rate: 10°C / min, test atmosphere: nitrogen); Figure 5-2 , is the DSC curve of the disazopyridine borate prepared in Example 1 of the present invention (the test temperature range is 20-180° C., the gas is nitrogen, and the heating rate is 10° C. / min); Figure 5-1It can be seen that the weight loss of disazo borate was 71.84% and it began to decompose at 330℃. Figure 5-2 It can be seen that the glass transition temperature of disazopyridinium borate is 84.18°C;

[0093] The disazopyridine and disazopyridine borate in Example 1 of the present invention were detected using a Cary UV-visible spectrophotometer. The sample was dissolved in an ethanol solution. Figure 6 As shown, the maximum absorption peak of disazopyridine borate in ethanol solvent is 405nm, and the maximum absorption peak of disazopyridine in ethanol solvent is 360nm. When disazopyridine is transformed into disazopyridine borate, the maximum absorption peak is red-shifted and the maximum absorption peak value becomes smaller.

[0094] The quenching effect of different concentrations of the aqueous solution of disazopyridinium borate on the water-soluble quantum dots in Example 5 of the present invention was measured using an F4700 fluorescence spectrometer. Fig. 9 As shown, as the concentration of the aqueous solution of disazopyridinium borate increases, the fluorescence intensity gradually decreases. When the concentration of the aqueous solution of disazopyridinium borate reaches 700μmoL / L, the fluorescence intensity of the quantum dots is almost completely quenched, and the fluorescence intensity is close to 0. Because the fluorescence brightness is completely dimmed when observed by the naked eye at a concentration of 100μmoL / L, in order to reduce costs, the concentration of the probe quenching quantum dots is 100μmoL / L.

[0095] Similarly, the effect of adding different concentrations of glucose aqueous solution on the fluorescence intensity of the quenched quantum dots in Example 6 was measured using an F4700 fluorescence spectrometer. Fig.10 As shown, the fluorescence intensity increases with the increase of glucose concentration;

[0096] like Fig.11 As shown in FIG. 6 , a linear variation diagram of FL intensity with glucose concentration and a variation diagram of (F-F0) with glucose concentration are shown when different concentrations of glucose are added in Example 6 of the present invention; Fig. 9 Combined with the formula detection limit = 3σ / k (σ is the standard deviation of the blank measurement value, k is the slope between the fluorescence intensity and the sample concentration), the detection limit was calculated to be 0.0059mMoL / L;

[0097] like Fig.12 As shown, when different concentrations of glucose were added in Example 6 of the present invention, (F-F0) / F0 was linearly related to the glucose concentration, and the correlation coefficient was 0.977.

[0098] Application Example 1

[0099] Application of Bis(azopyridinium)borate@Quantum Dot Fluorescent Probe in Glucose Sensing

[0100] In Example 1 of the present invention, a pyridine compound with a carbon chain length of 8, namely a bipyridine azo compound, is designed through a three-step experiment of diazo coupling, etherification reaction, and substitution reaction. Through the reaction, the boric acid group is combined with the azo compound to generate a bisazopyridine borate having multiple properties: it can be organically combined with quantum dots, has photoresponsiveness, and can form a cyclic borate ester with sugar molecules.

[0101] Since the current blood glucose sensor based on glucose oxidase has the problems of unstable enzyme-based system and high requirements for storage environment, the fluorescent probe in Example 5 of the present invention utilizes the luminescent characteristics of fluorescent quantum dots as a reporting unit in the probe; the synthesized bisazopyridine borate is used as a fluorescence quencher and glucose receptor at the same time to detect glucose, which has the characteristics of simple detection, low cost of production, stable results, reliable and sensitive, and portability. In the absence of enzymes, glucose is detected by affinity and electrostatic attraction, solving the problems of enzyme system sensors.

[0102] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail, but do not limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a disazopyridine borate, comprising the following steps: (1) 4-aminopyridine and phenol undergo a diazo coupling reaction in the presence of nitrite to obtain the intermediate 4-hydroxyphenylazopyridine; (2) the intermediate 4-hydroxyphenylazopyridine in step (1) undergoes an etherification reaction with 1,8-dibromooctane or 1,8-diiodooctane to generate a disazopyridine derivative; (3) The disazopyridine derivative of step (2) undergoes a substitution reaction with 2-bromomethylphenylboronic acid to generate a disazopyridinium salt compound.

2. The method for preparing the disazopyridine borate according to claim 1, characterized in that: Step (1) is as follows: sodium nitrite and phenol are placed in a sodium hydroxide aqueous solution, cooled to about 0°C, the mixture is added dropwise to a hydrochloric acid aqueous solution of 4-aminopyridine, stirred in an ice bath, and the pH is adjusted to 6-7 with a sodium hydroxide aqueous solution, filtered, the precipitate is washed with water, recrystallized, and dried to obtain the intermediate 4-hydroxyphenylazopyridine.

3. The method for preparing the disazopyridine borate according to claim 2, characterized in that: In step (1), a magnetic stirrer is used with a rotation speed of 400 r / min for 4-5 h; and the filtration equipment is a SHZ-D (III) circulating water vacuum pump and a Buchner funnel.

4. The method for preparing the disazopyridine borate according to claim 3, characterized in that: The specific steps of step (2) are as follows: dissolving 4-hydroxyphenylazopyridine, 1,8-diiodooctane or (1,8-dibromooctane, 1,10-dibromodecane, 1,12-dibromodecane), potassium iodide and potassium carbonate in N,N-dimethylformamide (DMF) solution; heating to 113-115° C., condensing and reflux for 4-5 hours, and introducing nitrogen; placing in water, filtering to obtain a pink-purple precipitate, washing with water, and drying to obtain a pink-purple solid; then, performing column chromatography with a petroleum ether-ethyl acetate mixed solvent, collecting the front fraction; and vacuum drying the solvent in the front fraction to obtain an orange-yellow powder of a disazopyridine derivative.

5. The method for preparing the disazopyridine borate according to claim 4, characterized in that: In step (2), the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixture is 1:2.

5.

6. The method for preparing the disazopyridine borate according to claim 5, characterized in that: The specific steps of step (3) are as follows: dissolving the bisazopyridine compound derivative and 2-(bromomethyl)phenylboronic acid in N,N-dimethylformamide solution, heating to 70° C., condensing and reflux for 4-5 hours, and introducing nitrogen; dripping deionized water, centrifuging, filtering, and vacuum drying to obtain black-brown powder bisazopyridine borate.

7. A method for preparing a disazopyridine borate and quantum dot fluorescent probe, comprising the following steps: (1) Using boric acid buffer as solvent, diluting water-soluble carboxyl quantum dots in proportion; (2) Dissolving the bis-azopyridine borate prepared in claims 1 to 6 in an ethanol solution and dissolving by ultrasonication; then adding the bis-azopyridine borate to the diluted quantum dot solution and mixing evenly to obtain bis-azopyridine borate and quantum dot fluorescent probe.

8. The method for preparing the bisazopyridinium borate and quantum dot fluorescent probe according to claim 7, characterized in that: In step (1), the pH of the boric acid buffer is 8-9.

9. Use of the bisazopyridinium borate and quantum dot fluorescent probe according to any one of claims 7 to 8 in the field of blood glucose detection sensing.

10. The use of the bisazopyridinium borate and quantum dot fluorescent probe in the field of blood glucose detection sensing according to claim 9, characterized in that: The specific steps of the application are as follows: (1) mixing at least 5 concentrations of glucose aqueous solutions with disazopyridinium borate and quantum dot fluorescent probe solutions, respectively, and then measuring the FL spectrum to obtain the fluorescence spectrum intensity; (2) According to the fluorescence spectrum intensity and the concentration of the biochemical analyte obtained in step (1), a working curve is prepared to obtain a linear equation.

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