Application of 2-(2-hydroxyphenyl) benzimidazole and ratio fluorescence analysis method for detecting boride by using 2-(2-hydroxyphenyl) benzimidazole

Abstract

The invention belongs to a fluorescent probe detection technology, and particularly relates to application of 2-(2-hydroxyphenyl) benzimidazole and a ratio fluorescence analysis method for detecting borides by using the 2-(2-hydroxyphenyl) benzimidazole. O-phenylenediamine and salicylaldehyde are used as raw materials to synthesize the probe 2-(2-hydroxyphenyl) benzimidazole (HBI) with blue fluorescence emission, after the probe is combined with the boride, the ESIPT mechanism of the probe is destroyed, fluorescence blue shift linearity appears, ratio type detection of the boride (BPA) is achieved, and the probe and / or the reagent and / or the element for boride detection can be used for preparing the probe and / or the reagent and / or the element for boride detection. When being used for detecting borides, the 2-(2-hydroxyphenyl) benzimidazole fluorescent probe has the advantages of quick response, high selectivity, interference resistance and low cost, realizes noninvasive detection of borides, and has important application value in the fields of environmental science, biological science and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probe detection, and specifically relates to a preparation method, application and ratio fluorescence analysis method of a fluorescent probe for specifically detecting boride, especially BPA. Background Art

[0002] Boric acid compounds have important applications in organic chemistry and other fields and are one of the most useful reagents in modern synthetic organic chemistry. Boric acid compounds can also be used as effective agents for boron neutron capture therapy (BNCT), which uses external irradiation of thermal neutrons with boron ( 10 B) nuclear (what we call alpha rays) and high linear energy transfer (LET) radiation produced by lithium nuclear reactions to treat cancer. In 2020, Japan developed a drug that can be absorbed by tumor cells, namely 4-boron-L-phenylalanine (BPA) drug, which is currently being used as Borofalan ( ) is marketed under the name of BPA for the treatment of locally advanced or locally recurrent unresectable head and neck cancer. Considering the therapeutic mechanism of BNCT, the concentration of 10B delivered to tumor cells directly affects the prognosis of cancer, so 10B must be monitored in the clinic to estimate the concentration of 10B in the tumor during BPA injection.

[0003] Currently, the most commonly used methods for detecting BPA in cells are inductively coupled plasma optical emission spectroscopy (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS), which require ashing of the samples and are not suitable for determining the localization of boron in cells. Although there are other methods for evaluating the localization of boron in cells, such as secondary ion mass spectrometry (SIMS) and PET imaging, these methods are usually too expensive and require complex pretreatment. Compared with the above methods, the fluorescent probe method is popular because of its simple operation, rapid analysis, low cost and non-invasive detection.

[0004] Therefore, developing a rapid, sensitive and specific method for detecting BPA has important research significance in the field of biological sciences. Summary of the invention

[0005] The present invention provides a simple and efficient fluorescent probe and a preparation method and application thereof, which can be used to detect boride, especially BPA, and solve the problems of long detection time and narrow detection range of BPA metabolized through urine after human body is injected with BPA drugs. It has broad application prospects in the fields of environmental science and life science.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides the use of 2-(2-hydroxyphenyl)benzimidazole having the structural formula (I) shown below.

[0008]

[0009] The invention provides a preparation method of 2-(2-hydroxyphenyl)benzimidazole, comprising the following steps: o-phenylenediamine and salicylaldehyde are subjected to a reflux reaction in an ethanol solution containing silicon dioxide to obtain the 2-(2-hydroxyphenyl)benzimidazole.

[0010]

[0011] The molar ratio of o-phenylenediamine, salicylaldehyde and silicon dioxide is 1:0.8-1.5:0.5-1, preferably 1:1:0.82.

[0012] The ethanol solution is preferably anhydrous ethanol.

[0013] The reaction is refluxed at room temperature for 24 hours, preferably at 25°C for 24 hours.

[0014] After the reaction is completed, the precipitate of the fluorescent probe 2-(2-hydroxyphenyl)benzimidazole (HBI) is separated and purified by washing three times, and the washing solvent is an ethanol solution, preferably anhydrous ethanol.

[0015] The 2-(2-hydroxyphenyl)benzimidazole (HBI) provided by the present invention generates tautomers through excited state intramolecular proton transfer (ESIPT), and emits characteristic fluorescence with a large Stokes shift. After HBI is combined with 4-boron-L-phenylalanine (BPA), its own ESIPT mechanism is destroyed, and the linearity of fluorescence blue shift appears. As a fluorescent probe for BPA detection, the "ratio-type" fluorescence detection and analysis of BPA is realized, and the fluorescence quantum yield is high, the response is fast, the photostability is good, the selectivity is high, and the anti-interference performance is strong. The excellent fluorescence performance produced after the HBI provided by the present invention is combined with BPA confirms that HBI can be used for the detection of boride, especially the detection of BPA. The detection method using HBI as a fluorescent probe has the industrial advantages of non-invasive, simple operation, low cost, fast and efficient, and provides a new detection probe and detection method for the detection of boride, especially BPA, and has important application value in the fields of environmental science and life science.

[0016] The present invention provides an application of 2-(2-hydroxyphenyl)benzimidazole (HBI) in a probe for boride detection, especially as a probe for 4-boron-L-phenylalanine (BPA) detection, to realize boride detection by ratio fluorescence analysis. The detection probe containing 2-(2-hydroxyphenyl)benzimidazole can also be used to prepare a reagent or element for boride detection, especially BPA detection. The reagent or element can be flexibly designed according to the requirements of a specific detection scene. The reagent can be a solution containing the HBI probe configured on site, or a stock solution containing the HBI probe that has been configured. The element can be a carrier or device in various forms loaded with the HBI probe, including but not limited to a probe, a magnetic bead, a panel, a chip, a pipeline, a passage, etc., to meet the immediacy, accuracy, simplicity, operability, etc. of the detection, meet the industrial detection needs, and facilitate industrial promotion and application.

[0017] The invention provides a ratio fluorescence analysis method for detecting boride, comprising the following steps: using 2-(2-hydroxyphenyl)benzimidazole as a probe, mixing it with a sample to be tested to form a detection system, recording the ratio of the 372nm fluorescence intensity of the 2-(2-hydroxyphenyl)benzimidazole to the 445nm fluorescence intensity, and performing qualitative and / or quantitative determination of the boride concentration in the sample.

[0018] The ratio fluorescence analysis method of the present invention using 2-(2-hydroxyphenyl) benzimidazole as a probe has many methodological characteristics such as good photostability, fast response, good reproducibility, high recovery rate, selectivity and strong anti-interference ability, etc., with the boride concentration and the ratio of the 372nm fluorescence intensity of 2-(2-hydroxyphenyl) benzimidazole to the 445nm fluorescence intensity as horizontal and vertical coordinates, and the boride concentration in the sample is qualitatively and / or quantitatively determined by an external standard method, an internal standard method or a standard addition method, preferably, the boride concentration in the sample is qualitatively and / or quantitatively determined by an external standard method or a standard addition method, wherein the external standard method includes a standard curve method and a direct comparison method. As in the embodiment of the present invention, the boride concentration and the ratio of the 372nm fluorescence intensity of 2-(2-hydroxyphenyl) benzimidazole to the 445nm fluorescence intensity are respectively horizontal and vertical coordinates, and a standard curve is drawn to perform qualitative and / or quantitative determination of the boride concentration in the sample.

[0019] The detection system contains an organic solvent, including but not limited to a combination of one or more of toluene, dichloromethane, tetrahydrofuran, ethanol, N,N-dimethylformamide, acetonitrile and dimethyl sulfoxide, preferably dimethyl sulfoxide.

[0020] The detection system also contains a buffer solution, including but not limited to acetic acid-acetate buffer solution, phosphate buffer solution, borate buffer solution, ammonia-ammonium chloride buffer solution, etc., preferably a phosphate buffer solution, more preferably a PBS buffer. A buffer solution is added to the detection system to adjust the pH value of the detection system to meet the detection environment requirements of the sample to be detected. According to different samples to be detected, different buffer solutions are selected to stabilize the detection environment and the target compound without affecting subsequent detection, that is, the detection system of adding a buffer solution simulates the sample environment to be detected, maintains the stability of the sample to be detected and the target compound, and ensures the reliability and accuracy of the results. As in the embodiment of the present invention, a dimethyl sulfoxide (DMSO) detection system containing a PBS buffer solution is used when detecting urine samples, and its pH value is about 7.4, simulating the urine environment, and trying to avoid the influence of the change of the sample environment to be detected during the detection process on the detection of the target compound, and ensuring the accuracy and reliability of the test results.

[0021] The volume content of the organic solvent in the detection system is 0.01%-99.9%, preferably 40%-90%, more preferably 55%-80%, more preferably 50%-75%. In a preferred embodiment of the present invention, the volume content of the organic solvent in the detection system is 50%.

[0022] The boron compound is an organic boron compound and / or a metal boron compound, and the organic boron compound includes but is not limited to a combination of one or more of boric acid and its derivatives, boric esters and their derivatives, boric alcohols and their derivatives, boric aldehydes and their derivatives, boric amides and their derivatives, borate salts and their derivatives, and boron heterocyclic compounds and their derivatives, preferably 4-boron-L-phenylalanine (BPA) and / or phenylboronic acid.

[0023] In the detection system, the detection reaction time of the boride and 2-(2-hydroxyphenyl)benzimidazole is 1-20 minutes, preferably 5-15 minutes, more preferably 8-12 minutes, and more preferably 10 minutes.

[0024] The concentration of 2-(2-hydroxyphenyl)benzimidazole in the detection system is 10-50 μM, preferably 15-25 μM, and more preferably 20 μM, to ensure the linear relationship of the results obtained by using 2-(2-hydroxyphenyl)benzimidazole as a probe for detection. As in the embodiment of the present invention, the concentration of 2-(2-hydroxyphenyl)benzimidazole is 20 μM, the linear response range of the boron-containing compound is 0-220 μM, and the detection limit is 7.11 μM.

[0025] The samples include all collectible samples in the fields of environmental science and life science, including but not limited to liquid, solid, semi-solid, etc., such as water samples, soil samples, etc. in the field of environmental science, and urine samples, blood samples, tissue samples, etc. in the field of life science.

[0026] The beneficial effects of the present invention are:

[0027] The present invention uses 2-(2-hydroxyphenyl)benzimidazole (HBI) as a fluorescent probe to perform qualitative and / or quantitative detection of boride, especially 4-boron-L-phenylalanine (BPA) concentration, and has the following advantages: (1) HBI as a fluorescent probe has high fluorescence quantum yield, fast response, and good photostability, and realizes "ratio-type" fluorescence detection of boron-containing compounds, especially BPA; (2) It has strong selectivity and strong anti-metal interference ability, and the influence of metal ions on the detection results can be ignored; (3) The linear response range is wide from 0 to 220 μM, the linear relationship is good, the detection limit is 7.11 μM, and the sensitivity is high; (4) The detection time is short, the conditions are simple, the operation is easy, the cost is low, and the detection is efficient and fast; the reagent consumption is small, and it is green and environmentally friendly; (5) It is applied in the field of life sciences to realize non-invasive detection of boride (such as BPA). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The following is a synthetic route map of the probe described in the present invention.

[0029] Figure 2 This is a fluorescence picture of the fluorescent probe HBI solution before and after adding BPA, under 365nm ultraviolet light.

[0030] Figure 3 The UV absorption spectra of the compound HBI of the present invention in toluene (Tol), dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EA), ethanol (EtOH), N,N-dimethylformamide (DMF), acetonitrile (ACN) and dimethyl sulfoxide (DMSO) were studied.

[0031] Figure 4 The fluorescence spectra of the compound HBI of the present invention in toluene (Tol), dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EA), ethanol (EtOH), N,N-dimethylformamide (DMF), acetonitrile (ACN) and dimethyl sulfoxide (DMSO) were studied.

[0032] Figure 5 This is the fluorescence intensity diagram of the probe HBI to BPA in PBS / DMSO solutions with different volume ratios.

[0033] Figure 6 Job's Plot curve of the binding of probe HBI and BPA.

[0034] Figure 7 The fluorescence intensity graph of the probe HBI to BPA in PBS / DMSO (1 / 1, v / v) solution at different reaction times.

[0035] Figure 8 Schematic diagram (A) showing the change in fluorescence intensity of the fluorescent probe HBI of the present invention in PBS / DMSO (1 / 1, v / v) solution as a function of BPA concentration.

[0036] Fig. 9 I for probe HBI 372nm / I 445nm Linear relationship plot of intensity versus BPA concentration.

[0037] Fig.10 The fluorescence intensity bar graph of the fluorescent probe BHI detecting BPA under the interference of various metal ions. Among them, other analytes are the corresponding metal stock solutions.

[0038] Fig.11 I is the fluorescent probe HBI in urine sample 1 372nm / I 445nm Working curve between intensity and different BPA concentrations (0-220 μM).

[0039] Fig.12 I of the fluorescent probe HBI in urine sample 2 372nm / I 445nm Working curve between intensity and different BPA concentrations (0-220 μM).

[0040] Fig.13 I of the fluorescent probe HBI in urine sample 3 372nm / I 445nm Working curve between intensity and different BPA concentrations (0-220 μM).

[0041] Fig.14 The fluorescent probe HBI of the present invention 1 H NMR spectrum (A), using DMSO-d6 as solvent. The fluorescence probe HBI is in the range of 7-8 ppm. 1 Enlarged view of the H NMR spectrum (B).

[0042] Fig.15 The fluorescent probe HBI of the present invention 13 C NMR spectrum, the solvent used was DMSO-d6.

[0043] Fig.16 These are fluorescence images before and after adding PBA to the fluorescent probe HBI solution. DETAILED DESCRIPTION

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Example 1: Synthesis of fluorescent probe HBI

[0046] Synthesis route such as Figure 1 As shown, in a three-necked flask, o-phenylenediamine (108 mg, 1 mmol), salicylaldehyde (122 mg, 1 mmol) and silicon dioxide (50 mg, 0.82 mmol) were dissolved in 20 mL of ethanol solution and the reaction was stirred at room temperature for 24 hours.

[0047] The reaction was monitored by thin layer chromatography. After the reaction was completed, the mixture was cooled to room temperature, filtered three times with anhydrous ethanol, and dried to obtain white solid HBI (188 mg, yield 90%). Fig.14 As shown, 1 H NMR (400MHz, DMSO-d6) δppm: 7.00-7.08(m,2H),7.29(d,J=3.06Hz,2H),7.36-7.43(m,1H),7.67(br,s,2H),8.06(m,J=1.00Hz,1H),13.17(br,s,2H). Fig.15 As shown, 13 C NMR (101MHz, DMSO-d6) δppm: 165.83, 156.76, 151.91, 134.65, 132.96.129.00, 126.95, 125.59, 122.58, 122.46, 120.23, 118.76, 117.46.

[0048] Example 2: Study on UV absorption and fluorescence spectra of probe molecules in solvents of different polarities

[0049] (1) HBI (11.0 mg) was dissolved in 5 mL DMSO to prepare HBI test stock solution (10 mM). The stock solution was stored in a 4°C refrigerator away from light and had a shelf life of 3 months.

[0050] (2) In a 3.5 mL four-sided light-transmitting quartz cuvette, 1.98 mL of toluene (ToL), dichloromethane (DCM), tetrahydrofuran (THF), ethanol (EtOH), N,N-dimethylformamide (DMF), acetonitrile (ACN), and dimethyl sulfoxide (DMSO) were added, and 4 μL of the probe stock solution was added to obtain a test solution with a concentration of 20 μM.

[0051] (3) Place the working solution in a UV-visible absorption spectrum with a scanned baseline, set the scanning range to 250-500 nm, and obtain the UV-visible absorption spectra of HBI in different solutions. Read the absorbance at the maximum absorption peak of HBI in different solvents. The spectra are as follows: Figure 3 The calculation results are shown in Table 1.

[0052] (4) Place the working solution in a fluorescence spectrometer, set the excitation wavelength to 320 nm, and the scanning range to 340 nm to 650 nm to obtain the fluorescence spectra of BHI in different solutions. Figure 4 The relevant absorption and emission parameters are shown in Table 1.

[0053] Table 1 Absorption and emission of probe molecules in different solvents

[0054] Tol DCM EA THF EtOH DMF ACN DMSO <![CDATA[λ ab / nm]]> 280 / 320 281 / 318 287 / 325 318 / 319 279 / 329 285 / 322 283 / 320 275 / 323 <![CDATA[λ em / nm]]> 462 460 377 / 451 462 451 460 460 462

[0055] Example 3: Study on the detection system of BPA by probe molecules

[0056] In order to test the optimal detection system of probe molecules for detecting BPA, the changes in the fluorescence intensity of the probe molecules and BPA in the system of different volumes of PBS / DMSO were studied. The specific operation is as follows:

[0057] (1) Add 2 mL of PBS / DMSO solutions with different volume ratios (1 / 99, 1 / 9, 2 / 8, 3 / 7, 4 / 6, 5 / 5, 6 / 4, 7 / 3, 8 / 2, 9 / 1, 99 / 1) to 11 3.5 mL four-sided light-transmitting quartz cuvettes, and then add 4 μL of the probe stock solution to obtain a test solution with a probe concentration of 20 μM.

[0058] (2) Add 20 μL of BPA stock solution to each of the above cuvettes to obtain a test solution with a BPA concentration of 0.2 mM.

[0059] (3) Place the working solution in a fluorescence spectrometer, set the excitation wavelength to 320 nm, and the scanning range to 340 nm. Record the fluorescence intensity of the probe HBI at 372 nm and 445 nm, respectively. 372nm / I 445nm The results are shown in Figure 2. Figure 5 shown.

[0060] (4) The experimental results show that from pure DMSO solution to DMSO / PBS mixture containing 50% PBS, I 372nm / I 445nm When the PBS content increased from 60% to 99%, I372nm / I 445nm The fluorescence enhancement factor observed in the solution with PBS concentration of 50% was the highest (1.98 times), so PBS / DMSO (1 / 1, v / v) was selected as the test system.

[0061] Example 4: Job's Plot Curve Study of Probe Molecule Binding to BPA

[0062] The complexation ratio of probe HBI and BPA was determined by measuring the Job's Plot working curve of probe HBI binding to BPA. The specific operation is as follows:

[0063] (1) HBI (11.0 mg) was dissolved in 5 mL DMSO to prepare HBI test stock solution (10 mM). The stock solution was stored in a 4°C refrigerator away from light and had a shelf life of 3 months.

[0064] (2) Add different concentrations of probe and detection substance BPA to 2 mL of DMSO solution in 11 3.5 mL four-sided transparent quartz cuvettes, keep the total concentration of probe and BPA at 50 μM, and change the ratio of fluorescent probe HBI to BPA. Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 320 nm and the detection wavelength range to 340 nm to 650 nm. Obtain the fluorescence spectrum of the probe.

[0065] (3) Record the fluorescence intensity of the above fluorescence spectrum at 372nm and 445nm to obtain a series of I values ​​related to BPA concentration. 372nm / I 445nm With this ratio as the ordinate and [BPA] / ([BPA]+[HBI]) as the abscissa, the Job's Plot curve is fitted, as shown in Figure 6 shown.

[0066] (4) Figure 6 It shows that when [BPA] / ([BPA]+[HBI]) is 0.56, I 372nm / I 445nm The ratio reaches the maximum, indicating that the complexation ratio of the fluorescent probe HBI and BPA is 1:1.

[0067] Example 4: Study on the Kinetics of BPA by Probe Molecules

[0068] In order to test the optimal reaction time of the probe molecule to detect BPA, the changes in the fluorescence intensity of the probe molecule and BPA at different times were studied. The specific operation is as follows:

[0069] (1) Take BPA (4.1 mg) and dissolve it in 1 mL of deionized water with a pH of 9-10 to prepare a BPA test stock solution (20 mM). The stock solution is stored in a refrigerator at 4°C away from light and has a shelf life of 3 days.

[0070] (2) Add 4 μL of the probe stock solution to 2 mL of PBS / DMSO (1 / 1, v / v) solution in two 3.5 mL four-sided light-transmitting quartz cuvettes to obtain a test solution with a concentration of 20 μM.

[0071] (3) Add 10 μL of BPA stock solution to each of the two cuvettes to obtain a test solution with a BPA concentration of 0.1 mM.

[0072] (4) The working solution was placed in a fluorescence spectrometer, and the excitation wavelength was set to 320 nm, the scanning range was 340 nm to 650 nm, and the temperature was set to 25°C. The fluorescence intensity of the probe BHI at 372 nm and 445 nm after the addition of BPA was obtained over time. The fluorescence intensity of the probe HBI at 372 nm and 445 nm was recorded respectively, and the fluorescence intensity was expressed as I 372nm / I 445nm The result is as follows: Figure 7 shown.

[0073] (5) The experimental results show that at 25°C, the fluorescence at 445nm weakened, the fluorescence at 372nm increased, and 5 minutes after adding BPA, I 372nm / I 445nm The fluorescence ratio at 100 μm / s reached 1.06 times. The enhancement factor after 10 minutes was about 1.24 times. Therefore, the optimal detection time was 10 minutes.

[0074] Example 5: Study on the change of fluorescence intensity of probe molecules under different concentrations of BPA

[0075] In order to test the fluorescence response characteristics of the probe molecule to different concentrations of BPA, the experimental process is described in detail using PBS / DMSO (1 / 1, v / v) solution as an example.

[0076] (1) Place 2 mL of PBS / DMSO (1 / 1, v / v) solution in a 3.5 mL four-sided transparent quartz cuvette. Add 4 μL of HBI stock solution (10 mM) to the cuvette and shake well. Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 320 nm and the detection wavelength range to 340 nm to 650 nm. Obtain the initial fluorescence spectrum of the probe.

[0077] (2) Add 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 μL of BPA stock solution (20 mM) to the cuvette to obtain test solutions containing 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 and 220 μM BPA. Test the fluorescence spectra of the above test solutions (e.g. Figure 8 shown).

[0078] (3) Record the fluorescence intensity of the above fluorescence spectrum at 372nm and 445nm to obtain a series of I values ​​related to BPA concentration. 372nm / I 445nm The ratio of BPA concentration is plotted using this ratio, such as Fig. 9 shown.

[0079] (4) Yes Fig. 9 Linear fitting was performed in the linear range (0-220 μM), and the detection limit of probe HBI in PBS / DMSO (1 / 1, v / v) solution was calculated according to 3σ / k.

[0080] (5) The experimental results show that: 372nm / I 445nm The ratio showed a good linear relationship with the concentration of BPA, and the detection limit was 7.11 μM.

[0081] Figure 2 The actual fluorescence pictures of the fluorescent probe HBI solution before and after adding BPA under 365nm ultraviolet light are shown.

[0082] Example 6: Study on the selectivity of probe molecules for BPA

[0083] To test the selectivity of the probe molecule to BPA, the fluorescence response of the probe molecule to different types of metal ions was tested. The test objects included Na + , K + , Ca 2+ Mg 2+ , Fe 2+ , Fe 3+ 、Co 2+ 、Zn 2+ 、Cd 2+ 、Ni 2+ , Cu 2+ The detection concentration of metal ions is 0.4mM, and the working concentration of the probe molecule is 20μM. The specific operation process is as follows:

[0084] (1) Prepare a 20 mM metal ion stock solution. Weigh the corresponding stoichiometric ratio of the substance into a 5 mL volumetric flask, add about 4 mL of deionized water to dissolve, continue to add deionized water to the scale line of the volumetric flask, shake well, and place in a refrigerator for later use.

[0085] (2) Place 2 mL of PBS / DMSO (1 / 1, v / v) solution in a 3.5 mL four-sided transparent quartz cuvette. Add 4 μL of HBI stock solution (10 mM) to the cuvette and shake well. Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 320 nm and the detection wavelength range to 340-650 nm. Obtain the initial fluorescence spectrum of the probe and record the fluorescence intensity at 372 nm and 445 nm as I. 372nm / I 445nm The ratio of is taken as I0.

[0086] (3) Add 40 μL of each metal ion to the cuvette in (2) to obtain a series of test solutions containing 0.4 mM metal ions. Test the fluorescence spectra of the test solutions.

[0087] (4) Record the fluorescence intensity at 372 nm and 445 nm in the above fluorescence spectrum to obtain the I of the probe HBI in various interference environments. 372nm / I 445nm The fluorescence intensity value is used to make a ratio of I0 in (2). The I / I0 value of each sample is obtained and a bar graph is drawn. The result is as follows: Fig.10 shown.

[0088] (5) The experimental results show that: Fe 3+ , Cu 2+ These two metals can cause slight fluorescence quenching. However, considering that Fe 3+ and Cu 2+ The content in the human body is very small, so the influence of these two metal ions can be ignored. Therefore, the probe HBI has good selectivity and anti-interference for BPA.

[0089] Example 7: Detection of BPA in actual urine samples using probe molecules

[0090] The probe HBI can be directly tested after the urine sample is diluted 10 times with PBS (the urine samples are from 3 healthy people). The specific detection scheme is as follows:

[0091] (1) Take 1 mL of urine sample diluted 10 times with PBS / DMSO (1 / 1, v / v) solution and 1 mL of DMSO and place them in a 3.5 mL four-sided transparent quartz cuvette. Add 4 μL of HBI stock solution (10 mM) to the cuvette and shake well. Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 320 nm and the detection wavelength to 340-650 nm. Obtain the initial fluorescence intensity of the probe at 372 nm and 445 nm in the urine sample.

[0092] (2) Add 1, 3, 6, 9 and 12 μL of BPA stock solution (20 mM) to the cuvette to obtain 10, 30, 60, 90 and 120 μM BPA test solutions. Test the fluorescence intensity of the above test solutions at 372 nm and 445 nm.

[0093] (3) Record the fluorescence intensity at 372nm and 445nm in the above fluorescence spectrum respectively, and calculate I 372nm / I 445nm , and a series of I related to BPA concentration were obtained. 372nm / I 445nm Linear relationship. 372nm / I 445nm As the vertical axis, the BPA concentration is used as the horizontal axis to make a scatter plot, such as Fig.11 , 12 , as shown in 13.

[0094] (4) Yes Fig.11 Linear fitting was performed in the linear range (0-120 μM) to obtain the standard curve equation (urine sample 1); Fig.12 Linear fitting was performed in the linear range (0-120 μM) (urine sample 2) to obtain the standard curve equation; Fig.13 Linear fitting was performed in the linear range (0-120 μM) (urine sample 3) to obtain the standard curve equation.

[0095] (5) Add BPA standard solutions of known concentrations (15, 45, and 75 μM) to the urine sample diluted 10 times, record the fluorescence intensity of the probe, and calculate I 372nm / I 445nm The ratio of 2 and 4 was used to calculate the actual BPA content in urine according to the standard linear equation. This step was repeated three times. The test results are shown in Table 2.

[0096] Table 2 Probe molecules for detection of BPA in urine

[0097]

[0098] It can be seen from the table that the calculated BPA concentration in the actual sample is in good agreement with the concentration of added BPA, the recovery is in the range of 93.14-99.09%, and the RSD is in the range of 0.26-2.54%.

[0099] Example 8: Probe molecule sensing of PBA (phenylboronic acid)

[0100] In order to test the response of the probe molecule HBI to other boric acid compounds such as PBA (phenylboronic acid), the change of the fluorescence intensity of the probe molecule in the PBS / DMSO (1 / 1, v / v) system was studied. The specific operation is as follows:

[0101] (1) Accurately weigh 12.2 mg of PBA (phenylboronic acid) and dissolve it in 5 mL of DMSO solution to prepare a 20 mM PBA stock solution.

[0102] (2) Add 2 mL of a solution with a volume ratio of PBS / DMSO (1 / 1, v / v) to a 3.5 mL four-sided light-transmitting quartz cuvette, and then add 4 μL of the probe stock solution to obtain a test solution with a probe concentration of 20 μM.

[0103] (3) Add 20 μL of PBA stock solution to the above cuvette to obtain a test solution with a PBA concentration of 0.2 mM.

[0104] (4) Place the working solution into a fluorescence spectrometer, set the excitation wavelength to 320 nm, and the scanning range to 340 nm-650 nm. Obtain the spectrum Fig.16 .

[0105] (5) The experimental results show that the emission peak of the original probe molecule HBI is located at 450nm. After adding PBA, its fluorescence spectrum blue-shifts from 450nm to 375nm, indicating that the probe molecule can also detect PBA in a “ratio-type” manner.

[0106] Finally, it should be noted that the above description of the embodiments is for the convenience of understanding and use by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described below to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Application of 2-(2-hydroxyphenyl)benzimidazole in the preparation of probes and / or reagents and / or elements for boride detection.

2. A probe and / or reagent and / or element for detecting boride, characterized in that: Contains 2-(2-hydroxyphenyl)benzimidazole.

3. A ratio fluorescence analysis method for detecting boride, characterized in that: The following steps are involved: 2-(2-hydroxyphenyl)benzimidazole is used as a probe and mixed with a sample to be tested to form a detection system. The ratio of the 372nm fluorescence intensity to the 445nm fluorescence intensity of 2-(2-hydroxyphenyl)benzimidazole is recorded to qualitatively and / or quantitatively determine the boride concentration in the sample.

4. A ratio fluorescence analysis method for detecting boride according to claim 3, characterized in that: With the boride concentration and the ratio of the fluorescence intensity of 2-(2-hydroxyphenyl)benzimidazole at 372 nm to that at 445 nm as the horizontal and vertical coordinates, respectively, the boride concentration in the sample is qualitatively and / or quantitatively determined by the external standard method, the internal standard method or the standard addition method.

5. A ratio fluorescence analysis method for detecting boride according to claim 3 or 4, characterized in that: The boron-containing compound is an organic boron compound and / or a metal boron compound, and the organic boron compound includes a combination of one or more of boric acid and its derivatives, boric esters and their derivatives, boric alcohols and their derivatives, boric aldehydes and their derivatives, boric amides and their derivatives, borate salts and their derivatives, and boron heterocyclic compounds and their derivatives.

6. A ratio fluorescence analysis method for detecting boride according to claim 3 or 4, characterized in that: The detection system contains an organic solvent.

7. A ratio fluorescence analysis method for detecting boride according to claim 6, characterized in that: The detection system contains a buffer solution.

8. A ratio fluorescence analysis method for detecting boride according to claim 6 or 7, characterized in that: The volume content of the organic solvent in the detection system is 0.1%-99.9%.

9. A ratio fluorescence analysis method for detecting boride according to any one of claims 3 to 8, characterized in that: The detection reaction time of the boron compound and 2-(2-hydroxyphenyl)benzimidazole is 1-20 minutes.

10. A ratio fluorescence analysis method for detecting boride according to any one of claims 3 to 8, characterized in that: The concentration of 2-(2-hydroxyphenyl)benzimidazole in the detection system is 10-50 μM.

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