A hydrogen peroxide fluorescent probe and its preparation method and application

Abstract

The present invention discloses a hydrogen peroxide fluorescent probe, a preparation method, and an application thereof. The present invention relates to the field of analytical detection technology. The hydrogen peroxide fluorescent probe has the following structure: #imgabs0# wherein R1 is an alkyl group, an alkoxy group, or a substituted or unsubstituted aromatic group; wherein R2 is one of the following structural formulas: #imgabs1# The present invention proposes introducing a dual ESIPT and AIE mechanism, utilizing the advantages of the ESIPT mechanism, such as large Stokes shift and low background interference, combined with aggregation-induced emission properties, to develop a highly sensitive and rapidly responsive hydrogen peroxide fluorescent probe.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a hydrogen peroxide fluorescent probe and a preparation method and application thereof. Background Art

[0002] Hydrogen peroxide (H2O2) is a key member of the ROS family and has a certain regulatory effect on many physiological processes of organisms.

[0003] First, hydrogen peroxide (H2O2) is associated with cell migration, proliferation, and differentiation. Second, it plays a significant role in angiogenesis and regulating vascular diameter. Furthermore, H2O2 can reversibly oxidize proteins involved in the reaction, thereby activating or inactivating the pathway. Furthermore, it can effectively modulate the body's immune defense mechanisms.

[0004] Currently, the main methods for detecting hydrogen peroxide (H2O2) include electrochemical methods, chromatography, and colorimetry. However, these methods suffer from limitations such as slow detection speed, low sensitivity, complex equipment, and high cost. Therefore, the development of a new class of fluorescent probes with good selectivity, significant efficacy, low cost, fast response, and high sensitivity is urgently needed.

[0005] Fluorescence analysis is convenient, intuitive, real-time, and rapid, and the research on hydrogen peroxide (H2O2) fluorescent probes has also attracted considerable attention. In recent years, hydrogen peroxide (H2O2) fluorescent probes have received continued attention from researchers, with excellent fluorescent probes designed and synthesized based on mechanisms such as intramolecular charge transfer (ICT), excited-state intramolecular proton transfer (ESIPT), and aggregation-induced emission (AIE).

[0006] Although many fluorescent probes for detecting hydrogen peroxide (H2O2) based on different mechanisms have been reported, there are problems such as low sensitivity and long response time in the hydrogen peroxide (H2O2) detection process, which are not conducive to the analysis and detection of hydrogen peroxide (H2O2) in vivo and in vitro.

[0007] Based on this, there is an urgent need to develop a hydrogen peroxide (H2O2) fluorescent probe with high sensitivity and rapid response. Summary of the Invention

[0008] The purpose of the present invention is to develop a hydrogen peroxide (H2O2) fluorescent probe with high sensitivity and rapid response.

[0009] A first aspect of the present invention is:

[0010] Provided is a hydrogen peroxide fluorescent probe.

[0011] The second aspect of the present invention is:

[0012] Provided is a method for preparing a hydrogen peroxide fluorescent probe.

[0013] The third aspect of the present invention is:

[0014] Application of the hydrogen peroxide fluorescent probe.

[0015] Specifically, the technical solution adopted according to the first aspect of the present invention is:

[0016] A hydrogen peroxide fluorescent probe having the following structure:

[0017]

[0018] Wherein, R1 is one of an alkyl group, an alkoxy group, and a substituted or unsubstituted aromatic group;

[0019] Wherein, R2 is one of the following structural formulas:

[0020]

[0021] According to the embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0022] The present invention proposes to introduce the dual mechanisms of ESIPT and AIE, utilizing the advantages of the ESIPT mechanism such as large Stokes shift and small background interference, and combining it with the aggregation-induced emission properties, thereby developing a highly sensitive and rapidly responsive hydrogen peroxide (H2O2) fluorescent probe.

[0023] To construct a highly sensitive, rapidly responsive fluorescent probe for hydrogen peroxide (H2O2), the present invention introduces a benzothiazole group into the molecular design and places the benzothiazole in an ortho position to the recognition group, thereby ensuring that the hydroxyl group generated after the probe reacts with hydrogen peroxide can undergo an ESIPT effect with the benzothiazole nitrogen atom. This process, through the transformation from an enol form to a keto form, gives the probe a large Stokes shift, effectively avoiding the limitations of traditional organic fluorescent probes' self-absorption. Furthermore, the introduction of a tetraphenylethylene group into the molecular structure imparts the probe with AIE properties. This invention achieves the simultaneous introduction of ESIPT and AIE mechanisms within a single probe, further enhancing the fluorescence quantum yield of the probe after its reaction with hydrogen peroxide, thereby enabling highly sensitive monitoring of hydrogen peroxide.

[0024] In the structure of the hydrogen peroxide fluorescent probe of the present invention, sodium phenylboronic acid ester with strong reducing ability serves as an efficient H2O2 recognition site. In the presence of low concentration of H2O2, sodium phenylboronic acid ester is detached, thereby generating a strong excited state proton transfer process. At the same time, aggregation-induced luminescent molecules enhance the fluorescence emission intensity, accompanied by a dual change in the color and fluorescence of the probe solution, thereby achieving the purpose of highly sensitive, naked-eye detection of H2O2.

[0025] According to one embodiment of the present invention, R1 is C 1-6 alkyl.

[0026] According to one embodiment of the present invention, R1 is C 1-4 alkyl.

[0027] According to one embodiment of the present invention, the R1=-(CH2) n CH3, n=0-5.

[0028] According to one embodiment of the present invention, the R1=-O(CH2) n CH3, n=0-5.

[0029] According to one embodiment of the present invention, R1 is one of the following structural formulas:

[0030]

[0031] n=0-21.

[0032] Specifically, the technical solution adopted according to the second aspect of the present invention is:

[0033] A method for preparing the hydrogen peroxide fluorescent probe comprises the following steps:

[0034] S1: Under acidic conditions, a hydroxy-substituted benzaldehyde, an amino-substituted thiophenol, and an oxidant are mixed in an organic solvent, stirred, and then filtered to obtain a solid reagent; the solid reagent and a nitrogen source are dissolved in an acidic solution, refluxed, and extracted to obtain compound 1;

[0035] S2: Compound 1 reacts with Compound 2 or Compound 4 to obtain an intermediate product;

[0036] S3: mixing the intermediate product, phenylboronic acid pinacol ester with a halogen substituent, and an alkaline reagent in an organic solvent, and heating the mixture to react to obtain the hydrogen peroxide fluorescent probe;

[0037] The structural formula of the compound 2 is:

[0038] The structural formula of the compound 4 is:

[0039] According to the embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0040] The preparation method of the hydrogen peroxide fluorescent probe of the present invention has simple synthesis process, cheap and easily available raw materials, and can be produced on a large scale.

[0041] According to one embodiment of the present invention, the hydroxy-substituted benzaldehyde includes 2-hydroxy-5-methylbenzaldehyde.

[0042] According to one embodiment of the present invention, the nitrogen source includes hexamethylenetetramine. When the nitrogen source is hexamethylenetetramine, it can serve as a donor of formaldehyde, causing a duff reaction. Specifically, hexamethylenetetramine decomposes under acidic conditions to generate formaldehyde and ammonia, and the generated formaldehyde reacts with phenol to form hydroxymethylphenol.

[0043] According to one embodiment of the present invention, in step S2, the step of preparing the intermediate product by using compound 1 and compound 2 includes:

[0044] Compound 1, a basic catalyst and compound 2 are mixed, refluxed and extracted to obtain an intermediate product.

[0045] According to one embodiment of the present invention, in step S2, the step of preparing an intermediate product by using compound 1 and compound 4 includes:

[0046] Compound 1, a basic catalyst and compound 4 are mixed, refluxed and extracted to obtain an intermediate product.

[0047] According to one embodiment of the present invention, in step S3, the alkaline reagent includes K2CO3. The main function of K2CO3 is to provide an alkaline environment to help generate a strong nucleophile, thereby promoting the Williamson etherification reaction.

[0048] Another aspect of the present invention provides a biosensor comprising the hydrogen peroxide fluorescent probe described in the embodiment of the first aspect. Because this application utilizes all of the technical solutions of the hydrogen peroxide fluorescent probe described above, it at least has all the beneficial effects brought about by the technical solutions of the above embodiment.

[0049] The present invention also provides an application of a fluorescent probe based on ESIPT and AIE effects, which has high fluorescence quantum yield, large Stokes shift, high sensitivity, and can detect hydrogen peroxide (H2O2) with the naked eye.

[0050] According to one embodiment of the present invention, the fluorescent probe of the present invention is mixed with hydrogen peroxide (H2O2) in a solvent to react, and then ultraviolet or fluorescence spectrum detection is performed.

[0051] According to one embodiment of the present invention, the solvent is a mixture of DMSO and PBS buffer solution.

[0052] According to one embodiment of the present invention, the concentration of the probe is 10 -7 -10 -2 mol / L.

[0053] According to one embodiment of the present invention, the reaction time is 2-25 minutes.

[0054] According to one embodiment of the present invention, the upper limit of detection of hydrogen peroxide (H2O2) is 100 μmol / L.

[0055] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0057] Figure 1 This is a flow chart of the method for preparing the hydrogen peroxide fluorescent probe in Example 1.

[0058] Figure 2 This is a flow chart of the method for preparing a hydrogen peroxide fluorescent probe in Example 2.

[0059] Figure 3 This is the high-resolution mass spectrum of the hydrogen peroxide fluorescent probe prepared in Example 1.

[0060] Figure 4 This is the high-resolution mass spectrum of the hydrogen peroxide fluorescent probe prepared in Example 2.

[0061] Figure 5 This is the ultraviolet spectrum of hydrogen peroxide detected by the hydrogen peroxide fluorescent probe in Example 1.

[0062] Figure 6 This is a fluorescence spectrum diagram of the hydrogen peroxide fluorescent probe in Example 1 detecting hydrogen peroxide.

[0063] Figure 7 This is a test chart of the detection limit of hydrogen peroxide detected by the hydrogen peroxide fluorescent probe in Example 1. DETAILED DESCRIPTION

[0064] The terms "preferably," "more preferably," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present invention.

[0065] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0066] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of the present invention.

[0067] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0068] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0069] In the present invention, the term "alkyl" refers to a saturated hydrocarbon containing primary (normal) carbon atoms, or secondary carbon atoms, or tertiary carbon atoms, or quaternary carbon atoms, or a combination thereof. Phrases containing this term, for example, "C 1-8"Alkyl" refers to an alkyl group containing 1 to 8 carbon atoms. Suitable examples include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH2CH(CH3)2), u, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl 2-Methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3 )CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3 and octyl (-(CH2)7CH3).

[0070] In the embodiment, “C 1-6 "Alkyl" refers to an alkyl group having 1 to 6 carbon atoms, and means a saturated aliphatic hydrocarbon group including a branched and straight chain having the specified number of carbon atoms. For example, in "C 1-6 "Alkyl" is defined as including groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched chain structure. For example, "C 1-6 The term "alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl and the like.

[0071] In the embodiment, “C 1～4The term "alkyl" refers to an alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0072] In the embodiment, "alkenyl" refers to a group containing at least one unsaturated site, i.e., a carbon-carbon sp 2 A hydrocarbon containing a double-bonded positive, secondary, tertiary, or cyclic carbon atom. Phrases containing this term, such as "C 2-8 "Alkenyl" refers to an alkenyl group containing 2-8 carbon atoms. Suitable examples include, but are not limited to, ethenyl (-CH=CH2), propenyl (-CH2CH=CH2), cyclopentenyl (-C5H7) and 5-hexenyl (-CH2CH2CH2CH2CH=CH2).

[0073] In the embodiments, "halogen" or "halo" refers to F, Cl, Br or I.

[0074] In the embodiments, "halogen substituted" means that any number of H at any selected position on the corresponding group is substituted by halogen, such as fluoromethyl, including monofluoromethyl, difluoromethyl, and trifluoromethyl.

[0075] The term "aryl" refers to a monovalent carbon ring having one or more rings, preferably 1 or 2 rings, wherein at least one ring is aromatic and the other rings (if present) may be aromatic or non-aromatic. 6-10 aryl), more preferably 6 ring carbon atoms (i.e. C6 aryl or phenyl). Representative examples of aryl include, but are not limited to, phenyl, naphthyl or tetrahydronaphthyl, etc.

[0076] Example 1

[0077] A hydrogen peroxide fluorescent probe with the following structural formula:

[0078]

[0079] The method for preparing the above hydrogen peroxide fluorescent probe is shown in the flow chart. Figure 1 Specifically, the following steps are included:

[0080] S1: 2-Hydroxy-5-methylbenzaldehyde (2000 mg, 14.7 mmol) and 2-aminothiophenol (1838 mg, 14.7 mmol) were dissolved in dry ethanol (50 mL), and HCl (7%, 4 mL) and H2O2 (30%, 10 mL) were added. The mixture was stirred at room temperature for 1 h, filtered, and the filter cake was washed with 50% ethanol. After drying, 2280 mg of white powder was obtained. Hexamethylenetetramine (1000 mg, 6.21 mmol) and the above white powder (500 mg, 6.21 mmol) were dissolved in CF3COOH and refluxed at 80°C for 6 h. The reaction was monitored by LC. The crude product obtained after removing the solvent was extracted three times with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, and evaporated to dryness to obtain a mixture. The mixture was separated by column chromatography to obtain compound 1 as a bright yellow powder (491 mg, 88%).

[0081] S2: Compound 1 (50 mg, 0.13 mmol) and piperidine (100 μL) were dissolved in ultra-dry C2H5OH and activated for 0.5 h. After the reaction was complete, 50 mg of compound 2 was added in several portions and refluxed for 36 h. The reaction was monitored by TLC, extracted with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, and evaporated to dryness to obtain a mixture. The mixture was purified to obtain compound 3.

[0082] S3: Compound 3, 4-bromomethylphenylboronic acid pinacol ester (115 mg, 0.39 mmol), and K2CO3 (33 mg, 0.24 mmol) were dissolved in DMF (2 mL), and the mixture was fully reacted at 60°C for 1.5 h. After cooling, water was added to precipitate the product, and the product was dissolved in CH2Cl2 to obtain a mixture. The mixture was separated and purified to obtain the hydrogen peroxide fluorescent probe.

[0083] Among them, the structural formula of compound 2 is:

[0084]

[0085] Among them, the nuclear magnetic resonance hydrogen spectrum of compound 1 is:

[0086] 1 H NMR (500MHz, Chloroform-d) δ13.03 (s, 1H), 10.49 (s, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.94 (d, J = 9. 1Hz, 1H), 7.90 (s, 1H), 7.71 (s, 1H), 7.54 (t, J = 7.1Hz, 1H), 7.45 (t, J = 7.6Hz, 1H), 2.41 (s, 3H).

[0087] Among them, the nuclear magnetic resonance hydrogen spectrum of compound 3 is:

[0088] 1 H NMR(500MHz,Chloroform-d)δ13.33(s,1H),8.06(d,J=15.8Hz,1H),8.02(d,J=8.1Hz,1H),7.93(d,J=7.9Hz,1H),7.88(d,J=15.8Hz,1H ),7.83(d,J=8.1Hz,2H),7.56–7.52(m,2H),7.49(s,1H),7.44(t,J=7.6Hz,1H),7.19–7.10(m,11H),7.05(t,J=4.6Hz,6H),2.39(s,3H).

[0089] Among them, the nuclear magnetic resonance hydrogen spectrum of the hydrogen peroxide fluorescent probe is:

[0090] 1 H NMR(500MHz,Chloroform-d)δ8.27(s,1H),8.12(s,1H),8.09(d,J=6.1Hz,1H),7.90(d,J=7.9Hz,1H),7.82(d,J=7.6Hz,2H),7.70(d,J=8.3Hz,2H),7. 57(d,J=16.0Hz,2H),7.49(d,J=7.9Hz,3H),7.40(t,J=7.5Hz,1H),7.12(d, J=8.3Hz,11H),7.06–7.01(m,6H),4.91(s,2H),2.47(s,3H),1.35(s,12H).

[0091] Among them, the high-resolution mass spectrum of hydrogen peroxide fluorescent probe is as follows Figure 3 shown.

[0092] Example 2

[0093] A hydrogen peroxide fluorescent probe with the following structural formula:

[0094]

[0095] The method for preparing the above hydrogen peroxide fluorescent probe is shown in the flow chart. Figure 2 Specifically, the following steps are included:

[0096] S1: Compound 1 (50 mg, 0.19 mmol) prepared in Example 1 and piperidine (100 mL) were dissolved in C2H5OH and stirred. A total of 50 mg of compound 4 was added in small amounts and several times. The mixture was refluxed at 90°C for 36 h. The reaction was monitored by TLC. The mixture was extracted with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, and evaporated to dryness to obtain a mixture, which was separated by column chromatography to obtain compound 5 (16.04 mg, 15.7%).

[0097] S2 Solid powder 5 (50 mg, 0.09 mmol) and 4-bromomethylphenylboronic acid pinacol ester (110 mg, 0.37 mmol) and K2CO3 (37 mg, 0.24 mmol) were dissolved in DMF (2 mL) and fully reacted at 60°C. After cooling, water (8 mL) was added to precipitate the product, which was dissolved in CH2Cl2 to obtain a mixture, which was separated by column to obtain a hydrogen peroxide fluorescent probe (15.8 mg, 23.2%).

[0098] Among them, the structural formula of compound 4 is:

[0099]

[0100] Among them, the nuclear magnetic resonance hydrogen spectrum of compound 5 is:

[0101] 1H NMR(500MHz,Chloroform-d)δ13.30(s,1H),8.07(d,J=15.8Hz,1H),8.00(d,J=8.1Hz,1H),7.98–7.95(m,2H),7.95–7.90(m,2H),7.54–7.50( m,2H),7.48(d,J=2.1Hz,1H),7.45–7.40(m,1H),7.35–7.30(m,4H),7. 18(d,J=7.3Hz,4H),7.16–7.12(m,2H),7.08–7.04(m,2H),2.38(s,3H).

[0102] Among them, the nuclear magnetic resonance hydrogen spectrum of the hydrogen peroxide fluorescent probe is:

[0103] 1H NMR(500MHz,Chloroform-d)δ13.30(s,1H),8.07(d,J=15.8Hz,1H),8.00(d,J=8.1Hz,1H),7.98–7.95(m,2H),7.95–7.90(m,2H),7.54–7.50( m,2H),7.48(d,J=2.1Hz,1H),7.45–7.40(m,1H),7.35–7.30(m,4H),7. 18(d,J=7.3Hz,4H),7.16–7.12(m,2H),7.08–7.04(m,2H),2.38(s,3H).

[0104] Among them, the high-resolution mass spectra of hydrogen peroxide fluorescent probes, such as Figure 4 shown.

[0105] Performance testing:

[0106] The hydrogen peroxide fluorescent probe prepared in Example 1 was used for hydrogen peroxide (H2O2) fluorescence detection, and the changes in the ultraviolet spectrum were observed. Specifically, the hydrogen peroxide fluorescent probe prepared in Example 1 (named Probe-1) was dissolved in PBS (80% DMSO) solution to obtain a probe mother solution (10 -3 mol / L), and then diluted with PBS to obtain 10 -5 mol / L Probe-1 solution, add H2O2 solution (40×10 -5 mol / L), and after 30 minutes of testing, the following Figure 5 The UV spectrum shown in Figure 5 It can be seen that a new absorption peak is generated at 550 nm, and the color of the solution changes from colorless to pink.

[0107] The hydrogen peroxide fluorescent probe prepared in Example 1 was used for hydrogen peroxide (H2O2) fluorescence detection, and the change in fluorescence intensity was observed. Specifically, the hydrogen peroxide fluorescent probe prepared in Example 1 (named Probe-1) was dissolved in PBS (80% DMSO) solution to obtain a probe mother solution (10 -3 mol / L), and then diluted with PBS to obtain 10 -5 mol / L Probe-1 solution, add H2O2 solution (40×10 -5 mol / L), such as Figure 6 As shown, the change of fluorescence intensity at 380nm was tested. Figure 6 It can be seen that the fluorescence increased by nearly 70% at 3 minutes.

[0108] The hydrogen peroxide fluorescent probe prepared in Example 1 was used for hydrogen peroxide (H2O2) fluorescence detection, and the detection limit was tested. Specifically, the hydrogen peroxide fluorescent probe prepared in Example 1 (named Probe-1) was dissolved in PBS (80% DMSO) solution to obtain a probe mother solution (10 -3 mol / L), and then diluted with PBS to obtain 10 -5 mol / L Probe-1 solution, add different concentrations of H2O2 solution, such as Figure 7 The changes in fluorescence intensity were tested as shown in Figure 7 It can be seen that there is a good linear relationship between the emission peak intensity at 652 nm and the H2O2 concentration. Based on this linear relationship, the detection limit of the probe can be obtained as low as 0.58 nM. The nM-level detection limit of the probe indicates that it has very high sensitivity to hydrogen peroxide.

[0109] In summary, compared with the prior art, the hydrogen peroxide fluorescent probe of the present invention has the following advantages:

[0110] (1) The present invention overcomes the shortcomings of existing H2O2 fluorescent probes, such as small Stokes shift, large background interference, and weak solid-state emission fluorescence. The hydrogen peroxide fluorescent probe provided by the present invention has the characteristics of simple operation, rapid reaction, good selectivity, high sensitivity, large Stokes shift, and colorimetric and fluorescence dual-mode detection in H2O2 detection;

[0111] (2) The fluorescent probe provided by the present invention has the characteristics of colorimetric and fluorescence dual-mode detection in H2O2 detection;

[0112] (3) This method has practical application value in the field of H2O2 detection research and can be used for H2O2 monitoring in organisms, the environment, and food;

[0113] (4) The synthesis process of the probe molecule in the present invention is simple, the raw materials are cheap and readily available, and large-scale production can be carried out.

[0114] The above are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A hydrogen peroxide fluorescent probe, characterized in that: It has the following structure: Wherein, R1 is one of an alkyl group, an alkoxy group, and a substituted or unsubstituted aromatic group; Wherein, R2 is one of the following structural formulas:

2. A hydrogen peroxide fluorescent probe according to claim 1, characterized in that: R1=-(CH2) n CH3, n=0-5.

3. A hydrogen peroxide fluorescent probe according to claim 1, characterized in that: R1=-O(CH2) n CH3, n=0-5.

4. A hydrogen peroxide fluorescent probe according to claim 1, characterized in that: The R1 is one of the following structural formulas: n＝0-21。 5. A method for preparing a hydrogen peroxide fluorescent probe according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: Under acidic conditions, a hydroxy-substituted benzaldehyde, an amino-substituted thiophenol, and an oxidant are mixed in an organic solvent, stirred, and then filtered to obtain a solid reagent; the solid reagent and a nitrogen source are dissolved in an acidic solution, refluxed, and extracted to obtain compound 1; S2: Compound 1 reacts with Compound 2 or Compound 4 to obtain an intermediate product; S3: mixing the intermediate product, phenylboronic acid pinacol ester with a halogen substituent, and an alkaline reagent in an organic solvent, and heating the mixture to react to obtain the hydrogen peroxide fluorescent probe; The structural formula of the compound 2 is: The structural formula of the compound 4 is:

6. The method according to claim 5, characterized in that: The hydroxy-substituted benzaldehyde includes 2-hydroxy-5-methylbenzaldehyde.

7. The method according to claim 5, characterized in that: The nitrogen source includes hexamethylenetetramine.

8. The method according to claim 5, characterized in that: In step S2, the step of preparing the intermediate product by using compound 1 and compound 2 includes: Compound 1, a basic catalyst and compound 2 are mixed, refluxed and extracted to obtain an intermediate product.

9. The method according to claim 5, characterized in that: In step S2, the step of preparing the intermediate product by using compound 1 and compound 4 includes: Compound 1 and a catalyst are mixed in an activation reagent, and compound 4 is added after activation. The mixture is refluxed, extracted, and dried to obtain an intermediate product.

10. A biosensor, characterized in that: The method comprises a hydrogen peroxide fluorescent probe according to any one of claims 1 to 4.

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