Fluorescent probe for detecting carboxyl as well as preparation method and application of fluorescent probe

Abstract

The invention provides a fluorescent probe for detecting carboxyl as well as a preparation method and application of the fluorescent probe. The fluorescent probe is NBD-NHOH. The fluorescent probe belongs to an off-on type fluorescent probe, fluorescence can be turned on only when the fluorescent probe is covalently connected with carboxyl on glass and is excited, and when the fluorescent probe which is non-specifically adsorbed with the glass is in a closed loop state, the fluorescent probe is excited and cannot emit fluorescence.

Description

Technical Field

[0001] The invention belongs to the field of biosensors and relates to a fluorescent probe for detecting carboxyl groups and a preparation method and application thereof. Background Art

[0002] Fluorescent labeling technology is a technology that identifies the object of study based on the covalent bond between fluorescent substances and the molecules being studied. It is widely used in the field of molecular detection and quantitative analysis due to its advantages such as high sensitivity, low detection limit, low toxicity and no radioactive pollution.

[0003] Functional group-modified slides are widely used in biosensors. These slides primarily utilize reactions between chemical functional groups and biomolecules to immobilize proteins, antibodies, DNA, RNA, and other biomolecules. These biosensors have broad application prospects in medical diagnostics, environmental monitoring, food safety, and other fields. For example, in medical diagnostics, carboxyl-modified slides can be used to immobilize specific antibodies or proteins for the detection of pathogens or biomarkers in patient samples. Therefore, being able to quantitatively measure the content of functional groups on the slide is particularly important.

[0004] Currently, there are many fluorescent reagents on the market that can be used to chemically react with chemical functional groups to detect the content of functional groups. However, such fluorescent reagents are prone to non-specific adsorption with glass and then adhere to the glass surface, and are difficult to clean. Such fluorescent reagents will undergo non-specific adsorption with glass, and will emit fluorescence when excited even if they do not react with chemical functional groups, resulting in abnormal fluorescence images and high fluorescence intensity values ​​during photography, which cannot accurately reflect the distribution of chemical functional groups on the glass.

[0005] Therefore, it is of great significance to study a fluorescent probe that can accurately detect specific chemical functional groups. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention provides an "off-on" type fluorescent probe for detecting carboxyl groups on the glass surface, as well as its preparation method and application, which solves the problem of quantitative analysis errors caused by nonspecific adsorption between glass and fluorescent molecules.

[0007] To achieve this object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a fluorescent probe for detecting carboxyl groups, wherein the structural formula of the fluorescent probe is as follows: .

[0008] As a preferred embodiment of the present invention, the maximum absorption wavelength range of the fluorescent probe is 450nm-500nm.

[0009] As a preferred embodiment of the present invention, the maximum emission wavelength of the fluorescent probe is 620nm-650nm.

[0010] In a second aspect, the present invention provides a method for preparing the aforementioned fluorescent probe, the preparation method comprising: (1) dissolving the compound of formula I and halogenated p-benzaldehyde in an organic solvent and reacting them under alkaline conditions to obtain a compound of formula II; (2) dissolving the compound of formula II with hydroxylamine hydrochloride and an alkaline regulator in an organic solvent for reaction to obtain the fluorescent probe NBD-NHOH; Wherein, Formula I: ; Formula II: .

[0011] As a preferred embodiment of the present invention, the halogenated p-benzaldehyde in step (1) includes any one of p-fluorobenzaldehyde, p-chlorobenzaldehyde or p-bromobenzaldehyde, preferably p-fluorobenzaldehyde.

[0012] As a preferred embodiment of the present invention, the molar ratio of the compound of formula I to the halogenated p-benzaldehyde in step (1) is 1:(1-1.5), preferably 1:1.2.

[0013] As a preferred embodiment of the present invention, the organic solvent in step (1) includes any one of acetonitrile, ethanol or methanol, or a combination of at least two of them.

[0014] As a preferred embodiment of the present invention, the solubility of the compound of formula I in step (1) in an organic solvent is 40 mg / mL-60 mg / mL.

[0015] As a preferred embodiment of the present invention, the alkaline environment in step (1) is a reaction environment with a pH of 9-11.

[0016] As a preferred embodiment of the present invention, the alkaline environment in step (1) is adjusted by adding an alkaline regulator.

[0017] As a preferred embodiment of the present invention, the alkaline regulator includes any one of triethylamine, triethylenediamine, piperidine, tetramethylethylenediamine, sodium dihydrogen phosphate or sodium bicarbonate, or a combination of at least two thereof.

[0018] As a preferred embodiment of the present invention, the reaction temperature of the reaction in step (1) is 60°C-110°C.

[0019] As a preferred embodiment of the present invention, the reaction time of the reaction in step (1) is 12h-48h.

[0020] As a preferred embodiment of the present invention, the molar ratio of the compound of formula II to hydroxylamine hydrochloride and the alkaline regulator in step (2) is 1:(1-2):(1-5).

[0021] As a preferred embodiment of the present invention, the organic solvent in step (2) includes any one of acetonitrile, ethanol or methanol, or a combination of at least two of them.

[0022] As a preferred embodiment of the present invention, the solubility of the compound of formula II in step (2) in the organic solvent is 10 mg / mL-30 mg / mL.

[0023] As a preferred embodiment of the present invention, the alkaline regulator in step (2) includes any one of triethylamine, triethylenediamine, piperidine, tetramethylethylenediamine, sodium dihydrogen phosphate or sodium bicarbonate, or a combination of at least two thereof.

[0024] As a preferred embodiment of the present invention, the reaction temperature of the reaction in step (2) is 60°C-100°C.

[0025] As a preferred embodiment of the present invention, the reaction time of the reaction in step (2) is 2 h to 10 h.

[0026] As a preferred embodiment of the present invention, the reaction product in step (2) is purified by silica gel column chromatography.

[0027] In a third aspect, the present invention provides a method for detecting carboxyl groups using the aforementioned fluorescent probe, the method comprising: The substrate to be detected is immersed in a solution containing a fluorescent probe; Cleaning the substrate to be detected after immersion; The fluorescence distribution of the substrate to be detected is detected to obtain the carboxyl distribution of the substrate to be detected.

[0028] As a preferred embodiment of the present invention, the substrate includes any one of a glass substrate, a plastic substrate or a silicon wafer, or a combination of at least two of them.

[0029] As a preferred embodiment of the present invention, detecting the fluorescence distribution of the substrate to be detected includes: Exciting the fluorescent probe with the excitation light of the fluorescent probe; The fluorescence distribution of the substrate to be detected is obtained using a detection device.

[0030] As a preferred embodiment of the present invention, the wavelength range of the excitation light of the fluorescent probe is 480nm-520nm.

[0031] As a preferred solution of the present invention, the detection device includes a fluorescence detection device.

[0032] As a preferred embodiment of the present invention, the method for preparing the solution containing the fluorescent probe comprises: The fluorescent probe is dissolved in a solvent to prepare a mother solution, the mother solution is dissolved in a buffer solution and then ultrasonicated to obtain a solution containing the fluorescent probe.

[0033] As a preferred embodiment of the present invention, the solvent includes any one of dimethyl sulfoxide, DMF, methanol or ethanol, or a combination of at least two thereof.

[0034] As a preferred embodiment of the present invention, the concentration of the fluorescent probe in the mother solution is 50 μmol / L-100 μmol / L.

[0035] As a preferred embodiment of the present invention, the buffer comprises PBS buffer or KPi buffer.

[0036] As a preferred embodiment of the present invention, the pH of the buffer solution is 5-9.

[0037] As a preferred embodiment of the present invention, the ultrasonic time is 10 min-20 min; As a preferred embodiment of the present invention, the concentration of the fluorescent probe in the solution containing the fluorescent probe is 3 nmol / L-10 nmol / L; As a preferred embodiment of the present invention, the soaking time is 10 hours to 20 hours.

[0038] As a preferred embodiment of the present invention, the soaking temperature is 25°C-50°C.

[0039] As a preferred embodiment of the present invention, the washing is performed using a buffer solution, and the buffer solution includes any one of PBS solution, KPi buffer solution or SSC buffer solution, or a combination of at least two of them.

[0040] As a preferred embodiment of the present invention, the pH of the buffer solution is 6-8.

[0041] Compared with the prior art, the present invention has the following beneficial effects: The fluorescent probe described in this invention is an "off-on" fluorescent probe. It only fluoresces when covalently bound to carboxyl groups on the glass and excited. Fluorescent probes that are nonspecifically adsorbed to the glass remain in a "closed loop" state and do not fluoresce. Therefore, even if the cleaning reagent is applied, fluorescent probes not covalently bound to carboxyl groups are not completely washed away, and this does not affect the accuracy of detecting the distribution of carboxyl groups on the glass.

[0042] The fluorescent probe of the present invention can greatly eliminate the interference of detection background on the results during detection, improve the accuracy of detection, has a good response to carboxyl groups, has high sensitivity, is easy to store, and has good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the action mechanism of the fluorescent probe of the present invention; Figure 2 This is a fluorescence detection image of the carboxyl-free glass A-1 in Example 5 of the present invention using the fluorescent labeling reagent Cy3-NH2 in Comparative Example 1; Figure 3 This is a fluorescence detection image of the carboxyl-free glass A-2 in Example 5 of the present invention using the fluorescent labeling reagent in Example 1; Figure 4 This is a fluorescence detection image of carboxyl glass B-1 using the fluorescent labeling reagent Cy3-NH2 in Example 5 of the present invention; Figure 5 This is a fluorescence detection image of the carboxyl glass B-2 in Example 5 of the present invention using the fluorescent labeling reagent in Example 1. DETAILED DESCRIPTION

[0044] To better illustrate the present invention and facilitate understanding of the technical solution of the present invention, the present invention is further described in detail below. However, the following embodiments are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0045] In the first aspect, the specific embodiment of the present invention provides a fluorescent probe for detecting carboxyl groups, and the structural formula of the fluorescent probe is as follows: .

[0046] The fluorescent probe for detecting carboxyl groups described in the present invention is an "off-on" type fluorescent probe, which solves the problem of quantitative analysis errors caused by nonspecific adsorption between glass and fluorescent molecules. The fluorescence will only be turned on when the fluorescent probe is covalently linked to the carboxyl group on the glass and excited. When the fluorescent probe nonspecifically adsorbed to the glass is in a "closed loop" state, it will not emit fluorescence when excited. Therefore, when detecting the distribution of carboxyl groups on the glass, after the cleaning reagent is introduced, even if the fluorescent probe that has not reacted with the carboxyl group is not completely cleaned off, it will not be excited to emit fluorescence, and will not affect the accuracy of the detection of the distribution of carboxyl groups on the glass. The reaction of the above-mentioned fluorescent probe with the carboxyl group refers to the covalent linkage of the fluorescent probe with the carboxyl group. The distribution of the above-mentioned carboxyl groups includes but is not limited to: the presence or absence of carboxyl groups, the density, quantity, and distribution uniformity of the carboxyl groups, etc.

[0047] When the fluorescent probe of the present invention interacts with the carboxyl group, the hydroxylamine group interacts with the carboxyl group to cause fluorescence to turn on. The specific mechanism of action is as follows Figure 1 shown.

[0048] As a preferred embodiment of the present invention, the maximum absorption wavelength range of the fluorescent probe is 450nm-500nm, such as 450nm, 460nm, 470nm, 480nm, 490nm or 500nm, but is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable.

[0049] As a preferred embodiment of the present invention, the maximum emission wavelength of the fluorescent probe is 620nm-650nm, such as 620nm, 630nm, 640nm or 650nm, but is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable.

[0050] In a second aspect, the present invention provides a method for preparing the aforementioned fluorescent probe, which comprises: Dissolving the compound of formula I and halogenated p-benzaldehyde in an organic solvent and reacting them under alkaline conditions to obtain a compound of formula II; Taking p-fluorobenzaldehyde as an example, the reaction formula is as follows:

[0051] The compound of formula II is dissolved in an organic solvent with hydroxylamine hydrochloride and an alkaline regulator to react to obtain the fluorescent probe NBD-NHOH;

[0052] Wherein, Formula I: ; Formula II: .

[0053] In the present invention, the compound of formula I is prepared by dissolving 4-(diethylamino) salicylaldehyde and ethyl acetoacetate in anhydrous ethanol. For the specific preparation process, please refer to the document "Molecular Design of Coumarin / Indene Dione Dyes and Research on Fluorescence On-Type Recognition Performance" (Meng Yu, Tianjin University of Technology).

[0054] As a preferred embodiment of the present invention, the halogenated p-benzaldehyde in step (1) includes any one of p-fluorobenzaldehyde, p-chlorobenzaldehyde or p-bromobenzaldehyde, preferably p-fluorobenzaldehyde.

[0055] As a preferred embodiment of the present invention, the molar ratio of the compound of formula I to the halogenated p-benzaldehyde in step (1) is 1:(1-1.5), for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5, etc., but is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable, preferably 1:1.2.

[0056] As a preferred embodiment of the present invention, the organic solvent in step (1) includes any one of acetonitrile, ethanol or methanol, or a combination of at least two of them.

[0057] As a preferred embodiment of the present invention, the solubility of the compound of formula I in step (1) in the organic solvent is 40 mg / mL to 60 mg / mL, for example, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, or 60 mg / mL, but is not limited to the listed values. Other values ​​not listed within this range are also applicable. The amount of the organic solvent used is such that the compound of formula I is completely dissolved therein.

[0058] As a preferred embodiment of the present invention, the alkaline environment in step (1) is a reaction environment with a pH of 9-11, for example, a pH of 9, 10, 11, etc.

[0059] As a preferred embodiment of the present invention, the alkaline environment in step (1) is adjusted by adding an alkaline regulator.

[0060] As a preferred embodiment of the present invention, the alkaline regulator includes any one of triethylamine, triethylenediamine, piperidine, tetramethylethylenediamine, sodium dihydrogen phosphate or sodium bicarbonate, or a combination of at least two thereof.

[0061] As a preferred embodiment of the present invention, the reaction temperature of the reaction in step (1) is 60°C-110°C, for example, 60°C, 70°C, 80°C, 90°C, 100°C or 110°C, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0062] As a preferred embodiment of the present invention, the reaction time of the reaction in step (1) is 12 h to 48 h, for example, 12 h, 14 h, 16 h, 18 h, 20 h, 24 h, 26 h, 30 h, 34 h, 36 h, 40 h, 42 h, 44 h, 46 h or 48 h, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0063] As a preferred embodiment of the present invention, the molar ratio of the compound of formula II to hydroxylamine hydrochloride and the alkaline regulator in step (2) is 1:(1-2):(1-5), for example, 1:1:(1-5), 1:1.2:(1-5), 1:1.4:(1-5), 1:1.6:(1-5), 1:1.8:(1-5), 1:(1-2):2, 1:(1-2):3, 1:(1-2):4 or 1:(1-2):5, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0064] As a preferred embodiment of the present invention, the organic solvent in step (2) includes any one of acetonitrile, ethanol or methanol, or a combination of at least two of them.

[0065] As a preferred embodiment of the present invention, the solubility of the compound of Formula II in step (2) in the organic solvent is 10 mg / mL to 30 mg / mL, for example, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or 30 mg / mL, but is not limited to the listed values. Other values ​​not listed within this range are also applicable. The amount of the organic solvent used is sufficient to completely dissolve the compound of Formula II therein.

[0066] As a preferred embodiment of the present invention, the alkaline regulator in step (2) includes any one of triethylamine, triethylenediamine, piperidine, tetramethylethylenediamine, sodium dihydrogen phosphate or sodium bicarbonate, or a combination of at least two thereof.

[0067] As a preferred embodiment of the present invention, the reaction temperature of the reaction in step (2) is 60°C-100°C, for example, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or 100°C, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0068] As a preferred embodiment of the present invention, the reaction time of the reaction in step (2) is 2 h to 10 h, for example, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h or 10 h, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0069] As a preferred embodiment of the present invention, the reaction product in step (2) is purified by silica gel column chromatography.

[0070] In a third aspect, the present invention provides a method for detecting carboxyl groups using the aforementioned fluorescent probe, the method comprising: The substrate to be detected is immersed in a solution containing a fluorescent probe; Cleaning the substrate to be detected after immersion; The fluorescence distribution of the substrate to be detected is detected to obtain the carboxyl distribution of the substrate to be detected.

[0071] In the present invention, after the fluorescent probe is excited by the excitation light of the fluorescent probe, the fluorescent probe that reacts with the carboxyl group emits fluorescence, while the fluorescent probe that does not react with the carboxyl group does not emit light.

[0072] As a preferred embodiment of the present invention, the substrate includes any one of a glass substrate, a plastic substrate or a silicon wafer, or a combination of at least two of them.

[0073] As a preferred embodiment of the present invention, detecting the fluorescence distribution of the substrate to be detected includes: Exciting the fluorescent probe with the excitation light of the fluorescent probe; The fluorescence distribution of the substrate to be detected is obtained using a detection device.

[0074] As a preferred embodiment of the present invention, the wavelength range of the excitation light of the fluorescent probe is 480nm-520nm, such as 480nm, 490nm, 500nm, 510nm or 520nm, etc., but is not limited to the listed values. Other unlisted values ​​within the numerical range are also applicable.

[0075] As a preferred solution of the present invention, the detection device includes a fluorescence detection device.

[0076] As a preferred embodiment of the present invention, the method for preparing the solution containing the fluorescent probe comprises: The fluorescent probe is dissolved in a solvent to prepare a mother solution, the mother solution is dissolved in a buffer solution and then ultrasonicated to obtain a solution containing the fluorescent probe.

[0077] As a preferred embodiment of the present invention, the solvent includes any one of dimethyl sulfoxide (DMSO), DMF, methanol or ethanol, or a combination of at least two thereof.

[0078] As a preferred embodiment of the present invention, the concentration of the fluorescent probe in the mother solution is 50 μmol / L-100 μmol / L, for example, 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, 90 μmol / L or 100 μmol / L, etc., but is not limited to the listed values, and other unlisted values ​​within this numerical range are also applicable.

[0079] As a preferred embodiment of the present invention, the buffer comprises PBS buffer or KPi buffer.

[0080] As a preferred embodiment of the present invention, the pH of the buffer solution is 5-9.

[0081] As a preferred embodiment of the present invention, the ultrasonic time is 10 min-20 min, for example, 10 min, 12 min, 14 min, 16 min, 18 min or 20 min, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0082] As a preferred embodiment of the present invention, the concentration of the fluorescent probe in the solution containing the fluorescent probe is 3nmol / L-10nmol / L, for example, 3nmol / L, 4nmol / L, 5nmol / L, 6nmol / L, 7nmol / L, 8nmol / L, 9nmol / L or 10nmol / L, etc., but is not limited to the listed values, and other unlisted values ​​within this numerical range are also applicable.

[0083] As a preferred embodiment of the present invention, the soaking time is 10h-20h, such as 10h, 12h, 14h, 16h, 18h or 20h, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0084] As a preferred embodiment of the present invention, the soaking temperature is 25°C-50°C, such as 25°C, 30°C, 35°C, 40°C, 45°C or 50°C, but is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable.

[0085] As a preferred embodiment of the present invention, the washing is performed using a buffer solution, and the buffer solution includes any one of PBS solution, KPi buffer solution or SSC buffer solution, or a combination of at least two of them.

[0086] As a preferred embodiment of the present invention, the pH of the buffer solution is 8.

[0087] The following are typical but non-limiting examples of the present invention: The structures of the compounds of formula I used in the following examples are: ; The preparation method of the compound of formula I is as follows: Mix 4-(diethylamino)salicylaldehyde (3.73 g, 19.3 mmol) and ethyl acetoacetate (2.92 g, 23.16 mmol), add 20 mL of anhydrous ethanol and 0.1 mL of piperidine, and stir at room temperature for 16 h. Cool in an ice-water bath and filter to obtain a yellow powder, which is the compound of formula I.

[0088] Example 1: This embodiment provides a fluorescent probe for detecting carboxyl groups and a preparation method thereof. The structure of the fluorescent probe is: .

[0089] The preparation method of the fluorescent probe is as follows: (1) The compound of formula I (1 mmol, 259 mg) and p-fluorobenzaldehyde (1.5 mmol, 186 mg) were dissolved in 10 mL of acetonitrile. 1-2 drops of piperidine were added dropwise and the mixture was reacted at 90°C for 24 h. After completion of the reaction, the mixture was recrystallized from ethanol / ether to obtain 328 mg of an orange-red solid, which was the compound of formula II. The yield was 90%. ESI: m / z [M + H]+ = 366.14; calculated: 365.14. Mp: 120-122°C. 1H NMR (400 MHz, CDCl3-CD3OD, 5:1, v / v): 8.83(s, 1H), 7.66 (s, 1H),7.45(d, J = 7.6 Hz, 1H), 7.28(d, J = 7.6 Hz, 2H), 7.07 (d, J = 15.6 Hz, 2H), 6.92 (d, J = 7.6 Hz, 2H), 6.55 (s, 1H), 6.53 (d, J = 7.6 Hz, 1H), 3.50 (q, J =6.8 Hz, 4H), 1.51 (t, J = 6.8 Hz, 6H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v): 187.90, 162.67, 161.21, 160.11, 151.71, 150.43, 144.12, 134.21, 130.23,129.87, 127.86, 127.56, 117.28, 115.43, 109.87, 105.96, 48.88, 13.39.

[0090] (2) Compound II (0.55 mmol, 200 mg), hydroxylamine hydrochloride (1 mmol, 33 mg), and sodium acetate (1.65 mmol, 135.34 mg) were dissolved in 50 mL of ethanol and heated under reflux for 4 h. After the reaction, the mixture was filtered and separated by dichloromethane / methanol column chromatography to obtain 41 mg of an orange-red solid, NBD-NHOH, in a yield of 20%. ESI: m / z [M + H]+ = 379.42; calculated: 378.40. Mp: 114-115°C. 1H NMR (400 MHz, DMSO-d6): 8.75(s, 1H), 7.66 (d, J = 15.6 Hz, 2H), 7.45 (d, J = 7.6 Hz, 1H), 6.53 (d, J = 7.6 Hz, 2H), 6.41(d, J = 7.6 Hz, 2H), 4.17(s,1H), 3.50 (q, J = 6.8 Hz, 4H), 2.0(s,1H), 1.51 (t, J = 6.8 Hz, 6H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v):194.32, 162.21, 160.11, 151.77, 150.36, 149.27, 144.11, 134.25, 129.88,129.69, 127.56, 124.00, 117.35, 115.34, 109.89, 105.66, 50.37, 20.33.

[0091] Example 2: This embodiment provides a fluorescent probe for detecting carboxyl groups and a preparation method thereof. The structure of the fluorescent probe is: .

[0092] The preparation method of the fluorescent probe is as follows: (1) The compound of formula I (1 mmol, 259 mg) and p-fluorobenzaldehyde (1.2 mmol, 149 mg) were dissolved in 10 mL of acetonitrile. 1-2 drops of piperidine were added dropwise. The mixture was reacted at 100°C for 24 h. After completion of the reaction, the mixture was recrystallized from ethanol / ether to obtain 340 mg of an orange-red solid, which was the compound of formula II. The yield was 93%. ESI: m / z [M + H]+ = 366.14; calculated: 365.14. Mp: 120-122°C. 1H NMR (400 MHz, CDCl3-CD3OD, 5:1, v / v): 8.83(s, 1H), 7.66 (s, 1H),7.45(d, J = 7.6 Hz, 1H), 7.28(d, J = 7.6 Hz, 2H), 7.07 (d, J = 15.6 Hz, 2H), 6.92 (d, J = 7.6 Hz, 2H), 6.55 (s, 1H), 6.53 (d, J = 7.6 Hz, 1H), 3.50 (q, J =6.8 Hz, 4H), 1.51 (t, J = 6.8 Hz, 6H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v): 187.90, 162.67, 161.21, 160.11, 151.71, 150.43, 144.12, 134.21, 130.23,129.87, 127.86, 127.56, 117.28, 115.43, 109.87, 105.96, 48.88, 13.39.

[0093] (2) Compound II (0.55 mmol, 200 mg), hydroxylamine hydrochloride (0.6 mmol, 20 mg), and sodium acetate (1.5 mmol, 135.34 mg) were dissolved in 30 mL of ethanol and heated under reflux for 6 h. After the reaction, the mixture was filtered and separated by dichloromethane / methanol column chromatography to obtain 56 mg of an orange-red solid, NBD-NHOH, in a yield of 27%. ESI: m / z [M + H]+ = 379.42; calculated: 378.40. Mp: 114-115°C. 1H NMR (400 MHz, DMSO-d6): 8.75(s, 1H), 7.66 (d, J = 15.6 Hz, 2H), 7.45 (d, J = 7.6 Hz, 1H), 6.53 (d, J = 7.6 Hz, 2H), 6.41(d, J = 7.6 Hz, 2H), 4.17(s,1H), 3.50 (q, J = 6.8 Hz, 4H), 2.0(s,1H), 1.51 (t, J = 6.8 Hz, 6H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v):194.32, 162.21, 160.11, 151.77, 150.36, 149.27, 144.11, 134.25, 129.88,129.69, 127.56, 124.00, 117.35, 115.34, 109.89, 105.66, 50.37, 20.33.

[0094] Example 3: This embodiment provides a fluorescent probe for detecting carboxyl groups and a preparation method thereof. The structure of the fluorescent probe is: .

[0095] The preparation method of the fluorescent probe is as follows: (1) The compound of formula I (1 mmol, 259 mg) and p-fluorobenzaldehyde (1.3 mmol, 162 mg) were dissolved in 10 mL of acetonitrile. 1-2 drops of piperidine were added dropwise and the mixture was reacted at 60°C for 30 h. After completion of the reaction, the product was recrystallized from ethanol / ether to obtain 250 mg of an orange-red solid, which was the compound of formula II. The yield was 68%. ESI: m / z [M + H]+ = 366.14; calculated: 365.14. Mp: 120-122°C. 1H NMR (400 MHz, CDCl3-CD3OD, 5:1, v / v): 8.83(s, 1H), 7.66 (s, 1H),7.45(d, J = 7.6 Hz, 1H), 7.28(d, J = 7.6 Hz, 2H), 7.07 (d, J = 15.6 Hz, 2H), 6.92 (d, J = 7.6 Hz, 2H), 6.55 (s, 1H), 6.53 (d, J = 7.6 Hz, 1H), 3.50 (q, J =6.8 Hz, 4H), 1.51 (t, J = 6.8 Hz, 6H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v): 187.90, 162.67, 161.21, 160.11, 151.71, 150.43, 144.12, 134.21, 130.23,129.87, 127.86, 127.56, 117.28, 115.43, 109.87, 105.96, 48.88, 13.39.

[0096] (2) Compound II (0.55 mmol, 200 mg), hydroxylamine hydrochloride (1 mmol, 33 mg), and sodium acetate (1 mmol, 83 mg) were dissolved in 50 mL of ethanol and heated under reflux for 4 h. After the reaction, the mixture was filtered and separated by dichloromethane / methanol column chromatography to obtain 37 mg of orange-red solid NBD-NHOH in 18% yield. ESI: m / z [M + H]+ = 379.42; calculated: 378.40. Mp 114-115°C. 1H NMR (400 MHz, DMSO-d6): 8.75(s, 1H), 7.66 (d, J =15.6 Hz, 2H), 7.45 (d, J = 7.6 Hz, 1H), 6.53 (d, J = 7.6 Hz, 2H), 6.41(d, J= 7.6 Hz, 2H), 4.17 (s, 1H), 3.50 (q, J = 6.8 Hz, 4H), 2.0 (s, 1H), 1.51 (t, J =6.8 Hz, 6H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v): 194.32, 162.21, 160.11,151.77, 150.36, 149.27, 144.11, 134.25, 129.88, 129.69, 127.56, 124.00,117.35, 115.34, 109.89, 105.66, 50.37, 20.33.

[0097] Comparative Example 1: This comparative example provides a fluorescent labeling reagent Cy3-NH2 commonly used in the prior art.

[0098] Example 5: This example uses four pieces of glass, two of which are carboxyl-free glass, labeled A-1 and A2, and two of which are glass with carboxyl functional groups, labeled B-1 and B-2. This example uses the fluorescent probes described in Example 1 and Comparative Example 1 to detect the four pieces of glass, respectively, as follows: The fluorescent probe was dissolved in DMSO to prepare a mother solution, which was then dissolved in a PBS solution with a pH of 8. Ultrasonication was performed for 10-20 minutes to ensure that the fluorescent molecules were fully dissolved in the buffer solution. Four pieces of glass with carboxyl groups were prepared and immersed in two different fluorescent solutions at 50°C for 10-20 hours. After the reaction, the glass was washed with a PBS buffer with a pH of 8. Within 3 hours after washing, the fluorescence intensity value was detected and counted using a fluorescence detection device. The test results are shown in Table 1.

[0099] The density and uniformity of the carboxyl groups detected on the four pieces of glass were measured, as shown in Table 1.

[0100] The fluorescence excitation wavelength was 500 nm, and the results were as follows. Figure 2-5 As shown. Among them, Figure 2 Schematic diagram of fluorescence detection after the carboxyl-free glass A-1 is immersed in Cy3-NH2 fluorescent solution. Figure 3 Schematic diagram of fluorescence detection after the carboxyl-free glass A-2 is immersed in NBD-NHOH fluorescent solution. Figure 4 Schematic diagram of fluorescence detection after carboxyl glass B-1 is immersed in Cy3-NH2 fluorescent solution. Figure 5 Schematic diagram of fluorescence detection after carboxyl glass B-2 is immersed in NBD-NHOH fluorescent solution. Figure 2 and Figure 3 It can be seen that even for glass without carboxyl groups, there are obvious bright spots in the Cy3-NH2 detection results, indicating that some fluorescent reagents remain on the chip surface, indicating that such fluorescent reagents are prone to non-specific adsorption with glass and then adhere to the glass surface. Moreover, fluorescent reagents that undergo non-specific adsorption with glass will also emit fluorescence when excited, thereby causing detection errors. However, when using NBD-NHOH for detection, even if some fluorescent reagents remain, they will not emit fluorescence when excited because they have not reacted with carboxyl groups, and will not cause detection errors. Figure 4 and Figure 5 It can be seen that carboxyl glasses B-1 and B-2 emit fluorescence when excited (Picture 4- Figure 5 Medium fluorescence is presented in grayscale. This is because the density of carboxyl groups on the glass is very high, resulting in too dense fluorescent spots. Therefore, the image appears in grayscale rather than bright spots. No fluorescence appears in black, such as Figure 2-Figure 3 As shown in the figure, however, glass B-1 immersed in the Cy3-NH2 fluorescent solution exhibits fluorescent bright spots due to the high local fluorescence intensity. This is because some of the Cy3-NH2 fluorescent reagent is easily non-specifically adsorbed to the glass and adheres to the glass surface. Moreover, the Cy3-NH2 fluorescent reagent that is non-specifically adsorbed to the glass also emits fluorescence when excited, resulting in fluorescent bright spots and detection errors. However, even if some residual fluorescent probe NBD-NHOH described in the present invention remains, it does not react with the carboxyl group and does not emit fluorescence when excited. Therefore, there are no fluorescent bright spots in the detected image.

[0101] Conventional fluorescent reagent Cy3-NH2 does not react with carboxyl groups and will emit fluorescence when excited. Its fluorescence intensity value is generally high, and bright spots will be generated due to its aggregation on the glass surface where no carboxyl groups are present, which can easily cause detection errors. In other words, glass without carboxyl groups may be mistakenly judged to have carboxyl groups. However, the fluorescent probe NBD-NHOH used in the present invention does not react with carboxyl groups for glass without carboxyl groups and will not emit fluorescence when excited, thus preventing detection errors.

[0102] While conventional fluorescent reagent Cy3-NH2 can also be excited to fluoresce on glass surfaces with carboxyl groups, the weak interaction between it and the glass causes residual fluorescence to also be excited, resulting in high local fluorescence intensity and deviations in the accuracy of detecting carboxyl group density and uniformity. The fluorescent probe NBD-NHOH used in the present invention, due to its off-on properties, does not fluoresce when excited even if it remains on the glass, even though it does not react with carboxyl groups. This avoids the drawback of high local fluorescence and provides higher accuracy in detecting carboxyl group density and uniformity.

[0103] Example 6: This embodiment uses four pieces of glass, two of which are carboxyl-free glass and two of which are glass with carboxyl functional groups. This embodiment uses the fluorescent probes described in Example 1 and Comparative Example 1 to detect the four pieces of glass respectively, as follows: The fluorescent probe was dissolved in DMSO to prepare a mother solution, which was then dissolved in a KPi solution with a pH of 8. Ultrasonication was performed for 10-20 minutes to ensure that the reagent was fully dissolved in the buffer solution. Four pieces of glass were immersed in two different fluorescent solutions at 50°C for 10-20 hours. After the reaction, they were washed with 3×SSC buffer. Within 3 hours after washing, the fluorescence intensity value was detected using a fluorescence detection device.

[0104] The density and uniformity of the carboxyl groups detected on the four pieces of glass were measured, as shown in Table 1.

[0105] Example 7: This example uses four pieces of glass, two of which are carboxyl-free and two of which are glass with carboxyl functional groups. This example uses the fluorescent probes described in Example 1 and Comparative Example 1 to detect carboxyl groups on the glass surface, as follows: The fluorescent probe was dissolved in TE buffer to prepare a stock solution, which was then dissolved in KPi solution (pH 8). Ultrasonication was performed for 10-20 minutes to ensure full dissolution of the reagent in the buffer. Four glass sheets were then immersed in each of the two fluorescent solutions at 50°C for 10-20 hours. After the reaction, the samples were washed with 3×SSC buffer and fluorescence intensity was measured within 3 hours using a fluorescence detector. The sites on the glass surface were photographed using a fluorescence microscope and analyzed using image processing software such as ImageJ. The number of fluorescent spots per unit area was counted to represent the functional group density.

[0106] The carboxyl group density and fluorescence intensity detected on the four pieces of glass were measured, as shown in Table 1.

[0107] The test results of Examples 5-7 are shown in Table 1.

[0108] Table 1: Test results of Examples 5-7

[0109] As can be seen from Table 1, when Cy3-NH2 is used as a fluorescent probe, its fluorescence intensity value and functional group density are generally high. There are fluorescent bright spots in the picture, and the grayscale distribution of the image is uneven. The main reason for this phenomenon is that ordinary fluorescent dyes will fluoresce when excited, regardless of whether they react with carboxyl groups. They are adsorbed on the glass and aggregate to produce bright spots. The presence of bright spots causes the fluorescence intensity value to increase. However, NBD-NHOH will only fluoresce when it is covalently linked to the carboxyl group and excited. It will not produce fluorescence even if it is adsorbed on the glass, avoiding the disadvantage that ordinary dye residual reagents will be excited to fluoresce when adsorbed on the glass. As shown in the attached figure, Figure 4-5 As shown, compared with Cy3-NH2, the fluorescent probe of the embodiment has no fluorescent spots and better uniformity.

[0110] The applicant declares that the present invention uses the above-described embodiments to illustrate the detailed process equipment and process flow of the present invention. However, the present invention is not limited to the above-described detailed process equipment and process flow, and does not necessarily rely on the above-described detailed process equipment and process flow in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for the raw materials of the present invention's products, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. A fluorescent probe for detecting a carboxyl group, characterized in that The structural formula of the fluorescent probe is as follows: 。 2. The fluorescent probe according to claim 1, characterized in that The maximum absorption wavelength range of the fluorescent probe is 450nm-500nm; Preferably, the maximum emission wavelength of the fluorescent probe is 620nm-650nm.

3. A method for preparing a fluorescent probe according to claim 1, characterized in that: The preparation method comprises: (1) dissolving the compound of formula I and halogenated p-benzaldehyde in an organic solvent and reacting them under alkaline conditions to obtain a compound of formula II; (2) dissolving the compound of formula II with hydroxylamine hydrochloride and an alkaline regulator in an organic solvent for reaction to obtain the fluorescent probe NBD-NHOH; Wherein, Formula I: ; Formula II: .

4. The preparation method according to claim 3, characterized in that The halogenated p-benzaldehyde in step (1) includes any one of p-fluorobenzaldehyde, p-chlorobenzaldehyde or p-bromobenzaldehyde, preferably p-fluorobenzaldehyde; Preferably, the molar ratio of the compound of formula I to the halogenated p-benzaldehyde in step (1) is 1:(1-1.5), preferably 1:1.2; Preferably, the organic solvent in step (1) comprises any one of acetonitrile, ethanol or methanol, or a combination of at least two thereof; Preferably, the solubility of the compound of formula I in step (1) in an organic solvent is 40 mg / mL-60 mg / mL; Preferably, the alkaline environment in step (1) is a reaction environment with a pH of 9-11; Preferably, the alkaline environment in step (1) is adjusted by adding an alkaline regulator; Preferably, the alkaline regulator comprises any one or a combination of at least two of triethylamine, triethylenediamine, piperidine, tetramethylethylenediamine, sodium dihydrogen phosphate or sodium bicarbonate; Preferably, the reaction temperature of the reaction in step (1) is 60°C-110°C; Preferably, the reaction time of the reaction in step (1) is 12h-48h.

5. The preparation method according to claim 3, characterized in that The molar ratio of the compound of formula II, hydroxylamine hydrochloride and alkaline regulator in step (2) is 1:(1-2):(1-5); Preferably, the organic solvent in step (2) comprises any one of acetonitrile, ethanol or methanol, or a combination of at least two thereof; Preferably, the solubility of the compound of formula II in step (2) in the organic solvent is 10 mg / mL-30 mg / mL; Preferably, the alkaline regulator in step (2) comprises any one or a combination of at least two of triethylamine, triethylenediamine, piperidine, tetramethylethylenediamine, sodium dihydrogen phosphate or sodium bicarbonate; Preferably, the reaction temperature of the reaction in step (2) is 60°C-100°C; Preferably, the reaction time of the reaction in step (2) is 2 h to 10 h. Preferably, in step (2), the reaction product is purified by silica gel column chromatography.

6. A method for detecting a carboxyl group using a fluorescent probe as claimed in claim 1 or 2, characterized in that: The method comprises: The substrate to be detected is immersed in a solution containing a fluorescent probe; Cleaning the substrate to be detected after immersion; The fluorescence distribution of the substrate to be detected is detected to obtain the carboxyl distribution of the substrate to be detected.

7. The method according to claim 6, characterized in that The substrate comprises any one of a glass substrate, a plastic substrate or a silicon wafer, or a combination of at least two thereof; Preferably, detecting the fluorescence distribution of the substrate to be detected includes: Exciting the fluorescent probe with the excitation light of the fluorescent probe; Using a detection device to obtain the fluorescence distribution of the substrate to be detected; Preferably, the wavelength range of the excitation light of the fluorescent probe is 480nm-520nm; Preferably, the detection device comprises a fluorescence detection device.

8. The method according to claim 6, characterized in that The method for preparing the solution containing the fluorescent probe comprises: The fluorescent probe is dissolved in a solvent to prepare a mother solution, the mother solution is dissolved in a buffer solution and then ultrasonicated to obtain a solution containing the fluorescent probe.

9. The method according to claim 8, characterized in that The solvent includes any one or a combination of at least two of dimethyl sulfoxide, DMF, methanol or ethanol; Preferably, the concentration of the fluorescent probe in the mother solution is 50 μmol / L-100 μmol / L; Preferably, the buffer comprises PBS buffer or KPi buffer; Preferably, the pH of the buffer is 5-9; Preferably, the ultrasonic time is 10 min-20 min; Preferably, the concentration of the fluorescent probe in the solution containing the fluorescent probe is 3 nmol / L-10 nmol / L.

10. The method according to claim 6, characterized in that The soaking time is 10h-20h; Preferably, the soaking temperature is 25°C-50°C; Preferably, the washing is performed using a buffer solution, and the buffer solution includes any one of PBS solution, KPi buffer solution or SSC buffer solution, or a combination of at least two thereof; Preferably, the pH of the PBS buffer is 6-8.

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