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

Abstract

The invention provides a fluorescent probe for detecting an aldehyde group as well as a preparation method and application of the fluorescent probe. The fluorescent probe is Flu-N2H4. 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 an aldehyde group on the 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 does not emit fluorescence.

Description

Technical Field

[0001] The invention belongs to the field of biosensors and relates to a fluorescent probe for detecting aldehyde 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, aldehyde-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 slides 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 develop 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 aldehyde 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 aldehyde 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 490nm-530nm.

[0009] As a preferred embodiment of the present invention, the maximum emission wavelength of the fluorescent probe is 560-595 nm.

[0010] In a second aspect, the present invention provides a method for preparing the aforementioned fluorescent probe, the preparation method comprising: reacting 3,6-halogenated fluorescein and hydrazine hydrate in an organic solvent to obtain the fluorescent probe.

[0011] As a preferred embodiment of the present invention, the 3,6-halogenated fluorescein includes any one of 3,6-dichlorofluorescein, 3,6-dibromofluorescein or 3,6-difluorofluorescein, preferably 3,6-dichlorofluorescein.

[0012] As a preferred embodiment of the present invention, the molar ratio of the 3,6-halogenated fluorescein to hydrazine hydrate is 1:(1.5-10), preferably 1:5.

[0013] As a preferred embodiment of the present invention, the organic solvent includes any one of methanol, ethanol, DMSO or DMF, or a combination of at least two of them.

[0014] As a preferred embodiment of the present invention, the solubility of the 3,6-halogenated fluorescein in an organic solvent is 30 mg / mL-100 mg / mL.

[0015] As a preferred embodiment of the present invention, the mass ratio of the total mass of the 3,6-halogenated fluorescein and hydrazine hydrate to the organic solvent is 1:(9-15).

[0016] As a preferred embodiment of the present invention, the reaction temperature is 25°C-90°C.

[0017] As a preferred embodiment of the present invention, the reaction time is 3h-24h.

[0018] As a preferred embodiment of the present invention, the reaction product is purified by silica gel column chromatography.

[0019] As a preferred embodiment of the present invention, when the 3,6-halogenated fluorescein is 3,6-dichlorofluorescein, the preparation method of the 3,6-dichlorofluorescein comprises: Fluorescein, dimethylformamide, thionyl chloride and a solvent are mixed and reacted to obtain 3,6-dichlorofluorescein.

[0020] As a preferred embodiment of the present invention, the molar ratio of fluorescein to thionyl chloride is 1:(1.1-1.5), preferably 1:1.3.

[0021] As a preferred embodiment of the present invention, the amount of dimethylformamide used is 1 mL-2 mL.

[0022] As a preferred embodiment of the present invention, the solvent includes any one of sulfolane, ethylene glycol or nitrobenzene, or a combination of at least two thereof.

[0023] As a preferred embodiment of the present invention, the mass ratio of the total mass of the fluorescein, dimethylformamide and thionyl chloride to the solvent is 1:(20-30).

[0024] As a preferred embodiment of the present invention, the reaction temperature is 70°C-100°C.

[0025] As a preferred embodiment of the present invention, the reaction time is 3h-24h.

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

[0027] In a third aspect, the present invention provides a method for detecting aldehyde 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 aldehyde group 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.

[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 500nm-540nm.

[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 (DMSO), 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 ultrasonication time is 10 min-20 min.

[0038] 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.

[0039] As a preferred embodiment of the present invention, the soaking temperature is 25°C-50°C.

[0040] 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.

[0041] As a preferred embodiment of the present invention, the pH of the buffer solution is 6-8.

[0042] 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 aldehyde groups on the glass and excited. Fluorescent probes that nonspecifically adsorb to the glass remain in a "closed loop" state and do not fluoresce. Therefore, even if the cleaning reagent is not completely removed after the passage of fluorescent probes not covalently bound to aldehyde groups, they will not be excited to fluoresce, thus maintaining the accuracy of the detection of aldehyde group distribution on the glass.

[0043] 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 aldehyde groups, is highly sensitive, is easy to store, and has good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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 aldehyde-free glass A-1 in Example 5 of the present invention using the fluorescent labeling reagent Cy3-NH2; Figure 3This is a fluorescence detection image of the aldehyde-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 the aldehyde-based 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 aldehyde-based glass B-2 in Example 5 of the present invention using the fluorescent labeling reagent in Example 1. DETAILED DESCRIPTION

[0045] 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.

[0046] In a first aspect, the specific embodiment of the present invention provides a fluorescent probe for detecting aldehyde groups, and the structural formula of the fluorescent probe is as follows: .

[0047] The present invention uses fluorescein as a parent structure, obtains an intermediate 3,6-halogenated fluorescein through a halogenation reaction, and utilizes the high reactivity of the halogen to undergo a nucleophilic substitution reaction with hydrazine hydrate to obtain a fluorescent probe Flu-N2H4.

[0048] The fluorescent probe for aldehyde group detection 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 turn on when the fluorescent probe is covalently linked to the aldehyde 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 aldehyde groups on the glass, after the cleaning reagent is passed through, even if the fluorescent probe that has not reacted with the aldehyde group is not completely cleaned off, it will not be excited to emit fluorescence, and will not affect the accuracy of the aldehyde group distribution detection on the glass. The reaction of the above-mentioned fluorescent probe with the aldehyde group refers to the covalent linkage of the fluorescent probe with the aldehyde group. The distribution of the above-mentioned aldehyde groups includes but is not limited to: the presence or absence of aldehyde groups, the density, quantity, and distribution uniformity of aldehyde groups, etc.

[0049] The maximum absorption wavelength of the fluorescent probe described in the present invention is around 490nm-530nm, and the maximum emission wavelength is 560-595nm. Its recognition mechanism is intramolecular induced electron transfer (PET). When the fluorescent probe interacts with the aldehyde group, the hydrazine hydrate becomes a cyano group, the electron-donating ability is weakened, the PET mechanism is blocked, and the fluorescence is turned on. The specific mechanism of action is as follows Figure 1 shown.

[0050] As a preferred embodiment of the present invention, the maximum absorption wavelength range of the fluorescent probe is 490nm-530nm, such as 490nm, 500nm, 510nm, 520nm or 530nm, but is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable.

[0051] As a preferred embodiment of the present invention, the maximum emission wavelength of the fluorescent probe is 560nm-595nm, for example, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm or 595nm, but is not limited to the listed values. Other values ​​not listed within this numerical range are also applicable.

[0052] In a second aspect, the specific embodiments of the present invention provide a method for preparing the aforementioned fluorescent probe, which comprises: reacting 3,6-halogenated fluorescein and hydrazine hydrate in an organic solvent to obtain the fluorescent probe.

[0053] Taking 3,6-dichlorofluorescein as an example, the reaction formula is as follows:

[0054] As a preferred embodiment of the present invention, the 3,6-halogenated fluorescein includes any one of 3,6-dichlorofluorescein, 3,6-dibromofluorescein or 3,6-difluorofluorescein, preferably 3,6-dichlorofluorescein.

[0055] As a preferred embodiment of the present invention, the molar ratio of the 3,6-halogenated fluorescein to hydrazine hydrate is 1:(1.5-10), for example, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, etc., but is not limited to the listed values. Other values ​​not listed within this numerical range are also applicable, preferably 1:5.

[0056] As a preferred embodiment of the present invention, the organic solvent includes any one of methanol, ethanol, DMSO or DMF, or a combination of at least two of them.

[0057] As a preferred embodiment of the present invention, the solubility of the 3,6-halogenated fluorescein in the organic solvent is 30 mg / mL-100 mg / mL, for example, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL, but is not limited to the listed values. Other values ​​not listed within this numerical range are also applicable. In the present invention, the amount of the organic solvent used is sufficient to completely dissolve the 3,6-halogenated fluorescein.

[0058] As a preferred embodiment of the present invention, the mass ratio of the total mass of the 3,6-halogenated fluorescein and hydrazine hydrate to the organic solvent is 1:(9-15), for example, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15, etc., but is not limited to the listed values, and other unlisted values ​​within this numerical range are also applicable.

[0059] As a preferred embodiment of the present invention, the reaction temperature of the reaction is 25°C-90°C, for example, 25°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C or 90°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0060] As a preferred embodiment of the present invention, the reaction time of the reaction is 3h-24h, for example, 3h, 5h, 7h, 10h, 13h, 15h, 17h, 20h, 22h or 24h, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0061] As a preferred embodiment of the present invention, the reaction product is purified by silica gel column chromatography.

[0062] As a preferred embodiment of the present invention, when the 3,6-halogenated fluorescein is 3,6-dichlorofluorescein, the preparation method of the 3,6-dichlorofluorescein comprises: Fluorescein, dimethylformamide, thionyl chloride and a solvent are mixed and reacted to obtain 3,6-dichlorofluorescein.

[0063] As a preferred embodiment of the present invention, the molar ratio of fluorescein to thionyl chloride is 1:(1.1-1.5), for example, 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 this numerical range are also applicable, preferably 1:1.3.

[0064] In the present invention, if the addition ratio of thionyl chloride (SOCl2) is small, the reaction time will be long and by-products will be easily generated; if the addition ratio of thionyl chloride is large, the reaction cost will be increased.

[0065] As a preferred embodiment of the present invention, the amount of dimethylformamide used is 1 mL-2 mL, for example, 1 mL, 1.2 mL, 1.4 mL, 1.6 mL, 1.8 mL or 2 mL, but is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable.

[0066] As a preferred embodiment of the present invention, the solvent includes any one of sulfolane, ethylene glycol or nitrobenzene, or a combination of at least two thereof.

[0067] As a preferred embodiment of the present invention, the mass ratio of the total mass of fluorescein, dimethylformamide and thionyl chloride to the solvent is 1:(20-30), for example, 1:20, 1:22, 1:24, 1:26, 1:28 or 1:30, etc., but is not limited to the listed values, and other values ​​not listed within this numerical range are also applicable.

[0068] As a preferred embodiment of the present invention, the reaction temperature of the reaction is 70°C-100°C, such as 70°C, 80°C, 90°C or 100°C, but is not limited to the listed values. Other unlisted values ​​within the numerical range are also applicable.

[0069] As a preferred embodiment of the present invention, the reaction time of the reaction is 3h-24h, for example, 3h, 5h, 7h, 10h, 13h, 15h, 17h, 20h, 22h or 24h, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0070] As a preferred embodiment of the present invention, the reaction product is purified by silica gel column chromatography.

[0071] In a third aspect, the present invention provides a method for detecting aldehyde 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 aldehyde group distribution of the substrate to be detected.

[0072] 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 aldehyde group emits fluorescence, while the fluorescent probe that does not react with the aldehyde group does not emit light.

[0073] As a preferred embodiment of the present invention, the substrate includes any one of a glass substrate, a plastic substrate or a silicon wafer.

[0074] 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.

[0075] As a preferred embodiment of the present invention, the wavelength range of the excitation light of the fluorescent probe is 500nm-540nm, such as 500nm, 510nm, 520nm, 530nm or 540nm, but is not limited to the listed values. Other unlisted values ​​within the numerical range are also applicable.

[0076] As a preferred solution of the present invention, the detection device includes a fluorescence detection device.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] As a preferred embodiment of the present invention, the buffer comprises PBS buffer or KPi buffer.

[0081] As a preferred embodiment of the present invention, the pH of the PBS buffer is 5-9, such as 5, 6, 7, 8 or 9, but is not limited to the listed values. Other values ​​not listed within the range are also applicable.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] As a preferred embodiment of the present invention, the pH of the buffer solution is 6-8.

[0088] The following are typical but non-limiting examples of the present invention: The 3,6-dichlorofluorescein Flu-Cl used in the following examples can be prepared using existing methods in the prior art, such as the method described in patent CN 106967304 A, but is not limited to the method described in the patent. Examples are as follows: 50 g (0.15 mol) of fluorescein (C 20 H 12 O5), 1.46g (0.02mol) dimethylformamide (DMF) and 200ml sulfolane (C4H8O2S) were added to a 500ml reactor, stirred and heated to 60°C, 70.8g (0.60mol) thionyl chloride (SOCl2) was added dropwise, and the addition was completed in about 30 minutes. The temperature was continued to rise to 80°C and the reaction was kept warm for 2h. After the reaction was completed, it was cooled to about 30°C and poured into 250ml of a mixture of acetone and water with a volume ratio of 1:1. After stirring and precipitation for 2h, it was filtered. The filter cake was rinsed with 50ml of a mixture of acetone and water with a volume ratio of 1:1 and dried to obtain a light yellow intermediate, which is 3,6-dichlorofluorescein.

[0089] Example 1: This embodiment provides a fluorescent probe for detecting aldehyde groups and a preparation method thereof. The structure of the fluorescent probe is: .

[0090] The preparation method of the fluorescent probe is as follows: 3,6-Dichlorofluorescein Flu-Cl (1 mmol, 368 mg) and hydrazine hydrate (N₂H₄.H₂O) (5 mmol, 250 μL) were dissolved in 10 mL of methanol in a 50 mL round-bottom flask. The mixture was refluxed at room temperature for 6 hours, and the solvent was removed by vacuum distillation. The crude solid was purified by silica gel column chromatography to obtain 110 mg of an orange solid, the fluorescent probe Flu-N₂H₄. The yield was 30%. 1H NMR (400 MHz, CDCl3-CD3OD, 5:1, v / v): 7.96 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.07(d, J = 7.6 Hz, 1H), 6.91(d, J =7.6 Hz, 2H), 6.31(d, J = 7.6 Hz, 2H), 6.25(s, 2H), 3.50 (t, J = 6.4 Hz, 2H),2.03-1.98 (m, 4H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v): 171.01, 168.82,156.51, 144.23, 139.43, 135.46, 137.74, 132.63, 131.80, 130.4, 129.85,129.77, 129.15, 127.53, 126.96, 125.91, 122.22, 101.31, 87.32.

[0091] Example 2: This embodiment provides a fluorescent probe for detecting aldehyde groups and a preparation method thereof. The structure of the fluorescent probe is the same as that of Example 1.

[0092] The preparation method of the fluorescent probe is as follows: Flu-Cl (1 mmol, 368 mg) and N₂H₄.H₂O (1.5 mmol, 75 μL) were dissolved in 10 mL of methanol in a 50 mL round-bottom flask. The mixture was refluxed at 50°C for 6 h, and the solvent was removed by vacuum distillation. The crude solid was purified by silica gel column chromatography to yield 162 mg of an orange solid, the fluorescent probe Flu-N₂H₄. The yield was 45%. Mp 98-100 °C. ESI: m / z [M + H] + = 360.10; calculated value: 360.12. 1 H NMR (400 MHz, CDCl3-CD3OD, 5:1, v / v): 7.96(d, J = 7.6 Hz, 1H), 7.39 (t, J= 7.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.07(d, J = 7.6 Hz, 1H), 6.91(d, J = 7.6 Hz, 2H), 6.31(d, J = 7.6 Hz, 2H), 6.25(s, 2H), 3.50 (t, J = 6.4 Hz, 2H), 2.03-1.98 (m, 4H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v): 171.01, 168.82, 156.51, 144.23, 139.43, 135.46, 137.74,132.63, 131.80, 130.4, 129.85, 129.77, 129.15, 127.53, 126.96, 125.91,122.22, 101.31, 87.32.

[0093] Example 3: This embodiment provides a fluorescent probe for detecting aldehyde groups and a preparation method thereof. The structure of the fluorescent probe is the same as that of Example 1.

[0094] The preparation method of the fluorescent probe is as follows: Flu-Cl (1 mmol, 368 mg) and N₂H₄.H₂O (4 mmol, 200 μL) were dissolved in 10 mL of methanol in a 50 mL round-bottom flask. The mixture was refluxed at 70°C for 12 h, and the solvent was removed by distillation under reduced pressure. The crude solid was purified by silica gel column chromatography to yield 180 mg of an orange solid, the fluorescent probe Flu-N₂H₄. The yield was 50%. Mp 98-100 °C. ESI: m / z [M + H] + = 360.10; calculated value: 360.12. 1 H NMR (400 MHz, CDCl3-CD3OD, 5:1, v / v): 7.96(d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.07(d, J = 7.6 Hz, 1H), 6.91(d,J = 7.6 Hz, 2H), 6.31(d, J = 7.6 Hz, 2H), 6.25(s, 2H), 3.50 (t, J = 6.4 Hz, 2H), 2.03-1.98 (m, 4H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v): 171.01, 168.82, 156.51, 144.23, 139.43, 135.46, 137.74,132.63, 131.80, 130.4, 129.85, 129.77, 129.15, 127.53, 126.96, 125.91,122.22, 101.31, 87.32.

[0095] Example 4: This embodiment provides a fluorescent probe for detecting aldehyde groups and a preparation method thereof. The structure of the fluorescent probe is the same as that of Example 1.

[0096] The preparation method of the fluorescent probe is as follows: Flu-Cl (1 mmol, 368 mg) and N₂H₄.H₂O (5 mmol, 250 μL) were dissolved in 10 mL of methanol in a 50 mL round-bottom flask. The mixture was refluxed at 70°C for 12 h, and the solvent was removed by vacuum distillation. The crude solid was purified by silica gel column chromatography to yield 151.2 mg of an orange solid, the fluorescent probe Flu-N₂H₄. The yield was 41%. Mp 98-100 °C. ESI: m / z [M + H] + = 360.10; calculated value: 360.12. 1 H NMR (400 MHz, CDCl3-CD3OD, 5:1, v / v): 7.96(d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.07(d, J = 7.6 Hz, 1H), 6.91(d, J = 7.6 Hz, 2H), 6.31(d, J = 7.6 Hz, 2H), 6.25(s, 2H), 3.50 (t, J= 6.4 Hz, 2H), 2.03-1.98 (m, 4H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v): 171.01, 168.82, 156.51, 144.23, 139.43, 135.46, 137.74,132.63, 131.80, 130.4, 129.85, 129.77, 129.15, 127.53, 126.96, 125.91,122.22, 101.31, 87.32.

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

[0098] Example 5: In this example, four pieces of glass were selected, two of which were formaldehyde-free glass, labeled A-1 and A-2, and two of which were glass with formaldehyde functional groups, labeled B-1 and B-2. In this example, the fluorescent probes described in Example 1 and Comparative Example 1 were used 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 pH = 8 PBS solution. Ultrasonication was performed for 10-20 minutes to ensure that the fluorescent molecules were 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 a pH = 8 PBS buffer solution. 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 aldehyde groups detected on the four pieces of glass were measured, as shown in Table 1.

[0100] The four pieces of glass were excited with a fluorescence excitation wavelength of 520nm. The results are as follows: Figure 2-5 As shown. Among them, Figure 2 Schematic diagram of fluorescence detection after aldehyde-free glass A-1 is immersed in Cy3-NH2 fluorescent solution. Figure 3 Schematic diagram of fluorescence detection after aldehyde-free glass A-2 is immersed in Flu-N2H4 fluorescent solution. Figure 4 Schematic diagram of fluorescence detection after aldehyde-based glass B-1 is immersed in Cy3-NH2 fluorescent solution. Figure 5 Schematic diagram of fluorescence detection after aldehyde-based glass B-2 is immersed in Flu-N2H4 fluorescent solution. Figure 2 and Figure 3It can be seen that even for glass without aldehyde groups, there are obvious bright spots in the detection results of Cy3-NH2, indicating that some fluorescent reagents remain on the surface of the chip, indicating that such fluorescent reagents are easily non-specifically adsorbed with the glass and then attached to the glass surface. Moreover, fluorescent reagents that undergo non-specific adsorption with the glass will also emit fluorescence when excited, thereby causing detection errors. However, when using Flu-N2H4 detection, even if some fluorescent reagents remain, they will not emit fluorescence when excited because they have not reacted with aldehyde groups, and will not cause detection errors. Figure 4 and Figure 5 It can be seen that the aldehyde-based glasses B-1 and B-2 emit fluorescence when excited ( Figure 4-Figure 5 The image is presented in grayscale because the density of aldehyde groups on the glass is very high, resulting in too dense fluorescent spots. Therefore, the image appears in grayscale instead of bright spots. The image without fluorescence appears in black. 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 of the fluorescent probe Flu-N2H4 described in the present invention remains, it does not react with the aldehyde group and will not emit fluorescence when excited. Therefore, there are no fluorescent bright spots in the detected image.

[0101] Conventional fluorescent reagent Cy3-NH2 will emit fluorescence when excited without reacting with aldehyde groups, and will then produce bright spots due to its aggregation on the glass surface where no aldehyde groups are present, which can easily cause detection errors. That is, glass without no aldehyde groups will be mistakenly judged to have aldehyde groups. However, the fluorescent probe Flu-N2H4 used in the present invention does not react with aldehyde groups for glass without aldehyde groups, and will not emit fluorescence when excited, so it will not cause detection errors.

[0102] While conventional fluorescent reagent Cy3-NH2 can also be excited to fluoresce on glass surfaces with aldehyde groups, the weak interaction between it and the glass causes residual fluorescence to also be excited, resulting in high local fluorescence intensity and a deviation in the accuracy of detecting aldehyde group density and uniformity. The fluorescent probe Flu-N2H4 used in the present invention, due to its off-on property, does not fluoresce when excited even if it remains on the glass, even if it does not react with aldehyde groups. This avoids the drawback of high local fluorescence and provides higher accuracy in detecting aldehyde group density and uniformity.

[0103] Example 6: This embodiment uses four pieces of glass, two of which are formaldehyde-free glass and two of which are glass with formaldehyde 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 and counted using a fluorescence detection device. The test results are shown in Table 1.

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

[0105] Example 7: This embodiment uses four pieces of glass, two of which are formaldehyde-free glass and two of which are glass with formaldehyde 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 TE buffer to prepare a mother solution, which was then dissolved in KPi solution with a pH of 8. Ultrasonic treatment 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.

[0106] The density and uniformity of the aldehyde groups 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, the fluorescence intensity values ​​of Cy3-NH2 as a fluorescent probe are generally high and there are obvious fluorescent bright spots in the image. This is mainly due to the residual reagents of ordinary fluorescent dyes. Ordinary fluorescent dyes will fluoresce when excited regardless of whether they react with aldehyde groups. They are adsorbed on the glass and aggregate to produce bright spots. Flu-N2H4 will only fluoresce when it is covalently linked to aldehyde groups and excited, avoiding the disadvantage that the residual reagents of ordinary dyes adsorbed on the glass will also be excited to fluoresce. 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 aldehyde groups, characterized in that The structural formula of the fluorescent probe is as follows: 。 2. The fluorescent probe according to claim 1, wherein The maximum absorption wavelength range of the fluorescent probe is 490nm-530nm; Preferably, the maximum emission wavelength of the fluorescent probe is 560nm-595nm.

3. The method for preparing a fluorescent probe according to claim 1, wherein: The preparation method comprises: placing 3,6-halogenated fluorescein and hydrazine hydrate in an organic solvent to react to obtain a fluorescent probe; Preferably, the 3,6-halogenated fluorescein includes any one of 3,6-dichlorofluorescein, 3,6-dibromofluorescein or 3,6-difluorofluorescein, preferably 3,6-dichlorofluorescein; Preferably, the molar ratio of the 3,6-halogenated fluorescein to hydrazine hydrate is 1:(1.5-10), preferably 1:5; Preferably, the organic solvent comprises any one of methanol, ethanol, DMSO or DMF, or a combination of at least two thereof; Preferably, the solubility of the 3,6-halogenated fluorescein in the organic solvent is 30 mg / mL-100 mg / mL; Preferably, the mass ratio of the total mass of the 3,6-halogenated fluorescein and hydrazine hydrate to the organic solvent is 1:(9-15); Preferably, the reaction temperature of the reaction is 25°C-90°C; Preferably, the reaction time is 3h-24h; Preferably, the reaction product is purified by silica gel column chromatography.

4. The preparation method according to claim 3, characterized in that When the 3,6-halogenated fluorescein is 3,6-dichlorofluorescein, the preparation method of the 3,6-dichlorofluorescein comprises: Fluorescein, dimethylformamide, thionyl chloride and a solvent are mixed and reacted to obtain 3,6-dichlorofluorescein.

5. The preparation method according to claim 4, characterized in that The molar ratio of fluorescein to thionyl chloride is 1:(1.1-1.5), preferably 1:1.3; Preferably, the amount of dimethylformamide used is 1 mL-2 mL; Preferably, the solvent comprises any one or a combination of at least two of sulfolane, ethylene glycol or nitrobenzene; Preferably, the mass ratio of the total mass of the fluorescein, dimethylformamide and thionyl chloride to the solvent is 1:(20-30); Preferably, the reaction temperature is 70°C-100°C; Preferably, the reaction time is 3h-24h; Preferably, the reaction product is purified by silica gel column chromatography.

6. A method for detecting aldehyde groups using the fluorescent probe according to 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 aldehyde group distribution of the substrate to be detected.

7. The method according to claim 6, characterized in that The substrate includes any one of a glass substrate, a plastic substrate or a silicon wafer; 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 500nm-540nm; 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 buffer solution is 6-8.

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