Method for detecting functional groups on surface of substrate based on off-on fluorescent probe

Abstract

The invention provides a method for detecting functional groups on the surface of a substrate based on an off-on type fluorescent probe. The method comprises the following steps: soaking the substrate to be detected in a solution containing the fluorescent probe; the soaked substrate to be detected is cleaned; detecting the fluorescence distribution of the to-be-detected substrate to obtain the distribution condition of functional groups of the to-be-detected substrate; wherein the fluorescent probe is a'off-on 'type fluorescent probe. According to the invention, an off-on fluorescent probe is adopted to detect amino on a substrate, when the fluorescent probe is covalently connected with the amino on the substrate and is excited, fluorescence is turned on, and when the fluorescent probe which is non-specifically adsorbed with 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 method for detecting substrate surface functional groups based on an "off-on" type fluorescent probe. 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 functional groups. However, these fluorescent reagents are prone to non-specific adsorption with glass and then adhere to the glass surface. They are also difficult to clean. These fluorescent reagents will undergo non-specific adsorption with glass and emit fluorescence when excited even if they do not react with chemical functional groups. This will cause abnormal fluorescence images and high fluorescence intensity values ​​during photography, and cannot accurately reflect the distribution of chemical functional groups on the glass.

[0005] Therefore, it is of great significance to study how to accurately detect specific chemical functional groups on the substrate surface. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention provides a method for detecting substrate surface functional groups based on an "off-on" type fluorescent probe, 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 method for detecting functional groups on a substrate surface, 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; detecting the fluorescence distribution of the substrate to be detected to obtain the distribution of functional groups of the substrate to be detected; Wherein, the fluorescent probe is an "off-on" type fluorescent probe.

[0008] As a preferred embodiment of the present invention, the functional group is an amino group.

[0009] As a preferred embodiment of the present invention, the structural formula of the fluorescent probe is as follows: .

[0010] As a preferred embodiment of the present invention, the substrate includes any one of a glass substrate, a plastic substrate or a silicon wafer.

[0011] 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.

[0012] As a preferred embodiment of the present invention, the wavelength range of the excitation light of the fluorescent probe is 470nm-490nm.

[0013] As a preferred solution of the present invention, the detection device includes a fluorescence detection device.

[0014] As a preferred embodiment of the present invention, the method further comprises: preparing a solution containing a fluorescent probe, wherein the method for preparing the solution containing a 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.

[0015] 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.

[0016] 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.

[0017] As a preferred embodiment of the present invention, the buffer comprises PBS buffer or KPi buffer.

[0018] As a preferred embodiment of the present invention, the pH of the buffer solution is 5-9.

[0019] As a preferred embodiment of the present invention, the ultrasonication time is 10 min-20 min.

[0020] 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.

[0021] As a preferred embodiment of the present invention, the soaking time is 10 hours to 20 hours.

[0022] As a preferred embodiment of the present invention, the soaking temperature is 25°C-50°C.

[0023] 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.

[0024] As a preferred embodiment of the present invention, the pH of the buffer solution is 6-8.

[0025] As a preferred embodiment of the present invention, the method further comprises: preparing the fluorescent probe, and the method for preparing the fluorescent probe comprises: (1) dissolving the compound of formula I in an organic solvent and reacting with BF3.Et2O to obtain a compound of formula II; (2) dissolving the compound of formula II and p-nitrobenzaldehyde in an organic solvent and carrying out a condensation reaction under alkaline conditions to obtain a compound of formula III; (3) subjecting the compound of formula III to nitro reduction reaction to obtain a fluorescent probe; Wherein, the compound of formula I is ; The compound of formula II is ; The compound of formula III is .

[0026] As a preferred embodiment of the present invention, the molar ratio of the compound of formula I to BF3.Et2O in step (1) is 1:(0.8-2).

[0027] As a preferred embodiment of the present invention, the organic solvent in step (1) includes any one of dichloromethane, ethanol or acetonitrile, or a combination of at least two thereof.

[0028] 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-60 mg / mL.

[0029] As a preferred embodiment of the present invention, the reaction temperature of the reaction in step (1) is 25°C-50°C.

[0030] As a preferred embodiment of the present invention, the reaction time of the reaction in step (1) is 15 min-60 min.

[0031] As a preferred embodiment of the present invention, the molar ratio of the compound of formula II to p-nitrobenzaldehyde in step (2) is 1:(1-2).

[0032] As a preferred embodiment of the present invention, the organic solvent in step (2) includes any one of acetonitrile, ethanol, methanol, DMF or DMSO, or a combination of at least two thereof.

[0033] As a preferred embodiment of the present invention, the solubility of the compound of formula II in step (2) in an organic solvent is 10 mg / mL-30 mg / mL.

[0034] As a preferred embodiment of the present invention, the pH range of the alkaline condition in step (2) is 9-12.

[0035] As a preferred embodiment of the present invention, the reaction temperature of the reaction in step (2) is 60°C-100°C.

[0036] As a preferred embodiment of the present invention, the reaction time of the reaction in step (2) is 24h-48h.

[0037] As a preferred embodiment of the present invention, the nitro reduction reaction in step (3) is: mixing the compound of formula III with stannous chloride and concentrated hydrochloric acid to react to obtain a fluorescent probe.

[0038] As a preferred embodiment of the present invention, the molar ratio of the compound of formula III to stannous chloride and concentrated hydrochloric acid is 1:(1-10):(0.1-0.5).

[0039] As a preferred embodiment of the present invention, the concentration of the concentrated hydrochloric acid is 6 mol / L-10 mol / L.

[0040] As a preferred embodiment of the present invention, the reaction temperature is 60°C-100°C.

[0041] As a preferred embodiment of the present invention, the reaction time is 6h-24h.

[0042] As a preferred embodiment of the present invention, the reaction product is purified by silica gel column chromatography.

[0043] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses an "off-on" fluorescent probe to detect functional groups on the substrate. Fluorescence is activated only when the fluorescent probe is covalently bonded to the functional groups on the substrate and excited. Fluorescent probes nonspecifically adsorbed 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 fluorescent probes are introduced, they will not be excited to fluoresce, thus maintaining the accuracy of the detection of the amino group distribution on the glass.

[0044] The detection method 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 amino groups, is highly sensitive, is easy to store, and has good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the mechanism of action of the detection method of the present invention; Figure 2 This is a fluorescence detection image of amino-free glass A-1 in Example 2 of the present invention using the fluorescent labeling reagent Cy3-NHS in Comparative Example 1; Figure 3 This is a fluorescence detection image of the amino-free glass A-2 in Example 2 of the present invention using the fluorescent labeling reagent in Example 1; Figure 4 This is a fluorescence detection image of amino glass B-1 using fluorescent labeling reagent Cy3-NHS in Example 5 of the present invention; Figure 5 This is a fluorescence detection image of amino glass B-2 using the fluorescent labeling reagent in Example 1 in Example 5 of the present invention. DETAILED DESCRIPTION

[0046] 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.

[0047] In a first aspect, a method for detecting functional groups on a substrate surface is provided, 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; detecting the fluorescence distribution of the substrate to be detected to obtain the distribution of functional groups of the substrate to be detected; Wherein, the fluorescent probe is an "off-on" type fluorescent probe.

[0048] The "off-on" fluorescent probe is a fluorescent probe that emits fluorescence when excited when covalently bound to a functional group, but does not emit fluorescence when not covalently bound to the functional group. Specifically, when the "off-on" fluorescent probe is excited with excitation light from the fluorescent probe, the fluorescent probe emits fluorescence when covalently bound to a functional group, but does not emit fluorescence when not covalently bound to the functional group.

[0049] As a preferred embodiment of the present invention, the functional group is an amino group.

[0050] As a preferred embodiment of the present invention, the structural formula of the fluorescent probe is as follows: .

[0051] The detection method of the present invention adopts 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 functional 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 functional groups on the glass, after the cleaning reagent is introduced, even if the fluorescent probe that has not reacted with the functional group is not completely cleaned off, it will not be excited to fluoresce, and will not affect the accuracy of the detection of the functional group distribution on the glass. The reaction of the above-mentioned fluorescent probe with the functional group refers to the covalent connection of the fluorescent probe with the functional group. The distribution of the above-mentioned functional groups includes but is not limited to: the presence or absence of functional groups, the density, quantity, and distribution uniformity of the functional groups, etc.

[0052] When the fluorescent probe of the present invention interacts with the functional group (amino group), the boron difluoride group interacts with the amino group to cause fluorescence to turn on. The specific mechanism of action is as follows Figure 1 shown.

[0053] As a preferred embodiment of the present invention, the substrate includes any one of a glass substrate, a plastic substrate or a silicon wafer.

[0054] 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.

[0055] As a preferred embodiment of the present invention, the wavelength range of the excitation light of the fluorescent probe is 470nm-490nm, such as 470nm, 475nm, 480nm, 485nm or 490nm, but is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable.

[0056] As a preferred solution of the present invention, the detection device includes a fluorescence detection device.

[0057] As a preferred embodiment of the present invention, the method further comprises: preparing a solution containing a fluorescent probe, wherein the method for preparing the solution containing a 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.

[0058] 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.

[0059] 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.

[0060] As a preferred embodiment of the present invention, the buffer comprises PBS buffer or KPi buffer.

[0061] As a preferred embodiment of the present invention, the pH of the buffer solution 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 numerical range are also applicable.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] As a preferred embodiment of the present invention, the pH of the buffer solution is 6-8, such as 6, 6.5, 7, 7.5 or 8, but is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable.

[0068] As a preferred embodiment of the present invention, the method further comprises: preparing the fluorescent probe, and the method for preparing the fluorescent probe comprises: The compound of formula I is dissolved in an organic solvent and reacted with BF3.Et2O to obtain a compound of formula II;

[0069] Dissolving the compound of formula II and p-nitrobenzaldehyde in an organic solvent and carrying out a condensation reaction under alkaline conditions to obtain a compound of formula III;

[0070] The compound of formula III is subjected to a nitro reduction reaction to obtain a fluorescent probe;

[0071] Wherein, the compound of formula I is ; The compound of formula II is ; The compound of formula III is .

[0072] 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).

[0073] As a preferred embodiment of the present invention, the molar ratio of the compound of formula I to BF3.Et2O in step (1) is 1:(0.8-2), such as 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8 or 1:2, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0074] As a preferred embodiment of the present invention, the organic solvent in step (1) includes any one of dichloromethane, ethanol or acetonitrile, or a combination of at least two thereof.

[0075] 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.

[0076] As a preferred embodiment of the present invention, the reaction temperature of the reaction in step (1) is 25°C-50°C, for example, 25°C, 30°C, 35°C, 40°C, 45°C or 50°C, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0077] As a preferred embodiment of the present invention, the reaction time of the reaction in step (1) is 15 min-60 min, for example, 15 min, 20 min, 30 min, 40 min, 50 min or 60 min, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0078] As a preferred embodiment of the present invention, the molar ratio of the compound of formula II to p-nitrobenzaldehyde in step (2) is 1:(1-2), such as 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8 or 1:2, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0079] As a preferred embodiment of the present invention, the organic solvent in step (2) includes any one of acetonitrile, ethanol, methanol, DMF or DMSO, or a combination of at least two thereof.

[0080] 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.

[0081] As a preferred embodiment of the present invention, the pH range of the alkaline condition in step (2) is 9-12, such as 9, 10, 11 or 12, but is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable.

[0082] 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, 70°C, 80°C, 90°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.

[0083] As a preferred embodiment of the present invention, the reaction time of the reaction in step (2) is 24 h-48 h, for example, 24 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 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.

[0084] As a preferred embodiment of the present invention, the nitro reduction reaction in step (3) is: mixing the compound of formula III with stannous chloride and concentrated hydrochloric acid to react to obtain a fluorescent probe.

[0085] As a preferred embodiment of the present invention, the molar ratio of the compound of formula III to stannous chloride and concentrated hydrochloric acid is 1:(1-10):(0.1-0.5), for example, 1:2:(0.1-0.5), 1:4:(0.1-0.5), 1:6:(0.1-0.5), 1:8:(0.1-0.5), 1:(1-10):0.2, 1:(1-10):0.3 or 1:(1-10):0.4, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0086] As a preferred embodiment of the present invention, the concentration of the concentrated hydrochloric acid is 6 mol / L-10 mol / L, for example, 6 mol / L, 7 mol / L, 8 mol / L, 9 mol / L or 10 mol / L, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0087] As a preferred embodiment of the present invention, the reaction temperature of the reaction is 60°C-100°C, for example, 60°C, 70°C, 80°C, 90°C or 100°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0088] As a preferred embodiment of the present invention, the reaction time of the reaction is 6h-24h, for example, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h or 24h, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0089] As a preferred embodiment of the present invention, the reaction product is purified by silica gel column chromatography.

[0090] 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.

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

[0092] The preparation method of the fluorescent probe is as follows: Under a nitrogen atmosphere, the compound of Formula I (1 mmol, 259 mg) was dissolved in 10 mL of DCM. 100 μL of BF₃.Et₂O was added dropwise at room temperature for half an hour. The reaction was then quenched with 0.2 M aqueous NaOH and extracted with DCM. The crude product was separated by column chromatography using DCM / petroleum ether to obtain 266 mg of an orange-red solid, Compound II, with a yield of 76%. Mp 150-152°C. ESI: m / z [M + H]+ = 350.14; calculated: 349.14. 1H NMR (400 MHz, CDCl3-CD3OD, 5:1, v / v): 7.81 (s, 1H), 7.39 (d, J = 7.6 Hz, 1H), 6.31 (d, J = 7.6 Hz, 1H), 6.25 (s, 1H), 5.0 (s, 1H), 3.50 (q, J = 6.8 Hz, 4H), 1.38 (t, J = 6.8 Hz, 6H); 13 C NMR (100 MHz, CDCl3-CD3OD, 5:1, v / v): 162.00, 160.01, 151.72,144.11, 127.54, 117.31, 109.67, 105.48, 81.23, 48.8, 26.31, 13.32.

[0093] Dissolve the compound of Formula II (0.5 mmol, 133 mg) and p-nitrobenzaldehyde (0.75 mmol, 110 mg) in 10 mL of ethanol. Add 10 μL of piperidine solution dropwise and react at 90°C for 24 h. Recrystallize from acetonitrile to obtain 216 mg of an orange-red solid, which is the compound of Formula III, with a yield of 90%. Mp 110-112°C. ESI: m / z [M + H]+ = 483.15; calculated: 482.14. 1H NMR (400 MHz, CDCl3-CD3OD, 5:1, v / v): 8.14 (d, J = 7.6 Hz, 2H ), 7.81 (s, 1H), 7.56 (d, J = 7.6 Hz, 2H), 7.27 (d, J = 7.6 Hz, 1H), 6.76 (d, J = 15.6 Hz, 2H), 6.31 (d, J = 7.6 Hz, 1H), 6.25 (s, 1H), 5.0 (s, 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): 165.47, 161.16, 160.01, 151.72, 147.26, 144.11, 141.36,128.87, 127.54, 127.11, 126.42, 123.59, 117.31, 109.67, 105.48, 81.23, 48.8, 26.31, 13.32.

[0094] Compound III (200 mg, 0.41 mmol) was placed in a 50 mL round-bottom flask and completely dissolved in anhydrous ethanol (20 mL). SnCl₂·2H₂O (500 mg, 2 mmol) and concentrated hydrochloric acid (40 μL) were added and refluxed for 12 h. The reaction mixture was purified by silica gel column chromatography (SiO₂, CH₂Cl₂ / MeOH, v / v, 100 / 3) to obtain 74 mg of an orange-red solid, the fluorescent probe, in a 40% yield. Mp 120-122°C. ESI: m / z [M + H]+ = 453.15; calculated: 452.11. 1H NMR (400 MHz, CDCl3-CD3OD, 5:1, v / v): 8.10 (d, J = 7.6 Hz, 2H ), 7.72 (s, 1H), 7.51 (d, J = 7.6 Hz, 2H), 7.21 (d, J = 7.6 Hz, 1H), 6.53 (d, J = 15.6 Hz, 2H), 6.21 (d, J = 7.6 Hz, 1H), 6.12 (s, 1H), 5.0 (s, 1H), 4.41 (s, 2H), 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):171.47, 163.16, 161.01, 151.72, 150.26, 147.11, 134.36, 131.87, 130.54,127.11, 125.42, 123.59, 115.31, 109.67, 105.48, 85.23, 48.85, 26.31, 13.32.

[0095] Comparative Example 1: This comparative example provides a fluorescent labeling reagent Cy3-NHS commonly used in the prior art.

[0096] Example 2: This example uses four pieces of glass, two of which are ordinary glasses without amino groups, labeled A-1 and A2, and two of which are glasses with amino 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. The glass was immersed in two different fluorescent solutions at 50°C for 10-20 hours. After the reaction, it 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.

[0097] The density and uniformity of amino groups detected on the four pieces of glass were measured, as shown in Table 1.

[0098] The four pieces of glass were excited with a fluorescence excitation wavelength of 490nm. The results are as follows: Figure 2-5 As shown. Among them, Figure 2 Schematic diagram of fluorescence detection after amino-free glass A-1 is immersed in Cy3-NHS fluorescent solution. Figure 3 This is a schematic diagram of fluorescence detection after amino-free glass A-2 is immersed in the fluorescent solution of the fluorescent reagent in Example 1. Figure 4 Schematic diagram of fluorescence detection after amino glass B-1 is immersed in Cy3-NHS fluorescent solution. Figure 5 Schematic diagram of fluorescence detection after amino glass B-2 is immersed in the fluorescent solution of the fluorescent reagent in Example 1. Figure 2 and Figure 3 It can be seen that even for glass without amino groups, there are obvious bright spots in the Cy3-NHS detection results, indicating that some fluorescent reagents remain on the chip surface, indicating that such fluorescent reagents are easily non-specifically adsorbed with the glass and then attached to the glass surface, and fluorescent reagents that undergo non-specific adsorption with the glass will also emit fluorescence when excited, thereby causing detection errors. However, when using the fluorescent probe prepared by the present invention for detection, even if some fluorescent reagents remain, they will not emit fluorescence when excited because they do not react with the amino groups, and will not cause detection errors. Figure 4 and Figure 5 It can be seen that amino glasses B-1 and B-2 emit fluorescence when excited ( Figure 4-Figure 5 The image is presented in grayscale because the density of amino groups on the glass is very high, resulting in too dense fluorescent spots. Therefore, the image is presented in grayscale rather than bright spots. The image without fluorescence is presented in black. Figure 2-Figure 3As shown in the figure, however, glass B-1 immersed in the Cy3-NHS 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 described in the present invention remains, it does not react with the amino group and will not emit fluorescence when excited. Therefore, there are no fluorescent bright spots in the detected image.

[0099] Conventional fluorescent reagent Cy3-NHS does not react with amino 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 amino groups are present, which can easily cause detection errors. In other words, even glass without amino groups may be mistakenly judged to have amino groups. However, the fluorescent probe used in the present invention does not react with amino groups for glass without amino groups and will not emit fluorescence when excited, thus preventing detection errors.

[0100] While conventional fluorescent reagent Cy3-NHS can be excited to fluoresce on glass surfaces containing amino groups, its weak interaction with the glass causes residual fluorescence to also be excited, resulting in localized fluorescence intensity that is biased, leading to inaccurate detection of amino group density and uniformity. The fluorescent probe 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 amino groups. This avoids the drawback of high localized fluorescence and provides higher accuracy in detecting amino group density and uniformity.

[0101] Example 3: This example uses four pieces of glass, two of which are ordinary glass without amino groups, and two of which are glass with amino functional groups. 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 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.

[0102] The density and uniformity of amino groups detected on the four pieces of glass were measured, as shown in Table 1.

[0103] Example 4: This example uses four pieces of glass, two of which are ordinary glass without amino groups, and two of which are glass with amino functional groups. 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 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.

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

[0105] The test results of Examples 2-4 are shown in Table 1.

[0106] Table 1: Test results of Examples 2-4

[0107] As can be seen from Table 1, the fluorescence intensity values ​​of Cy3-NHS 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 amino groups. They are adsorbed on the glass and aggregate to produce bright spots. The fluorescent probes in the embodiment will only fluoresce when they are covalently linked to amino 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-NHS, the fluorescent probe of the embodiment has no fluorescent spots and better uniformity.

[0108] 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 method for detecting functional groups on a substrate surface, 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; detecting the fluorescence distribution of the substrate to be detected to obtain the distribution of functional groups of the substrate to be detected; Wherein, the fluorescent probe is an "off-on" type fluorescent probe.

2. The detection method according to claim 1, wherein The functional group is an amino group; Preferably, the structural formula of the fluorescent probe is as follows: 。 Preferably, the substrate comprises 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 470nm-490nm; Preferably, the detection device comprises a fluorescence detection device.

3. The detection method according to claim 1, wherein The method further comprises: preparing a solution containing a fluorescent probe, wherein the method for preparing the solution containing a 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.

4. The detection method according to claim 3, 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.

5. The detection method according to claim 1, wherein 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.

6. The detection method according to claim 1, characterized in that The method further comprises: preparing the fluorescent probe, and the method for preparing the fluorescent probe comprises:

7. Dissolve the compound of formula I in an organic solvent and react with BF3.Et2O to obtain a compound of formula II; 7. Dissolving the compound of formula II and p-nitrobenzaldehyde in an organic solvent and conducting a condensation reaction under alkaline conditions to obtain a compound of formula III; 7. subjecting the compound of formula III to nitro reduction reaction to obtain a fluorescent probe; in, The compound of formula I is ; The compound of formula II is ; The compound of formula III is .

7. The detection method according to claim 6, characterized in that the molar ratio of the compound of formula I to BF3.Et2O in step (1) is 1:(0.8-2); Preferably, the organic solvent in step (1) comprises any one of dichloromethane, ethanol or acetonitrile, or a combination of at least two thereof; Preferably, the solubility of the compound of formula I in step (1) in the organic solvent is 40 mg / mL-60 mg / mL; Preferably, the reaction temperature of the reaction in step (1) is 25°C-50°C; Preferably, the reaction time of the reaction in step (1) is 15 min-60 min.

8. The detection method according to claim 6, characterized in that In step (2), the molar ratio of the compound of formula II to p-nitrobenzaldehyde is 1:(1-2); Preferably, the organic solvent in step (2) comprises any one of acetonitrile, ethanol, methanol, DMF or DMSO, 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 pH range of the alkaline condition in step (2) is 9-12; 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 24h-48h.

9. The detection method according to claim 6, characterized in that The nitro reduction reaction in step (3) is as follows: the compound of formula III is mixed with stannous chloride and concentrated hydrochloric acid to react to obtain a fluorescent probe.

10. The detection method according to claim 9, characterized in that: The molar ratio of the compound of formula III to stannous chloride and concentrated hydrochloric acid is 1:(1-10):(0.1-0.5); Preferably, the concentration of the concentrated hydrochloric acid is 6 mol / L-10 mol / L; Preferably, the reaction temperature is 60°C-100°C; Preferably, the reaction time is 6h-24h; Preferably, the reaction product is purified by silica gel column chromatography.

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