A fluorescent molecular probe for identifying collagen and a preparation method thereof

Abstract

The present invention relates to a fluorescent molecular probe for identifying collagen and a preparation method thereof in the technical field of fluorescent probe technology. The fluorescent probe prepared by the present invention belongs to the field of small molecule fluorescent probes. This molecular probe exhibits different fluorescence responses in different collagen solutions. By reasonably designing a detection model, extracting the characteristic values of the fluorescence emission spectrum, and constructing a PCA analysis method, the identification of type I collagen and type II collagen is achieved. The basis for the present invention to distinguish collagen is that different collagens have different interaction forces with the probe, and the cumulative difference of these forces can cause the characteristic signal difference of the fluorescence emission spectrum of the prepared probe. By analyzing the spectral characteristic signals obtained by the principal element analysis method, the two types of collagens can be distinguished. The detection method for characterizing the target analyte by analyzing the change of the fluorescence signal before and after the action has the advantages of simple operation, low cost, and real-time performance, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of small molecule fluorescent probes, and specifically, to a fluorescent molecular probe for identifying collagen and a preparation method thereof. Background Art

[0002] Collagen is a protein commonly present in animals. In vertebrates alone, 28 different types of collagen have been discovered. Collagen is the main component of various connective tissues, maintaining the integrity of tissues and organs, and is closely related to functions such as human early development, organ formation, cell - cell connection, cell chemotaxis, platelet aggregation, and membrane permeability. Excessive or insufficient production of collagen, as well as defects in collagen structure, can lead to diseases. Therefore, establishing an effective detection method not only helps in the quality control of collagen - containing biological products but also provides basic support for in - depth research on collagen - related diseases.

[0003] Collagen is structurally divided into type I collagen and type II collagen. Type I collagen is a heterotrimeric molecule composed of two α1 chains (blue chains) and one α2 chain (gray chain), and type II collagen is a homotrimeric superhelical structure composed of three identical α1(II) chains; functionally: type I collagen is usually present in positions that resist tensile loads (e.g., tendons, ligaments, skin, vasculature, annulus fibrosus, cornea, sclera, and meniscus), and type II collagen is mainly distributed in cartilage and vitreous body, accounting for more than 90% of the total amount of collagen in adult cartilage matrix.

[0004] Currently, there are mainly four methods for identifying collagen protein types, namely: Fourier transform infrared spectroscopy detection method, ultraviolet spectroscopy analysis method, phosphate - buffered saline detection method, and histochemical staining method. Methods for detecting the content of collagen: ultraviolet spectrophotometry, hydroxyproline colorimetry, high - performance liquid chromatography, Sirius red staining method, and immunological detection method. All methods have disadvantages such as complex sample processing procedures, low precision and sensitivity, and narrow application ranges. The deficiencies of the above - mentioned methods are that currently, the types of collagen identified by fluorescence analysis are limited, and the data pre - processing is relatively cumbersome. Summary of the Invention

[0005] In view of the problems of complex technical sample processing procedures and limited identification types in the prior art, the present invention designs and prepares a preparation method of a fluorescent probe that can be qualitative and quantitative, easy to operate, low - cost, and real - time.

[0006] The object of the present invention is achieved as follows. A preparation method of a fluorescent molecular probe for identifying collagen includes the following steps:

[0007] (1) 4-Hydroxybenzophenone and K2CO3 were added to a flask, and acetonitrile was poured in. The mixture was stirred for 1 h. 1,12-Dibromododecane dissolved in acetonitrile was added to the reaction flask, and the reaction was carried out overnight in an oil bath at 80 °C. Then the reaction solution was added to water to quench the reaction, and it was extracted three times with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the organic solvent was removed by distillation under reduced pressure. The purified product, compound 1, was obtained by column chromatography. The reaction equation is as follows:

[0008]

[0009] (2) Under N2 protection, compound 1 was dissolved in anhydrous tetrahydrofuran and cooled to 0 °C. TiCl4 and Zn powder were slowly added. After the reaction stabilized, the reaction flask was placed in an oil bath at 70 °C and reacted overnight. After the reaction was completed, it was cooled to room temperature. The reaction solution was added to 10% Na2CO3 aqueous solution to quench the reaction, and it was extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the organic solvent was removed by distillation under reduced pressure. The purified product, compound 2, was obtained by column chromatography. The reaction equation is as follows:

[0010]

[0011] (3) Compound 2 and tetramethylethylenediamine were dissolved in chloroform, and the reaction flask was placed in an oil bath at 60 °C and reacted overnight. After the reaction was completed, the reaction solution was cooled to room temperature, and the organic solvent was removed by rotary evaporation under reduced pressure. The solid was washed with anhydrous ether to obtain product compound 3. The reaction equation is as follows:

[0012]

[0013] (4) Compound 3 was dissolved in acetonitrile, and methyl iodide was added. The reaction was stirred at room temperature. After the reaction was completed, recrystallization was carried out, and the product 1,2-bis-(p-(N,N,N,N,N,-pentamethyl-11,13-pentadecanediyl iodonium salt)phenoxy)-1,2-diphenylethylene, namely the fluorescent probe TPE-NN, was obtained by centrifugation. The reaction equation is as follows:

[0014]

[0015] Furthermore, in step (1), the molar ratio of 4-hydroxybenzophenone to 1,12-dibromododecane is 1:4; the reaction temperature is 80 °C; the reaction time is 12 h; the eluent is ethyl acetate:petroleum ether = 1:3 (v / v);

[0016] Furthermore, in step (2), the reaction temperature is 70 - 75 °C; the reaction time is 12 - 14 h; the eluent is dichloromethane:petroleum ether = 4:1 (v / v);

[0017] Further, in step (3), the reaction temperature is 60 - 65 °C and the reaction time is 12 h;

[0018] Further, in step (4), the molar ratio of compound 3 to methyl iodide is 1:20; the reaction temperature is 80 °C; the reaction time is 12 - 16 h.

[0019] Further, the conditions for vacuum distillation are distillation under a negative pressure of 0.1 MPa at a water bath temperature of 40 °C.

[0020] When the fluorescence probe prepared by the present invention is used for detection, the prepared probe is uniformly dispersed in water, and then type I collagen and type II collagen are added. At room temperature (20 - 25 °C), 0.1 mL of the mixed sample is taken, and a steady-state fluorescence spectrum scan is performed using a microplate reader, where the excitation wavelength is 350 nm and the detection range is 400 - 600 nm. The obtained spectrum is subjected to PCA analysis to compare and obtain the characteristic signals of the two collagens. Thus, the distinction between collagens can be achieved. The basis for distinguishing collagens in the present invention is that different collagens have different interaction forces with the probe, and the cumulative difference in these forces can cause differences in the characteristic signals of the fluorescence emission spectrum of the prepared probe. By analyzing the spectral characteristic signals obtained by the principal element analysis method, the two collagens can be distinguished. Fluorescence analysis is a detection method based on the interaction between a fluorescence probe molecule and a target substance, and by analyzing the change in the fluorescence signal before and after the interaction to characterize the target analyte. This method has the characteristics of simple operation, low cost, and real-time performance. Description of the Drawings

[0021] Figure 1 is the 1 1H NMR spectrum of probe TPE-NN;

[0022] Figure 2 is the 13 13C NMR spectrum of probe TPE-NN;

[0023] Figure 3 is the fluorescence emission spectrum of probe TPE-NN in type I collagen solutions with different concentrations;

[0024] Figure 4 is the fluorescence emission spectrum of probe TPE-NN in type II collagen solutions with different concentrations;

[0025] Figure 5 is the linear fitting graph of the fluorescence change of probe TPE-NN upon interaction with type I collagen at different concentrations;

[0026] Figure 6 is the linear fitting graph of the fluorescence change of probe TPE-NN upon interaction with type II collagen at different concentrations;

[0027] Figure 7 It is the scree plot of the PCA model constructed by the probe TPE-NN in response to two types of collagen;

[0028] Figure 8 It is the PCA score plot of the probe TPE-NN in response to two types of collagen. Detailed implementation manner

[0029] The present invention will be further analyzed, explained, and compared through specific examples and comparative examples below.

[0030] Example 1

[0031] Synthesis of the fluorescent probe TPE-NN is carried out according to the following steps:

[0032] (1) Dissolve 4-hydroxybenzophenone (0.99 g, 5 mmol) and K2CO3 (1.73 g, 12.5 mmol) in acetonitrile (20 mL) and stir for 1 h. Dropwise add a solution of 1,12-dibromododecane (6.56 g, 20 mmol) in acetonitrile (15 mL) into the above mixture slowly drop by drop. Stir the mixture overnight and cool the reaction solution to room temperature. Then add the reaction solution to water to quench the reaction, and extract it with dichloromethane three times. The organic phase is dried with anhydrous magnesium sulfate, filtered, and the organic solvent in the system is rotary evaporated under reduced pressure. The purified product compound 1 is obtained through column chromatography [silica gel, ethyl acetate / petroleum ether; 1:3 (v / v)]. The reaction formula is as follows:

[0033]

[0034] (2) Under N2 protection, dissolve compound 1 (0.89 g, 2 mmol) in anhydrous tetrahydrofuran (10 ml), cool it to 0 °C, add TiCl4 (0.38 g, 2 mmol) and Zn (0.26 g, 3.98 mmol), and reflux overnight. Cool it to room temperature. Add the reaction solution to 10% Na2CO3 aqueous solution to quench the reaction, extract it with dichloromethane, dry the organic phase with anhydrous magnesium sulfate, filter, and rotary evaporate the organic solvent in the system under reduced pressure. The purified product compound 2 is obtained through column chromatography [silica gel, dichloromethane / petroleum ether; 4:1 (v / v)]. The reaction formula is as follows:

[0035]

[0036] (3) Dissolve compound 2 (0.3 g, 0.35 mmol) and tetramethylethylenediamine (1 ml) in chloroform (5 ml), reflux overnight, cool the reaction solution to room temperature, rotary evaporate the organic solvent under reduced pressure, and wash the solid with anhydrous ether to obtain product compound 3.

[0037] The reaction formula is as follows:

[0038]

[0039] (3) Dissolve compound 3 (1.86 g, 2 mmol) in 20 mL of acetonitrile, and add methyl iodide (5.6 g, 39.5 mmol). Stir the reaction at room temperature for 12 hours. Recrystallize the reaction solution with ether and centrifuge to obtain a pale yellow solid TPE-NN with a yield of 85%.

[0040] The reaction formula is as follows:

[0041]

[0042] 1H NMR spectrum of TPE-NN 1 is as follows Figure 1 ; 13C NMR spectrum of TPE-NN 13 is as follows Figure 2 .

[0043] Example 2

[0044] Emission spectra of the fluorescent probe in different collagen solutions

[0045] (1) Prepare a test stock solution of the fluorescent probe TPE-NN obtained in Example 1 with a concentration of 10 -3 mol / L in dimethyl sulfoxide (DMSO) for use.

[0046] (2) Prepare a protein stock solution with a concentration of 0.2 mg / mL: Dissolve type I collagen and type II collagen separately in 0.1 M acetic acid solution to 2 mg / mL, and then dilute the solution with deionized water (v / v = 1:9) to obtain a protein stock solution with a final concentration of 0.2 mg / mL.

[0047] (3) Take ten EP tubes and add 5 μL, 25 μL, 50 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 750 μL, 1 mL of 0.2 mg / mL type I collagen stock solution respectively; add 10 μL of the fluorescent probe stock solution in S1 to each of the ten EP tubes; then add 995 μL, 975 μL, 950 μL, 900 μL, 800 μL, 700 μL, 600 μL, 500 μL, 250 μL of deionized water to the ten EP tubes as solvents to obtain test solutions with protein mass concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and the final concentration of the fluorescent probe is 1 μM. The preparation of the type II collagen test solution is the same as that of the type I collagen.

[0048] (4) Measure the fluorescence emission spectra of the test solutions obtained in the above steps respectively. Set the excitation wavelength to 350 nm and the detection range to 400 - 600 nm. Fluorescence emission spectra of test solutions with different protein concentrations are obtained respectively.

[0049] Example 3

[0050] Evaluating the ability of a fluorescent probe to identify collagen based on principal component analysis

[0051] (1) According to Example 2 above, measure the fluorescence emission spectra of the fluorescent probe in different collagen solutions;

[0052] (2) Record the maximum fluorescence emission intensity of each sample, and perform principal component analysis on the maximum fluorescence emission intensities of the probe for different concentrations of collagen (see Table 1).

[0053] Table 1 Variance interpretation table

[0054]

[0055]

[0056] In the table, only the first principal component of the model is greater than 1, and the variance contribution rate reaches 85%, indicating that the first principal component contains all the information of the ten indicators (ten different protein concentrations) and effectively extracts the information. In addition, from the scree plot (such as Figure 7 ) it can be seen that the second eigenvalue of the model is close to zero, that is, the change trend has become gentle.

[0057] (3) Through principal component analysis (PCA), the two types of collagen can be completely distinguished after 10 repeated experiments respectively. (Such as Figure 8 ) The above results show that the probe TPE-NN has good recognition ability for type I collagen and type II collagen.

[0058] Figure 1 is the 1 1H NMR spectrum of the probe TPE-NN; 1 1H NMR (400 MHz, DMSO-d6) δ 7.52–7.39 (m, 8H), 7.12–6.96 (m, 4H), 6.77–6.62 (m, 4H), 4.06 (t, J = 4.6 Hz, 4H), 3.78–3.61 (m, 4H), 3.58–3.45 (m, 4H), 2.93 (d, J = 27.7 Hz, 30H), 1.74 (tt, J = 7.9, 4.5 Hz, 4H), 1.55–1.36 (m, 8H), 1.34–1.21 (m, 31H).

[0059] Figure 2 of the probe TPE-NN 13 13C NMR spectrum; 13 13C NMR (101 MHz, DMSO-d6) δ 158.6, 131.6, 129.52, 128.54, 128.39, 114.27, 64.74, 57.26, 55.55, 53.85, 53.44, 51.10, 29.43, 29.29, 29.19, 28.98, 26.14, 26.00, 22.22. The fluorescence spectra of TPE-NN in type I collagen solutions with different concentrations were tested as a function of viscosity. The results showed that as the protein concentration increased continuously, the fluorescence emission intensity of TPE-NN at 488 nm also increased continuously,

[0060] As Figure 3 shown, the relationship between the fluorescence intensity of TPE-NN and the concentration of type I collagen was further studied. The logarithm of the fluorescence intensity at 488 nm and the logarithm of the protein concentration were linearly fitted,

[0061] As Figure 5 shown, the results indicated that there was a good linear relationship between the value of the fluorescence intensity of TPE-NN and the value of the concentration of type I collagen (R 2 = 0.9892). The fluorescence spectra of TPE-NN in type II collagen solutions with different concentrations were tested as a function of viscosity. The results showed that as the protein concentration increased continuously, the fluorescence emission intensity of TPE-NN at 488 nm also increased continuously, as Figure 4 shown. The relationship between the fluorescence intensity of TPE-NN and the concentration of type II collagen was further studied. The logarithm of the fluorescence intensity at 488 nm and the logarithm of the protein concentration were linearly fitted, as Figure 6 shown, the results indicated that there was a good linear relationship between the value of the fluorescence intensity of TPE-NN and the value of the concentration of type II collagen (R 2 = 0.9904). The maximum fluorescence emission intensity of each sample was recorded, and the maximum fluorescence emission intensities of the probe for different concentrations of collagen were subjected to principal component analysis (see Table 1). In the table, only the first principal component of the model was greater than 1, and the variance contribution rate reached 85%, indicating that the first principal component contained all the information of the ten indicators (ten different protein concentrations) and effectively extracted the information. In addition, from the scree plot (such as Figure 7 ) it can be seen that the second eigenvalue of the model was close to zero, that is, the change trend had become gentle.

[0062] Figure 8It is the PCA score plot of the probe TPE-NN in response to two types of collagen. The black dots represent the test samples of type I collagen, and the red dots represent the test samples of type II collagen. As shown in the figure, the two groups have significant separation. This shows that the probe TPE-NN has good recognition ability for type I collagen and type II collagen.

[0063] The present invention is not limited to the above embodiments. Based on the technical solutions disclosed in the present invention, those skilled in the art can make some substitutions and deformations to some of the technical features without creative labor according to the disclosed technical content, and these substitutions and deformations are all within the protection scope of the present invention.

Claims

1. A fluorescent molecular probe for identifying collagen, characterized in that, It has the following structural formula: 。 2. The preparation method of a fluorescent molecular probe for identifying collagen according to claim 1, wherein, It includes the following steps: (1) Add 4-hydroxybenzophenone and K2CO3 into a flask, pour in acetonitrile, and stir for 1 h; add 1,12-dibromododecane dissolved in acetonitrile into the reaction flask, place it in an oil bath at 80 °C and react overnight; then add the reaction solution into water to quench the reaction, and extract it three times with dichloromethane. The organic phase is dried with anhydrous magnesium sulfate, filtered, and the organic solvent is removed by distillation under reduced pressure. The purified product compound 1 is obtained by column chromatography separation. The reaction formula is as follows: ； (2) Under the protection of N2, dissolve compound 1 in anhydrous tetrahydrofuran, cool it to 0 °C, slowly add TiCl4 and Zn powder. After the reaction is stable, place the reaction flask in an oil bath at 70 °C and react overnight. After the reaction is completed, cool it to room temperature; add the reaction solution into 10% Na2CO3 aqueous solution to quench the reaction, extract it with dichloromethane. The organic phase is dried with anhydrous magnesium sulfate, filtered, and the organic solvent is removed by distillation under reduced pressure. The purified product compound 2 is obtained by column chromatography separation. The reaction formula is as follows: ； (3) Dissolve compound 2 and tetramethylethylenediamine in chloroform, place the reaction flask in an oil bath at 60 °C and react overnight. After the reaction is completed, cool the reaction solution to room temperature, spin-dry the organic solvent under reduced pressure, and wash the solid with anhydrous ether to obtain product compound 3. The reaction formula is as follows: ； (4) Dissolve compound 3 in acetonitrile, add methyl iodide; stir and react at room temperature. After the reaction is completed, perform recrystallization and centrifuge to obtain the product 1,2-bis-(p-(N,N,N,N,N-pentamethyl-11,13-pentadecanediyl iodonium salt)phenoxy)-1,2-distyrene, namely the fluorescent probe TPE-NN. The reaction formula is as follows: 。 3. The preparation method of a fluorescent molecular probe for identifying collagen according to claim 2, wherein: In step (1), the molar ratio of 4-hydroxybenzophenone to 1,12-dibromododecane is 1:4, the reaction temperature is 80 °C, the reaction time is 12 h, and the eluent is a mixed solution of ethyl acetate and petroleum ether, where the volume ratio of ethyl acetate to petroleum ether is 1:

3.

4. The preparation method of a fluorescent molecular probe for identifying collagen according to claim 2, characterized in that: In step (2), the reaction temperature is 70 - 75 °C, the reaction time is 12 - 14 h, and the eluent is a mixed solution of dichloromethane and petroleum ether, where the volume ratio of dichloromethane to petroleum ether is 4:

1.

5. The preparation method of a fluorescent molecular probe for identifying collagen according to claim 2, characterized in that: In step (3), the reaction temperature is 60 - 65 °C and the reaction time is 12 h.

6. The preparation method of a fluorescent molecular probe for identifying collagen according to claim 2, characterized in that: In step (4), the molar ratio of compound 3 to methyl iodide is 1:20; the reaction temperature is 80 °C; the reaction time is 12 - 16 h.

7. The preparation method of a fluorescent molecular probe for identifying collagen according to claim 2, characterized in that, The conditions for distillation under reduced pressure are distillation at a water bath temperature of 40 °C and a negative pressure of 0.1 MPa.

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