A method for determining the concentration of sulfated glycosaminoglycans by fluorescence spectroscopy

Abstract

This invention discloses a method for determining the concentration of sulfated glycosaminoglycans using fluorescence spectroscopy. The fluorescent probe carries a certain number of arginine residues. Utilizing the electrostatic interaction between the amino groups on the arginine residues and the sulfate ions on the sugar chains, a series of fluorescent probe molecules can align neatly along the sugar chains to form a stable fluorescent complex. Due to the small spatial distance between the fluorescent chromophores in the complex, the original fluorescence emission band shifts. By calculating the ratio of the fluorescence intensities at two points, the concentration of sulfated glycosaminoglycans can be determined. This invention provides accurate detection results, good reproducibility, and high sensitivity; the experimental materials are simple, saving a significant amount of time.

Description

Technical Field

[0001] This invention belongs to the field of drug analysis and detection, and specifically relates to a method for determining the concentration of sulfated glycosaminoglycans using a fluorescence method. Background Technology

[0002] LFG (Low Molecular Weight Fucosylated Glycosaminoglycan, hereinafter referred to as "LFG") is extracted from marine organisms of the genus Cucurbita and further refined and semi-synthesized. Fucosylated glycosaminoglycans (FGAG) are a class of glycosaminoglycan derivatives obtained from the body walls of echinoderms, with sulfated fucose side chains as substitutions. The commonalities among FGAGs from different sources are mainly reflected in their constituent monosaccharides, which include D-2-acetamido-2-deoxygalactose (D-GalNAc), D-glucuronic acid (D-GlcUA), and L-fucose (L-Fuc), as well as their sulfated esters. Among them, D-GlcUA and D-GalNAc are linked alternately by β-(1-3) and β-(1-4) glycosidic bonds to form the main chain of glycosaminoglycans, while L-Fuc is linked to the main chain as a side chain (Ricardo P. etal., JBC, 1988, 263 (34): 18176; Kenichiro Y. et al., Tetrahedron Lett, 1992, 33(34): 4959). Various sea cucumber-derived FGAGs possess potent anticoagulant activity and have important medicinal value for the prevention and clinical treatment of thrombotic diseases.

[0003] Sulfated glycosaminoglycans do not contain chromophores and cannot be detected by absorption spectroscopy. Therefore, the concentration of sulfated glycosaminoglycan drugs cannot be accurately measured to ensure their clinical quality. Summary of the Invention

[0004] To address the above problems, this invention aims to provide a method for determining the concentration of sulfated glycosaminoglycans using fluorescence spectroscopy, and the technical solution adopted is as follows:

[0005] A method for determining the concentration of sulfated glycosaminoglycans using fluorescence spectroscopy utilizes the electrostatic interaction between the amino group on arginine and the sulfate ion on the sugar chain. A series of fluorescent probe molecules can be neatly arranged along the sugar chain to form a stable fluorescent complex. Due to the small spatial distance between the fluorescent chromophores in the complex, the original fluorescence emission band is shifted. The concentration of sulfated glycosaminoglycans is determined by calculating the ratio of the fluorescence intensities at the two locations.

[0006] The molecular structure of the fluorescent probe is as follows:

[0007]

[0008] or or

[0009] or

[0010] The method for determining the concentration of sulfated glycosaminoglycans using fluorescence spectroscopy includes the following steps:

[0011] (1) Preparation of HEPES solution: Prepare HEPES solution and adjust pH to 7.4 with sodium hydroxide;

[0012] (2) Preparation of fluorescent probe molecule solution: Dissolve the fluorescent probe molecule in the HEPES solution of step (1);

[0013] (3) Preparation of sulfated glycosaminoglycan standard solution: dissolve sulfated glycosaminoglycan in the HEPES solution of step (1) and dilute to obtain sulfated glycosaminoglycan standard solution;

[0014] (4) Fluorescence spectral standard curve: Mix the fluorescent probe molecular solution from step (2) and the sulfated glycosaminoglycan standard solution from step (3) in equal volumes, add them to the quartz sample cell, and collect the fluorescence emission spectrum; record the fluorescence intensity at the newly added emission peak wavelength and the original maximum fluorescence emission peak wavelength on the fluorescence emission spectrum. Use the ratio I of (fluorescence intensity at the newly added emission peak wavelength) / (fluorescence intensity at the original maximum fluorescence emission peak wavelength) as the ordinate and the concentration of the sulfated glycosaminoglycan solution as the abscissa to make a standard curve and obtain the linear equation.

[0015] (5) Determine and calculate the concentration of the sulfated glycosaminoglycan to be tested; mix the sample solution to be tested with the fluorescent probe molecule solution from step (2) in equal volumes, add the mixture to the quartz sample cell, collect the fluorescence emission spectrum, and record the ratio I of (fluorescence intensity at the wavelength of the newly added emission peak) / (fluorescence intensity at the wavelength of the original maximum fluorescence emission peak). 样 Substituting into the linear equation obtained in step (4), the concentration of sulfated glycosaminoglycans in the sample to be tested can be calculated.

[0016] Preferably, the method for determining the concentration of sulfated glycosaminoglycans using fluorescence spectroscopy includes the following steps:

[0017] (1) Preparation of HEPES solution: Prepare a 10 mM / L HEPES solution and adjust the pH to 7.4 with sodium hydroxide;

[0018] (2) Preparation of fluorescent probe molecule solution: Dissolve the fluorescent probe molecule in the HEPES solution of step (1) to make the concentration of the probe molecule solution 20 μM / L;

[0019] (3) Preparation of sulfated glycosaminoglycan standard solution: dissolve sulfated glycosaminoglycan in the HEPES solution of step (1) and dilute to obtain a sulfated glycosaminoglycan standard solution of 0-11 μg / mL;

[0020] (4) Fluorescence spectral standard curve: The fluorescent probe molecular solution from step (2) and the sulfated glycosaminoglycan standard solution from step (3) are mixed in equal volumes (i.e., the final probe concentration is 10 μM / L and the sulfated glycosaminoglycan concentration is 0-5.5 μg / mL), and then added to the quartz sample cell to collect the fluorescence emission spectrum; the fluorescence intensity at the newly added emission peak wavelength and the original maximum fluorescence emission peak wavelength are recorded on the fluorescence emission spectrum. The ratio I of (fluorescence intensity at the newly added emission peak wavelength) / (fluorescence intensity at the original maximum fluorescence emission peak wavelength) is used as the ordinate, and the concentration of the sulfated glycosaminoglycan solution is used as the abscissa to make a standard curve and obtain the linear equation.

[0021] (5) Determine and calculate the concentration of the sulfated glycosaminoglycan to be tested; mix the sample solution to be tested with the fluorescent probe molecule solution from step (2) in equal volumes, add the mixture to the quartz sample cell, collect the fluorescence emission spectrum, and record the ratio I of (fluorescence intensity at the wavelength of the newly added emission peak) / (fluorescence intensity at the wavelength of the original maximum fluorescence emission peak). 样 Substituting into the linear equation obtained in step (4), the concentration of sulfated glycosaminoglycans in the sample to be tested can be calculated.

[0022] In steps (4) and (5), after each sample is collected, the sample cell is cleaned with acetone and ultrapure water.

[0023] The present invention provides accurate detection results, good reproducibility of test results, and high sensitivity; the test materials are simple and save a lot of time. Attached Figure Description

[0024] Figure 1 Fluorescence spectra of LFG concentrations in the range of 0-5.5 μg / mL when the probe concentration is fixed at 10 μM / L in HEPES.

[0025] Figure 2 LFG concentration and fluorescence intensity in HEPES 484nm / 376nm Linear relationship diagram Detailed Implementation

[0026] The following examples are used to illustrate the implementation of the present invention and to further understand the advantages and features of the present invention. They should not be construed as limiting the scope of the present invention.

[0027] Unless otherwise specified, all instruments and reagents used in the following examples are commercially available.

[0028] The relevant experimental instruments and reagents used in the following examples are as follows:

[0029] 1. Instruments and reagents

[0030] Hitachi F-2700FL spectrophotometer, excitation wavelength: 342nm, emission wavelength scan range: 360-600 nm, scan speed: 300 nm / min, excitation wavelength slit: 5.0nm, emission wavelength slit: 5.0nm. PMT voltage: 400Vo. Sartorius PB-20 standard pH meter (Beijing Sartorius Instrument Systems Co., Ltd.).

[0031] N-2-Hydroxyethyl-piperazine-N'-2-ehtanesulphonic acid (HEPES), purchased from Aladdin.

[0032] LFG (low molecular weight fucoidan, batch number: 20170922) was provided by Mudanjiang Youbo Pharmaceutical Co., Ltd.

[0033] 2. Experimental Procedure

[0034] (1) Preparation of HEPES solution: Prepare a 10 mM / L HEPES solution and adjust the pH to 7.4 with sodium hydroxide;

[0035] (2) Preparation of fluorescent probe molecule solution: Dissolve the fluorescent probe molecule in the HEPES solution of step (1) to make the concentration of the probe molecule solution 20 μM / L; the fluorescent probe used is Py–ArgArgArg–NH2, and its molecular structure is as follows.

[0036]

[0037] (3) Preparation of sulfated glycosaminoglycan standard solution: Sulfated glycosaminoglycan is dissolved in the HEPES solution of step (1) and diluted to obtain a sulfated glycosaminoglycan standard solution of 0-11 μg / mL; the sulfated glycosaminoglycan used is LFG (low molecular weight fucoidan glycosaminoglycan).

[0038] (4) Fluorescence spectral standard curve: Mix the fluorescent probe molecule solution from step (2) and the LFG standard solution from step (3) in equal volumes (i.e., the final probe concentration is 10 μM / L and the LFG solution concentration is 0-5.5 μg / mL), then add them to the quartz sample cell and collect the fluorescence emission spectrum (e.g., Figure 1As shown in the figure, at an excitation wavelength of 342 nm, the maximum fluorescence emission of the probe is at 376 nm (i.e., the original maximum fluorescence emission peak wavelength); while when the concentration of LFG is gradually increased in the range of 0-5.5 μg / mL, a new emission peak appears at 484 nm (i.e., the newly added emission peak). The fluorescence intensities at 484 nm and 376 nm on the fluorescence emission spectrum are recorded, and their ratio I is used to determine the fluorescence intensity. 484nm / 376nm A standard curve was constructed with the LFG sample solution concentration (final concentration) on the x-axis and the vertical axis on the y-axis, yielding a linear equation. Figure 2 It can be seen that, within the concentration range of 2.8 to 4.6 μg / mL, the ratio of fluorescence intensity at 484 nm to 376 nm is I. 484nm / 376nm It exhibits a good linear relationship with LFG concentration.

[0039] (5) Determine and calculate the concentration of the LFG solution to be tested; mix the LFG sample solution to be tested with the fluorescent probe molecule solution from step (2) in equal volumes, add the mixture to the quartz sample cell, collect the fluorescence emission spectrum, and record and calculate I. 样484nm / 376nm Substituting 0.811 into the equation in step (4), the final concentration of LFG in the test solution can be calculated as 3.482 μg / mL. Since the final concentration is the final concentration of the LFG test solution and the fluorescent probe molecule solution mixed in equal volumes, the concentration of LFG in the LFG sample solution is the final concentration * 2, i.e., 3.482 * 2 = 6.964 μg / mL.

[0040] In steps (4) and (5), after each sample is collected, the sample cell is cleaned with acetone and ultrapure water.

Claims

1. A method for determining the concentration of fucosylated glycosaminoglycans using fluorescence spectroscopy, characterized in that, By utilizing the electrostatic interaction between the amino group on arginine and the sulfate ion on the sugar chain, a series of fluorescent probe molecules can be neatly arranged along the sugar chain to form a stable fluorescent complex. Due to the small spatial distance between the fluorescent chromophores in the complex, the original fluorescence emission band is shifted. The concentration of fucosylated glycosaminoglycan is determined by calculating the ratio of the fluorescence intensities at two points. The structure of the fluorescent probe molecule is as follows: or or or , The method includes the following steps: (1) Preparation of HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) solution: Prepare HEPES solution and adjust pH to 7.4 with sodium hydroxide; (2) Preparation of fluorescent probe molecule solution: Dissolve the fluorescent probe molecule in the HEPES solution of step (1); (3) Preparation of fucosaccharified glycosaminoglycan standard solution: Fucosaccharified glycosaminoglycan is dissolved in the HEPES solution of step (1) and diluted to obtain fucosaccharified glycosaminoglycan standard solution; (4) Fluorescence spectral standard curve: Mix the fluorescent probe molecular solution from step (2) and the fucosyl glycosaminoglycan standard solution from step (3) in equal volumes, add them to the quartz sample cell, and collect the fluorescence emission spectrum; record the fluorescence intensity at the newly added emission peak wavelength and the original maximum fluorescence emission peak wavelength on the fluorescence emission spectrum, and use the ratio I of (fluorescence intensity at the newly added emission peak wavelength) / (fluorescence intensity at the original maximum fluorescence emission peak wavelength) as the ordinate and the concentration of the fucosyl glycosaminoglycan solution as the abscissa to make a standard curve and obtain the linear equation; (5) Determine and calculate the concentration of the fucosylated glycosaminoglycan to be tested; mix the sample solution to be tested with the fluorescent probe molecule solution from step (2) in equal volumes, add the mixture to the quartz sample cell, collect the fluorescence emission spectrum, and record the ratio I of (fluorescence intensity at the wavelength of the newly added emission peak) / (fluorescence intensity at the wavelength of the original maximum fluorescence emission peak). 样 Substituting into the linear equation obtained in step (4), the concentration of fucosylated glycosaminoglycans in the sample to be tested can be calculated.

2. The method for determining the concentration of fucosylated glycosaminoglycans by fluorescence spectroscopy according to claim 1, characterized in that, Includes the following steps: (1) Preparation of HEPES solution: Prepare a 10 mM / L HEPES solution and adjust the pH to 7.4 with sodium hydroxide; (2) Preparation of fluorescent probe molecule solution: Dissolve the fluorescent probe molecule in the HEPES solution of step (1) to make the concentration of the probe molecule solution 20 μM / L; (3) Preparation of fucosaccharified glycosaminoglycan standard solution: Fucosaccharified glycosaminoglycan is dissolved in the HEPES solution of step (1) and diluted to obtain a 0-11 μg / mL fucosaccharified glycosaminoglycan standard solution; (4) Fluorescence spectral standard curve: The fluorescent probe molecular solution from step (2) and the fucosyl glycosaminoglycan standard solution from step (3) are mixed in equal volumes (i.e., the final probe concentration is 10 μM / L and the fucosyl glycosaminoglycan concentration is 0-5.5 μg / mL), and then added to the quartz sample cell to collect the fluorescence emission spectrum; the fluorescence intensity at the newly added emission peak wavelength and the original maximum fluorescence emission peak wavelength are recorded on the fluorescence emission spectrum. The ratio I of (fluorescence intensity at the newly added emission peak wavelength) / (fluorescence intensity at the original maximum fluorescence emission peak wavelength) is used as the ordinate, and the concentration of the fucosyl glycosaminoglycan solution is used as the abscissa to construct a standard curve and obtain the linear equation. (5) Determine and calculate the concentration of the fucosylated glycosaminoglycan to be tested; mix the sample solution to be tested with the fluorescent probe molecule solution from step (2) in equal volumes, add the mixture to the quartz sample cell, collect the fluorescence emission spectrum, and record the ratio I of (fluorescence intensity at the wavelength of the newly added emission peak) / (fluorescence intensity at the wavelength of the original maximum fluorescence emission peak). 样 Substituting into the linear equation obtained in step (4), the concentration of fucosylated glycosaminoglycans in the sample to be tested can be calculated.

3. A method for determining the concentration of fucosylated glycosaminoglycans by fluorescence spectroscopy according to claim 1 or 2, characterized in that, In steps (4) and (5), after each sample is collected, the sample cell is cleaned with acetone and ultrapure water.

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