CN108195808B - Method for detecting oversulfated chondroitin sulfate impurities in heparin sodium - Google Patents
Method for detecting oversulfated chondroitin sulfate impurities in heparin sodium Download PDFInfo
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Abstract
The invention discloses a method for detecting oversulfated chondroitin sulfate impurities in heparin sodium, which is characterized in that two polypeptides of KRKC and GSH are used as stabilizing agents to synthesize gold nanoclusters with positive charges on the surfaces and fluorescence characteristics as fluorescence probes, the fluorescence probes are mixed with a heparin sodium standard solution and a heparin sodium solution containing the oversulfated chondroitin sulfate impurities respectively to have different fluorescence enhancement effects, and the oversulfated chondroitin sulfate impurities in a heparin sodium product are detected and determined. Provides a method for efficiently and rapidly detecting whether oversulfated chondroitin sulfate is contained in heparin sodium. The method has the advantages of low cost, high sensitivity, rapidness, high efficiency, strong operability and the like, and can realize the rapid detection of whether the oversulfated chondroitin sulfate impurity is contained in the heparin sodium.
Description
Technical Field
The invention belongs to the technical application field of functional nano materials, and relates to a method for detecting oversulfated chondroitin sulfate impurities in heparin sodium by using gold nanoclusters as a fluorescence indicator, in particular to a method for detecting oversulfated chondroitin sulfate impurities in heparin sodium by using gold nanoclusters which are synthesized by using two polypeptides of KRKC and GSH as stabilizers, have positive charges on the surfaces and can generate fluorescence and serve as fluorescence probes.
Background
Heparin sodium (Heparin sodium) is a common blood anticoagulant, is a polymer formed by connecting disaccharide units with negative charges, is a sodium salt of sulphated amino-polysaccharide, is a refined mucopolysaccharide medicament mainly extracted from the mucosa of the small intestine of a healthy pig, and is widely applied to the medical fields of venous thrombosis treatment, blood coagulation prevention and the like. In 2008, the quality of heparin raw material medicines and final finished products exported to the united states in China has problems, so that allergic reactions and other side effects occur in a plurality of patients, and adverse effects are caused on the health of the patients and normal medical care of hospitals. The reason for this event is that heparin sodium is doped with structurally similar polysaccharide polysulfated chondroitin sulfate (OSCS), which is a research by related departments. Because the structure, molecular weight, charge density and anticoagulant activity of polysaccharide molecules of various impurities including chondroitin sulfate are very similar to those of heparin sodium, the identification and detection of saccharide impurities are difficult to a certain degree. Therefore, how to quickly and accurately identify the oversulfated chondroitin sulfate polysaccharide impurities contained in the heparin sodium is a key for improving the detection level of the medicine and a key for improving the safety of the medicine and the personal safety of patients. The related detection method and regulation also rise to the quality standards of Chinese pharmacopoeia and United states pharmacopoeia.
Currently, the identification of heparin sodium mainly includes Capillary Electrophoresis (CE), Nuclear Magnetic Resonance (NMR), chromatography, and the like. The capillary method can test whether the oversulfated chondroitin sulfate exists in the heparin sodium, but the method can cause misjudgment because the main component peak and the oversulfated chondroitin sulfate peak of the atlas cannot be completely separated. According to the foreign literature, the nuclear magnetic resonance hydrogen spectrum (1H-nuclear magnetic resonance, 1H-NMR) can detect the N-acetyl methyl peak different from heparin from OSCS polluted heparin, and whether the heparin contains OSCS pollutants can be judged by detecting the characteristic peak. In addition, research teams abroad have conducted related studies on a method for detecting OSCS content in heparin by strong anion exchange-high performance liquid chromatography (SAX-HPLC). However, the methods have high requirements on instruments and high cost, so that the development of a simple, accurate and efficient method for distinguishing heparin sodium from other saccharide impurities has urgent market demands and good social benefits.
The metal nanocluster is a novel material between metal atoms and nanoparticles, has the advantages of strong fluorescence emission, good stability, good biocompatibility and the like, and can be applied to the field of biological small molecule detection. For example, the Chinese invention patent CN104865235B discloses a fluorescent silver nano-cluster based on in-situ preparationThe method for detecting the glucose concentration adopts the prepared fluorescent silver nanocluster fluorescence emission intensity to establish a quantitative relation with the glucose concentration, thereby realizing the detection of the glucose concentration. Chinese patent CN104330393B discloses a glucose detection method using gold nanoclusters as fluorescent probes. Both methods adopt glucose oxidase to oxidize glucose to generate H2O2,Fe2+Catalysis H2O2The detection methods only detect single saccharides and cannot distinguish two types of polysaccharide which have similar structures and are difficult to distinguish, and aiming at the problem, the gold nanoclusters which can realize different fluorescence responses to heparin sodium and oversulfated chondroitin sulfate are designed to realize qualitative and quantitative detection of the impurity oversulfated chondroitin sulfate contained in the heparin sodium.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and find a method for detecting oversulfated chondroitin sulfate impurities in heparin sodium and determining the purity of the heparin sodium. Solves the problems of high cost and complex method of the existing method for detecting the oversulfated chondroitin sulfate impurities in the heparin sodium.
In order to achieve the purpose, the invention provides a method for detecting oversulfated chondroitin sulfate impurities in heparin sodium, which adopts two polypeptides of KRKC and GSH as stabilizers to synthesize gold nanoclusters with positive charges on the surface and fluorescent characteristics as fluorescent probes, and the fluorescent probes are mixed with a heparin sodium standard solution and a heparin sodium solution containing the oversulfated chondroitin sulfate impurities respectively to have different fluorescence enhancement effects, so that the oversulfated chondroitin sulfate impurities in the heparin sodium product are detected and measured.
A method for detecting oversulfated chondroitin sulfate impurities in heparin sodium specifically comprises the following steps:
(1) respectively adding a certain amount of gold nanoclusters into a heparin sodium standard solution and a heparin sodium solution to be detected, uniformly mixing, and standing, wherein the mass ratio of the gold nanoclusters to the heparin sodium standard is (3-4) to 1;
(2) measuring fluorescence emission spectra of the two solutions obtained in the step (1) in a 420nm-780nm waveband by using a fluorescence spectrophotometer under excitation light with the wavelength of 400nm, and simultaneously comparing the fluorescence intensities of the two solutions at the wavelength of 600nm, wherein if the fluorescence intensities are the same, the heparin sodium solution to be detected does not contain impurities, otherwise, the heparin sodium solution to be detected contains impurities;
(3) adding a certain amount of gold nanoclusters into the mixed solution of the oversulfated chondroitin sulfate standard substance and the heparin sodium standard substance which are doped in different proportions, uniformly mixing, and standing;
(4) and (3) measuring the fluorescence emission spectrum of the solution obtained in the step (3) in a 420nm-780nm waveband by using a fluorescence spectrophotometer under the excitation light with the wavelength of 400nm, taking the fluorescence intensity value at 600nm, making a standard curve graph of the concentration of the oversulfated chondroitin sulfate and the fluorescence responsiveness, and calculating the content of the oversulfated chondroitin sulfate in the to-be-detected heparin sodium according to the standard curve graph and the fluorescence intensity measured by the to-be-detected heparin sodium in the step (2). The concentration of the heparin sodium standard solution used in the step (1), the concentration of the heparin sodium solution to be detected and the total concentration of the mixed solution of the heparin sodium standard solution and the oversulfated chondroitin sulfate standard solution in the step (3) are the same, and the addition amount of the gold nanoclusters in the step (1) and the step (3) is the same.
Further, the concentration of the gold nanoclusters in the solution in the step (1) is 100-200. mu.g/mL, preferably 150. mu.g/mL.
Further, the gold nanocluster is prepared by adopting a method disclosed in patent ZL201610389189.7, a preparation method for preparing the gold nanocluster by taking polypeptide as a reducing agent and a stabilizing agent.
Compared with the prior art, the invention takes the simple and easily obtained gold nanoclusters as a fluorescence detection reagent, and establishes the relation between the fluorescence change of the gold nanoclusters and the component change in the heparin sodium by utilizing the principle that different charge densities of the heparin sodium and oversulfated chondroitin sulfate molecules in the heparin sodium produce different fluorescence enhancement effects on the fluorescence of the gold nanoclusters, thereby providing a method for efficiently and quickly detecting whether the heparin sodium contains the oversulfated chondroitin sulfate. The method has the advantages of low cost, high sensitivity, rapidness, high efficiency, strong operability and the like, and can realize the rapid detection of whether the oversulfated chondroitin sulfate impurity is contained in the heparin sodium.
Description of the drawings:
FIG. 1 is a fluorescence emission spectrum of different solutions mixed with equal amount of gold nanoclusters in example 1 of the present invention.
Fig. 2 is a fluorescence emission spectrogram of the heparin sodium solution to be detected and the equivalent amount of gold nanoclusters mixed in embodiment 2 of the present invention.
FIG. 3 is a fluorescence emission spectrum of the mixed solution of oversulfated chondroitin sulfate standard and heparin sodium standard mixed with equal amount of gold nanoclusters in example 2 of the present invention.
Fig. 4 is a standard working curve of the impurity content of oversulfated chondroitin sulfate in example 2 of the present invention, in which the abscissa is the concentration of different oversulfated chondroitin sulfate, and the ordinate is the change value of the fluorescence intensity of the mixed solution of the oversulfated chondroitin sulfate standard substance and the heparin sodium standard substance doped in different proportions relative to the fluorescence intensity of the solution of the heparin sodium standard substance without the oversulfated chondroitin sulfate.
Detailed Description
The invention is further illustrated by the following detailed description and the accompanying drawings.
The spectrofluorometer used in the following examples is a F-2500 spectrofluorometer manufactured by Hitachi, Japan.
Example 1:
(1) preparation of gold nanoclusters
Preparation of HAuCl with ultrapure Water4Soaking a glass bottle with aqua regia (20mmol/L), KRKC (20mmol/L) and GSH (20mmol/L) solution, cleaning, and drying; adding 400 μ L KRKC solution and 120 μ L GSH solution into the treated glass bottle, mixing, adding HAuCl4The solution is 280 mu L, the color of the reaction system can be observed to change from colorless to light yellow, and finally, the ultrapure water is added into a glass bottle to ensure that the Au in the solution is dissolved+The final concentration of (a) is 2 mmol/L; placing the glass bottle added with the reactant in a constant-temperature water bath kettle, and setting the temperature at 70 ℃ for reaction for 12 hours; after the reaction is finished, transferring the sample into a centrifugal tube, and centrifuging to remove macromolecular substances to obtain goldNanoclusters; centrifuging to obtain supernatant, and filtering with ultrafiltration membrane with molecular weight cutoff of 10000 Dalton to obtain gold nanoclusters; the gold nanoclusters obtained have a concentration of C as measured by a quartz crystal microbalance0；
(2) Diluting the gold nanocluster solution obtained in the step (1) to 300 mu g/mL by using ultrapure water, and preparing a heparin sodium standard solution of 100 mu g/mL, a chondroitin polysulfate standard solution of 100 mu g/mL and a heparin sodium solution to be detected of 100 mu g/mL;
(3) mixing 100 mu L of the gold nanocluster solution obtained in the step (2) with 100 mu L of heparin sodium standard solution, 100 mu L of oversulfated chondroitin sulfate standard solution and 100 mu L of heparin sodium to be detected respectively, and standing for 5 min;
(4) measuring fluorescence emission spectra of the three mixed solutions obtained in the step (3) in a 420nm-780nm waveband by using a fluorescence spectrophotometer under excitation light with the wavelength of 400 nm;
(5) fig. 1 is a fluorescence spectrum diagram of different solutions, wherein 1 is a gold nanocluster solution of 150 μ g/mL, 2 is a mixed solution of the gold nanocluster solution and a heparin sodium standard solution in step (3), 3 is a mixed solution of the gold nanocluster solution and a oversulfated chondroitin sulfate standard solution in step (3), and 4 is a mixed solution of the gold nanocluster solution and a heparin sodium solution to be detected in step (3). From the figure, it can be seen that heparin sodium and oversulfated chondroitin sulfate with the same quality generate different fluorescence enhancement effects on the gold nanoclusters, the heparin sodium and the oversulfated chondroitin sulfate exhibit different fluorescence characteristics, particularly the fluorescence intensity at the wavelength of 600nm is obviously different, and the purity of the heparin sodium can be detected according to the characteristics.
Example 2:
(1) diluting the solution of the gold nanocluster obtained in the step (1) in the embodiment 1 to 300 mug/mL by using ultrapure water, and preparing a 100 mug/mL heparin sodium standard solution, a 100 mug/mL heparin sodium solution to be detected, 100 mug/mL mixed solution of different proportions of doped oversulfated chondroitin sulfate standard and heparin sodium standard (the mass contents of the oversulfated chondroitin sulfate are 6%, 12%, 16%, 22%, 28%, 34% and 40%, respectively);
(2) rapidly mixing 100 mu L of the gold nanocluster solution obtained in the step (1) with 100 mu L of a heparin sodium standard solution (the mass content of chondroitin polysulfate is 0%) and 100 mu L of a mixed solution of the chondroitin polysulfate standard and the heparin sodium standard (the mass content of the chondroitin polysulfate is 6%, 12%, 16%, 22%, 28%, 34% and 40%) doped in different proportions, and standing for 5 min;
(3) measuring the fluorescence spectrum (excitation wavelength is 400nm) of the 8 groups of mixed solutions in the step (2) by using a fluorescence spectrophotometer, scanning and recording the fluorescence emission spectrum of a 420nm-780nm waveband, and recording the fluorescence intensity at a 600nm wavelength;
(4) FIG. 3 is a fluorescence spectrum of different solutions, wherein A is a mixed solution of the gold nanocluster solution and the heparin sodium standard solution in step (2), B is a mixed solution of the gold nanocluster solution and the heparin sodium solution with the chondroitin polysulfate content of 6% in step (2), C is a mixed solution of the gold nanocluster solution and the heparin sodium solution with the chondroitin polysulfate content of 12% in step (2), D is a mixed solution of the gold nanocluster solution and the heparin sodium solution with the chondroitin polysulfate content of 16% in step (2), E is a mixed solution of the gold nanocluster solution and the heparin sodium solution with the chondroitin polysulfate content of 22% in step (2), F is a mixed solution of the gold nanocluster solution and the heparin sodium solution with the chondroitin polysulfate content of 28% in step (2), and G is a mixed solution of the gold nanocluster solution and the heparin sodium solution with the chondroitin polysulfate content of 34% in step (2), h is the mixed solution of the gold nanocluster solution and the heparin sodium solution with 40% of oversulfated chondroitin sulfate content in the step (2);
(5) the fluorescence intensity of each fluorescence emission spectrum shown in FIG. 3 at a wavelength of 600nm was recorded, and the fluorescence intensity of spectrum A at 600nm was P0The fluorescence intensity of spectrogram B at 600nm is P1Spectrum C has fluorescence intensity P at 600nm2Spectrum D fluorescence intensity at 600nm is P3Spectrum E has a fluorescence intensity of P at 600nm4Spectrum F has fluorescence intensity P at 600nm5Spectrum G has fluorescence intensity P at 600nm6The fluorescence intensity of spectrogram H at 600nm is P7Taking the concentration of oversulfated chondroitin sulfate in different solutions as the abscissa, the fluorescence intensity of each mixed solution is relative to P0Variation value (P)0-Pi)/P0As ordinate, the content of oversulfated chondroitin sulfate impurity is normalizedThe operating curve, as shown in fig. 4;
(6) quickly mixing the 100 mu L of gold nanocluster solution in the step (1) with 100 mu L of heparin sodium solution to be detected, and standing for 5 min;
(7) the fluorescence spectrum (excitation wavelength 400nm) of the mixed solution of step (6) was measured with a fluorescence spectrophotometer as shown in FIG. 2, and the fluorescence intensity at a wavelength of 600nm was recorded as Px；
(8) Will PxAnd (5) substituting the standard working curve of the content of the oversulfated chondroitin sulfate impurity obtained in the step (5) to calculate that the content of the oversulfated chondroitin sulfate impurity in the to-be-detected heparin sodium solution is 5.6% (2.8 mu g/mL). The limit of detection of the method is 3.8 percent (1.9 mu g/mL) of the impurity content of the oversulfated chondroitin sulfate.
Claims (5)
1. A method for detecting oversulfated chondroitin sulfate impurities in heparin sodium is characterized in that gold nanoclusters with positive charges on the surfaces and fluorescence characteristics are synthesized by using two polypeptides of KRKC and GSH as stabilizers and used as fluorescent probes, and the fluorescent probes are mixed with a heparin sodium standard solution and a heparin sodium solution containing the oversulfated chondroitin sulfate impurities respectively to have different fluorescence enhancement effects, so that the oversulfated chondroitin sulfate impurities in a heparin sodium product are detected and determined.
2. The method for detecting oversulfated chondroitin sulfate impurities in heparin sodium as claimed in claim 1, which comprises the following steps:
(1) respectively adding a certain amount of gold nanoclusters into the heparin sodium standard solution and the heparin sodium solution to be detected, uniformly mixing, and standing, wherein the mass ratio of the gold nanoclusters to the heparin sodium standard is (3-4) to 1;
(2) and (2) measuring the fluorescence emission spectra of the two solutions obtained in the step (1) at the wave band of 420nm-780nm by using a fluorescence spectrophotometer under the excitation light with the wavelength of 400nm, simultaneously comparing the fluorescence intensity of the two solutions at the wavelength of 600nm, if the fluorescence intensity is the same, the heparin sodium solution to be detected does not contain impurities, otherwise, the heparin sodium solution to be detected contains impurities.
3. The method for detecting oversulfated chondroitin sulfate impurities in heparin sodium as claimed in claim 2, further comprising the following steps:
(3) adding a certain amount of gold nanoclusters into the mixed solution of the oversulfated chondroitin sulfate standard substance and the heparin sodium standard substance which are doped in different proportions, uniformly mixing, and standing;
(4) and (3) measuring the fluorescence emission spectrum of the solution obtained in the step (3) in a 420nm-780nm waveband by using a fluorescence spectrophotometer under the excitation light with the wavelength of 400nm, taking the fluorescence intensity value at 600nm, making a standard curve graph of the concentration of the oversulfated chondroitin sulfate and the fluorescence responsiveness, and calculating the content of the oversulfated chondroitin sulfate in the to-be-detected heparin sodium according to the standard curve graph and the fluorescence intensity measured by the to-be-detected heparin sodium in the step (2).
4. The method as claimed in claim 3, wherein the concentration of gold nanoclusters in the solution of step (1) is 100-200 μ g/mL.
5. The method for detecting oversulfated chondroitin sulfate impurities in heparin sodium as claimed in claim 4, wherein the concentration of the gold nanoclusters in the solution in the step (1) is 150 μ g/mL.
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| CN102796511A (en) * | 2011-05-27 | 2012-11-28 | 中国科学院理化技术研究所 | Fluorescent probe for quantitatively detecting heparin, and synthesis method and application thereof |
| CN104076014A (en) * | 2014-06-30 | 2014-10-01 | 安徽师范大学 | Fluorescence sensor, as well as preparation method and application of sensor and method for detecting Heparin molecule |
| CN104678092B (en) * | 2015-01-28 | 2017-06-06 | 山东大学 | Application of the positive charge fluorescin high in glycosaminoglycan analysis detection |
| CN105154066A (en) * | 2015-08-28 | 2015-12-16 | 南京大学 | High-sensitivity fluorescent probe, method for preparing same and application of high-sensitivity fluorescent probe |
| CN106085420B (en) * | 2016-06-02 | 2018-02-16 | 中国石油大学(华东) | A kind of peptide modified gold nanoclusters and preparation method thereof |
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