阅读说明：本技术 一种采用荧光光谱定量检测胶原类蛋白的方法 (Method for quantitatively detecting collagen protein by adopting fluorescence spectrum ) 是由 李从虎 吴彦 龙槿彦 于 2020-06-29 设计创作，主要内容包括：本发明公开一种采用荧光光谱定量检测胶原类蛋白的方法,涉及胶原蛋白检测技术领域,本发明包括以下步骤：(1)将含有胶原类蛋白的原料粉碎后匀浆,在4℃条件下加入胃蛋白酶作用24h,然后经离心、盐析、透析处理后,冷冻干燥制得胶原类蛋白；(2)将胶原溶于0.1mol/L醋酸水溶液中,制成不同浓度梯度的胶原类蛋白溶液；(3)对胶原类蛋白溶液进行荧光扫描,绘制标准曲线；(4)将含有胶原蛋白的待测样品溶于0.1mol/L醋酸中,进行荧光扫描,将测得的吸收峰强度与标准曲线进行比对,即测得待测样品中胶原类蛋白的含量。本发明的有益效果在于：该方法具有准确、快速、灵敏和重现性好的优点,符合准确定量的要求。(The invention discloses a method for quantitatively detecting collagen by adopting fluorescence spectrum, relating to the technical field of collagen detection and comprising the following steps: (1) pulverizing raw materials containing collagen, homogenizing, adding pepsin at 4 deg.C for 24 hr, centrifuging, salting out, dialyzing, and freeze drying to obtain collagen; (2) dissolving collagen in 0.1mol/L acetic acid water solution to prepare collagen protein solutions with different concentration gradients; (3) performing fluorescence scanning on the collagen solution, and drawing a standard curve; (4) and dissolving a sample to be detected containing collagen in 0.1mol/L acetic acid, performing fluorescence scanning, and comparing the measured absorption peak intensity with a standard curve to obtain the content of the collagen in the sample to be detected. The invention has the beneficial effects that: the method has the advantages of accuracy, rapidness, sensitivity and good reproducibility, and meets the requirement of accurate quantification.)

1. A method for quantitatively detecting collagen by adopting fluorescence spectrum is characterized by comprising the following steps: the method comprises the following steps:

(1) pulverizing raw materials containing collagen, homogenizing, adding pepsin at 4 deg.C for 24 hr, centrifuging, salting out, dialyzing, and freeze drying to obtain collagen;

(2) dissolving collagen in 0.1mol/L acetic acid water solution to prepare collagen protein solutions with different concentration gradients, and standing at 4 ℃ for 24 h;

(3) performing fluorescence scanning on the collagen solutions with different concentration gradients in the step (2), and drawing a standard curve by taking the concentration of the collagen solution as a horizontal coordinate and the maximum absorption peak intensity as a vertical coordinate;

(4) and (2) treating a sample to be detected containing collagen according to the step (1), dissolving the sample to be detected in 0.1mol/L acetic acid, standing for 24 hours at the temperature of 4 ℃, performing fluorescence scanning, and comparing the measured absorption peak intensity with a standard curve to obtain the content of the collagen in the sample to be detected.

2. The method for quantitatively detecting collagen according to claim 1 by fluorescence spectroscopy, wherein: the collagen protein in the step (1) comprises collagen, gelatin or a collagen derivative.

3. The method for quantitatively detecting collagen according to claim 2 by fluorescence spectroscopy, wherein: the raw materials containing collagen in the step (1) comprise cow leather, pig leather or fish skin and the like.

4. The method for quantitatively detecting collagen according to claim 1 by fluorescence spectroscopy, wherein: the concentration of the collagen solution in the step (2) is 0.1-1.0 mg/mL.

5. The method for quantitatively detecting collagen according to claim 1 by fluorescence spectroscopy, wherein: the step of fluorescence scanning in step (3) comprises: and (3) moving the standing collagen solution into a four-way quartz cuvette, keeping the temperature at 20 ℃ for 30min, scanning the sample by using a fluorescence photometer, wherein the excitation wavelength range is 250-280nm, the scanning range is 235-450nm, and the slit width is 1-10 nm.

6. The method for quantitatively detecting collagen according to claim 5 by fluorescence spectroscopy, wherein: and scanning the sample by using a fluorescence photometer, wherein the excitation wavelength range is 253nm, the scanning range is 235-450nm, and the slit width is 5 nm.

7. The method for quantitatively detecting collagen according to claim 1 by fluorescence spectroscopy, wherein: the step of fluorescence scanning in step (3) comprises: and (3) moving the static collagen solution into a four-way quartz cuvette, keeping the temperature at 20 ℃ for 30min, scanning the sample by using a fluorescence photometer, wherein the difference range between the excitation wavelength and the emission wavelength is 10-30nm, the scanning range is 240-400nm, and the slit width is 1-10 nm.

8. The method for quantitatively detecting collagen according to claim 7 by fluorescence spectroscopy, wherein: and scanning the sample by using a fluorescence photometer, wherein the range of the difference between the excitation wavelength and the emission wavelength is 30nm, the scanning range is 240-400nm, and the slit width is 5 nm.

9. The method for quantitatively detecting collagen according to claim 1 by fluorescence spectroscopy, wherein: and (4) adding an exogenous fluorescent probe in the fluorescent scanning process in the step (3), wherein the exogenous fluorescent probe is 8-anilinonaphthalene-1-sulfonic acid or pyrene.

10. The method for quantitatively detecting collagen according to claim 9 by fluorescence spectroscopy, wherein: the exogenous fluorescent probe is 8-anilinonaphthalene-1-sulfonic acid, the fluorescence test conditions are that the excitation wavelength is 300-400nm, the emission wavelength is 400-650nm, and the slit width is 1-10 nm.

Technical Field

The invention relates to the technical field of collagen detection, in particular to a method for quantitatively detecting collagen by adopting fluorescence spectrum.

Background

Collagen, which is a major component of extracellular matrix (ECM), is a natural macromolecular protein widely distributed in organs and tissues such as skin, tendon, cartilage and blood vessels of animal organisms, and plays a role in supporting and protecting the animal organisms and organs, and its content is about one third of the total protein of the animal organisms. The collagen has the characteristics of good biocompatibility, biodegradability, low immunogenicity and the like, so that the collagen has important application in the fields of tissue engineering, biomedicine, food and the like.

At present, the quantitative detection method of the protein has related reports, such as TNBS method, Lorry method and Bergman method. However, the specific detection method for the content of collagen protein has not been systematically reported, and the detection is performed only by using the detection method of related protein as a standard, and the methods have the defects of complex operation and low sensitivity, for example, the TNBS method is a detection method for converting the amount of free amino groups into the content of collagen, and the TNBS method requires high purity of substances, and the whole course is protected from light, so that errors often occur in the test results.

Disclosure of Invention

The invention aims to solve the technical problem that errors are easy to occur in the quantitative detection method of the collagen protein in the prior art, and provides a method for quantitatively detecting the collagen protein by adopting a fluorescence spectrum.

The invention solves the technical problems through the following technical means:

a method for quantitatively detecting collagen by adopting fluorescence spectroscopy comprises the following steps:

(1) pulverizing raw materials containing collagen, homogenizing, adding pepsin at 4 deg.C for 24 hr, centrifuging, salting out, dialyzing, and freeze drying to obtain collagen;

(2) dissolving collagen in 0.1mol/L acetic acid water solution to prepare collagen protein solutions with different concentration gradients, and standing at 4 ℃ for 24 h;

(3) performing fluorescence scanning on the collagen solutions with different concentration gradients in the step (2), and drawing a standard curve by taking the concentration of the collagen solution as a horizontal coordinate and the maximum absorption peak intensity as a vertical coordinate;

(4) and (2) treating a sample to be detected containing collagen according to the step (1), dissolving the sample to be detected in 0.1mol/L acetic acid, standing for 24 hours at the temperature of 4 ℃, performing fluorescence scanning, and comparing the measured absorption peak intensity with a standard curve to obtain the content of the collagen in the sample to be detected.

Has the advantages that: the method comprises the steps of drawing a standard curve by collagen samples with different concentrations under the test condition of fluorescence spectrum, calculating a linear regression equation, simultaneously extracting and purifying the sample to be tested by the same method, and obtaining the concentration of the sample to be tested by the linear regression equation.

Preferably, the collagen of step (1) comprises collagen, gelatin or a collagen derivative.

Preferably, the collagen-containing raw material in step (1) includes cow leather, pig leather, fish skin, or the like.

Preferably, the concentration of the collagen solution in the step (2) is 0.1-1.0 mg/mL.

Preferably, the step of fluorescence scanning in step (3) comprises: and (3) moving the standing collagen solution into a four-way quartz cuvette, keeping the temperature at 20 ℃ for 30min, scanning the sample by using a fluorescence photometer, wherein the excitation wavelength range is 250-280nm, the scanning range is 235-450nm, and the slit width is 1-10 nm.

Preferably, the sample is scanned using a fluorescence spectrometer with an excitation wavelength range of 253nm, a scanning range of 235-450nm, and a slit width of 5 nm.

Preferably, the step of fluorescence scanning in step (3) comprises: and (3) moving the static collagen solution into a four-way quartz cuvette, keeping the temperature at 20 ℃ for 30min, scanning the sample by using a fluorescence photometer, wherein the difference range between the excitation wavelength and the emission wavelength is 10-30nm, the scanning range is 240-400nm, and the slit width is 1-10 nm.

Preferably, the sample is scanned by a fluorescence photometer, the difference between the excitation wavelength and the emission wavelength is in the range of 30nm, the scanning range is 240 nm and 400nm, and the slit width is 5 nm.

Has the advantages that: the fluorescence spectrum of the collagen solution with the wavelength difference of 30nm has a fluorescence absorption peak at 275nm, and after the fluorescence intensity is fitted, the fitting linearity of the fluorescence intensity is good, and the correlation coefficient is close to 1.

Preferably, an exogenous fluorescent probe is added in the fluorescent scanning process in the step (3), and the exogenous fluorescent probe is 8-anilinonaphthalene-1-sulfonic acid or pyrene.

Has the advantages that: the invention is based on endogenous fluorescent amino acids and exogenous probes of collagen, gelatin and derivatives thereof, and systematically and comprehensively adopts the fluorescent spectrometry to carry out quantitative detection on the collagen, the gelatin and the derivatives thereof, thereby laying a certain foundation for researching collagen proteins.

Preferably, the exogenous fluorescent probe is 8-anilinonaphthalene-1-sulfonic acid, the fluorescence test condition is that the excitation wavelength is 300-400nm, the emission wavelength is 400-650nm, and the slit width is 1-10 nm.

Preferably, the exogenous fluorescent probe is pyrene, and the pyrene is dissolved in absolute ethyl alcohol to obtain 1.0 × 10^-4The method comprises the following steps of mixing 0.1mL of pyrene ethanol solution with 10mL of collagen solutions with different concentrations by using a mol/L pyrene ethanol solution, volatilizing ethanol in the mixed solution, and scanning a sample by using a fluorescence photometer, wherein the scanning conditions are as follows: the excitation wavelength is 335nm, and the emission wavelength scanning range is 350-450 nm. The width of the slit is 1-10 nm.

The invention has the advantages that: by utilizing the optimized method, the collagen protein samples with different concentrations are used for making a standard curve under the test condition of the fluorescence spectrum, and a linear regression equation is calculated. Meanwhile, a sample to be detected is extracted and purified by the same method, and the concentration of the sample to be detected is obtained through a linear regression equation. The method has the advantages of accuracy, rapidness, sensitivity and good reproducibility, and meets the requirement of accurate quantification.

Drawings

FIG. 1 is a graph showing the fluorescence spectrum of collagen at an excitation wavelength of 253nm in example 1 of the present invention;

FIG. 2 is a graph showing a standard curve of a collagen solution in example 1 of the present invention;

FIG. 3 is a simultaneous fluorescence spectrum of collagen of example 2 according to the present invention at a wavelength difference of 30 nm;

FIG. 4 is a graph showing a standard curve of a collagen solution in example 2 of the present invention;

FIG. 5 is a fluorescence spectrum of collagen under an exogenous probe ANS in example 3 of the present invention;

FIG. 6 is a graph showing a standard curve of a collagen solution in example 3 of the present invention;

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.