阅读说明：本技术 一种拉曼显微镜测定雨生红球藻虾青素含量的方法 (Method for measuring astaxanthin content of haematococcus pluvialis by Raman microscope ) 是由 李珂 李清毅 李国能 郑友取 卜钟鸣 程军 于 2019-11-24 设计创作，主要内容包括：本发明涉及一种拉曼显微镜测定雨生红球藻虾青素含量的方法,其特征在于,包括下述测定步骤:a.取不同生长阶段的含有虾青素的红色雨生红球藻活体细胞,分为两组,一组取1mL离心得浓缩藻液用于拉曼光谱的测定,一组取50mL离心后冷冻干燥制备成雨生红球藻藻粉,用于高效液相色谱测定虾青素含量。本发明提供一种可以即时监测雨生红球藻活体细胞内部虾青素积累情况,所需样本量少,可以对单个细胞内的代谢物质含量进行检测,无需对样品进行破坏性预处理,可对活体细胞直接进行检测的拉曼显微镜测定雨生红球藻虾青素含量的方法。(The invention relates to a method for measuring astaxanthin content of haematococcus pluvialis by using a Raman microscope, which is characterized by comprising the following measuring steps of a, dividing red haematococcus pluvialis living cells containing astaxanthin at different growth stages into two groups, centrifuging 1mL of the red haematococcus pluvialis living cells to obtain concentrated algae liquid for measuring a Raman spectrum, centrifuging 50mL of the red haematococcus pluvialis living cells, and freeze-drying the centrifuged 50mL of the concentrated algae liquid to prepare haematococcus pluvialis algae powder for measuring the astaxanthin content by using a high performance liquid chromatography. The invention provides a method for measuring astaxanthin content of haematococcus pluvialis by using a Raman microscope, which can immediately monitor the astaxanthin accumulation condition in living cells of the haematococcus pluvialis, has small required sample amount, can detect the content of metabolic substances in single cells, does not need to perform destructive pretreatment on the sample, and can directly detect the living cells.)

1. A method for measuring astaxanthin content of haematococcus pluvialis by using a Raman microscope is characterized by comprising the following measuring steps of:

a. taking red haematococcus pluvialis living body cells containing astaxanthin at different growth stages, dividing the red haematococcus pluvialis living body cells into two groups, taking 1mL of the red haematococcus pluvialis living body cells for centrifugation to obtain concentrated algae liquid for Raman spectrum determination, taking 50mL of the red haematococcus pluvialis living body cells for centrifugation, and then carrying out freeze drying to prepare haematococcus pluvialis algae powder for high performance liquid chromatography to determine the astaxanthin content;

b. pretreatment of Raman spectrum samples: collecting red stage cells of haematococcus pluvialis, collecting Raman spectrum signals of living cells, fixing the cells by using low-temperature agar with the concentration of 4%, mixing concentrated algae liquid and low-temperature agar liquid to prepare a haematococcus pluvialis sample, dripping the haematococcus pluvialis sample on a glass slide, covering a cover glass, and placing the haematococcus pluvialis sample under a Raman microscope for observation after the low-temperature agar is cooled and solidified;

c. collecting Raman spectrum information: fixing the prepared haematococcus pluvialis sample on an objective table below an objective lens of a laser confocal Raman microscope, focusing a 532 nm laser beam on the surface of the haematococcus pluvialis single cell sample through a 20X objective lens, wherein the instantaneous laser intensity is 1mW, and the process is carried out at a constant temperature of 25 ℃ in the experimental process;

d. raman imaging extraction of spectral signals: drawing a grid with the step length of 0.6 mu m in haematococcus pluvialis cells to obtain points, and adopting a static scanning mode to 1525 cm^-1The center is 1250-1750 cm^-1Scanning within the range, wherein the exposure time is 5 s, and extracting the Raman spectrum of each sampling point;

e. preprocessing Raman spectrum data: firstly, baseline correction is carried out on original spectral data to eliminate the interference of a background value; then, processing the data after baseline correction by adopting a convolution smoothing method, and removing data noise caused by instruments, samples and human factors in the experimental process;

f. semi-quantitative astaxanthin content: in Raman spectrum of Haematococcus pluvialis cell, 1520 cm is taken^-1The characteristic peak is used for astaxanthinDiagnosis, the intensity of characteristic peak signal is in linear positive correlation with the content of substance, and can be used for quantifying astaxanthin content, and each sampling point is 1520 cm^-1The Raman intensity at the wave section is taken as an average value and represents the Raman intensity of astaxanthin in the whole haematococcus pluvialis cells;

g. astaxanthin content-raman intensity curve: determining astaxanthin content of haematococcus pluvialis at different growth stages by adopting a high performance liquid chromatography, corresponding to the Raman intensity one by one, drawing an astaxanthin content-Raman intensity curve by taking the Raman intensity as a horizontal coordinate and the astaxanthin content as a vertical coordinate, and calculating a fitting equation of the astaxanthin content to the Raman intensity;

h. determining the astaxanthin content by Raman spectroscopy: taking red stage cells of haematococcus pluvialis to be detected, collecting Raman spectrum signals of the cells, wherein the sample processing method is shown in the step (b); collecting Raman spectrum information as shown in step (c); the extraction of the single-point Raman spectrum is shown in step (d); preprocessing the Raman spectrum data as shown in step (e);

i. determination of astaxanthin content: taking the Raman data pretreated in the step (h) to be 1520 cm^-1Substituting the characteristic peak value into an astaxanthin content-Raman intensity curve, and calculating to obtain the astaxanthin content of the haematococcus pluvialis to be detected.

2. The method for measuring astaxanthin content of Haematococcus pluvialis by using Raman microscope as claimed in claim 1, wherein in step a, the step of centrifuging living cells of Haematococcus pluvialis to obtain concentrated algae solution comprises centrifuging microalgae biomass by using a high-speed refrigerated centrifuge at a rotation speed of 5000rpm-10000rpm/min for 5min-10 min.

3. The method for measuring astaxanthin content of Haematococcus pluvialis by using Raman microscope according to claim 1 or 2, wherein in the step (a), the step of freeze-drying living cells of Haematococcus pluvialis to obtain Haematococcus pluvialis powder comprises freezing the centrifuged algae paste in an ultra-low temperature refrigerator at-70 ℃ to-80 ℃ for 20h to 30h, and then placing the frozen algae paste in a freeze-drying machine for vacuum drying, wherein the freeze-drying machine is operated at-55 ℃ and the vacuum degree is 0.1 mbar.

4. The method for measuring astaxanthin content of Haematococcus pluvialis by Raman microscopy according to claim 1 or 2, wherein the low temperature agar of 4% concentration in step b is specifically prepared by the following method:

A. adding low-temperature agar powder into deionized water, heating to 40 ℃, and mixing according to the weight ratio of 1: 5, mixing the concentrated algae solution and the low-temperature agar solution, dripping the mixture onto a glass slide, covering the glass slide, and cooling and solidifying the low-temperature agar.

5. The method according to claim 1, wherein in step (c), the confocal laser raman microscope is a raney confocal laser raman instrument configured to:

1) confocal microscope, 5X, 20X, 50X, 100X, total luminous efficiency of raman spectrometer system is greater than 30%, 250 mm focal length, true confocal mode, under 100X objective, spatial resolution is better than 1 μm in transverse direction and better than 2 μm in longitudinal direction; the spectral repeatability is less than or equal to +/-0.2 cm^-1Three-point mechanical positioning of the instrument base plate; research grade come card microscope; 532 nm of semiconductor laser and 50 mW; the Renysha high-power semiconductor laser is 785 nm and larger than 250 mW; high spectral resolution is less than 0.65 cm^-1。

Technical Field

The invention relates to the technical field of nondestructive testing of cell bioactive substances, in particular to a method for measuring haematococcus pluvialis astaxanthin by using a Raman microscope.

Background

The content of astaxanthin in Haematococcus pluvialis is determined by solvent extraction, ultrasonic extraction, microwave extraction and supercritical CO₂Extracting by extraction method, and measuring by spectrophotometry, high performance gas chromatography and high performance liquid chromatography-mass spectrometry. The detection process is time-consuming and labor-consuming, and the requirement of rapid detection cannot be met. The extractant used in the solvent extraction process is generally toxic and is not healthy for the laboratory and operating personnel. High performance liquid chromatography and gas chromatography, while accurate and reliable, are complicated to operate and still require complicated pretreatment procedures. In addition, the above methods require extraction of a large amount of a target substance from a sample to measure it, are not suitable for measuring astaxanthin of a small amount of cultured Haematococcus pluvialis, or require destruction of cell structure, and cannot monitor the accumulation of astaxanthin in living cells of Haematococcus pluvialis in real time.

Disclosure of Invention

The invention mainly solves the technical problems that the existing methods in the prior art can be measured only by extracting a large amount of target substances from samples, are not suitable for measuring a small amount of cultured haematococcus pluvialis astaxanthin, or can not immediately monitor the accumulation of astaxanthin in living haematococcus pluvialis cells by destroying cell structures, and the like, and provides the method for measuring the astaxanthin content of the haematococcus pluvialis in the living cells by using the Raman microscope, which can immediately monitor the accumulation condition of the astaxanthin in the living cells of the haematococcus pluvialis, has small required sample amount, can detect the content of metabolic substances in single cells, does not need to destructively pre-treat the samples, and can directly detect the living cells.

In order to solve the above-mentioned problems and achieve the above-mentioned object, the present invention provides a method for measuring astaxanthin content in Haematococcus pluvialis by Raman microscopy, comprising the steps of:

a. taking red haematococcus pluvialis living body cells containing astaxanthin at different growth stages, dividing the red haematococcus pluvialis living body cells into two groups, taking 1mL of the red haematococcus pluvialis living body cells for centrifugation to obtain concentrated algae liquid for Raman spectrum determination, taking 50mL of the red haematococcus pluvialis living body cells for centrifugation, and then carrying out freeze drying to prepare haematococcus pluvialis algae powder for high performance liquid chromatography to determine the astaxanthin content;

b. pretreatment of Raman spectrum samples: collecting red stage cells of haematococcus pluvialis, collecting Raman spectrum signals of living cells, fixing the cells by using low-temperature agar with the concentration of 4%, mixing concentrated algae liquid and low-temperature agar liquid to prepare a haematococcus pluvialis sample, dripping the haematococcus pluvialis sample on a glass slide, covering a cover glass, and placing the haematococcus pluvialis sample under a Raman microscope for observation after the low-temperature agar is cooled and solidified;

c. collecting Raman spectrum information: fixing the prepared haematococcus pluvialis sample on an objective table below an objective lens of a laser confocal Raman microscope, focusing a 532 nm laser beam on the surface of the haematococcus pluvialis single cell sample through a 20X objective lens, wherein the instantaneous laser intensity is 1mW, and the process is carried out at a constant temperature of 25 ℃ in the experimental process;

d. raman imaging extraction of spectral signals: drawing a grid with the step length of 0.6 mu m in haematococcus pluvialis cells to obtain points, and adopting a static scanning mode to 1525 cm^-1The center is 1250-1750 cm^-1Scanning within the range, wherein the exposure time is 5 s, and extracting the Raman spectrum of each sampling point;

e. preprocessing Raman spectrum data: firstly, baseline correction is carried out on original spectral data to eliminate the interference of a background value; then, processing the data after baseline correction by adopting a convolution smoothing method, and removing data noise caused by instruments, samples and human factors in the experimental process;

f. semi-quantitative astaxanthin content: taking 1525 cm in Raman spectrum of haematococcus pluvialis cell^-1The characteristic peak is used for diagnosing astaxanthin, the intensity of the characteristic peak signal is in linear positive correlation with the content of the substance, the method can be used for quantifying the content of astaxanthin, and each sampling point is 1525 cm^-1The Raman intensity at the wave section is taken as an average value and represents the Raman intensity of astaxanthin in the whole haematococcus pluvialis cells;

g. astaxanthin content-raman intensity curve: determining astaxanthin content of haematococcus pluvialis at different growth stages by adopting a high performance liquid chromatography, corresponding to the Raman intensity one by one, drawing an astaxanthin content-Raman intensity curve by taking the Raman intensity as a horizontal coordinate and the astaxanthin content as a vertical coordinate, and calculating a fitting equation of the astaxanthin content to the Raman intensity;

h. determining the astaxanthin content by Raman spectroscopy: taking red stage cells of haematococcus pluvialis to be detected, collecting Raman spectrum signals of the cells, wherein the sample processing method is shown in the step (b); collecting Raman spectrum information as shown in step (c); raman imaging extracting spectral signals as shown in step (d); preprocessing the Raman spectrum data as shown in step (e);

i. determination of astaxanthin content: and (h) substituting the Raman data processed in the step (h) into an astaxanthin content-Raman intensity curve, and calculating to obtain the astaxanthin content of the haematococcus pluvialis to be detected.

In the step a, the step of centrifuging living cells of the haematococcus pluvialis to obtain concentrated algae solution comprises the step of centrifuging the living cells of the haematococcus pluvialis to obtain microalgae biomass by adopting a high-speed refrigerated centrifuge, wherein the rotation speed is 5000-10000 rpm/min, and the centrifugation is carried out for 5-10 min.

In the step (a), the haematococcus pluvialis living body cells are frozen and dried to obtain haematococcus pluvialis powder, the centrifuged algae mud is frozen in an ultra-low temperature refrigerator at-70 ℃ to-80 ℃ for 20h to 30h, and then is placed in a freeze dryer for vacuum drying, wherein the operating temperature of the freeze dryer is-55 ℃, and the vacuum degree is 0.1 mbar.

As the method for measuring the astaxanthin content of haematococcus pluvialis by using a manmicroscope, the method for preparing the low-temperature agar with the concentration of 4% in the step b specifically comprises the following steps:

A. adding low-temperature agar powder into deionized water, heating to 40 ℃, and mixing according to the weight ratio of 1: 5, mixing the concentrated algae solution and the low-temperature agar solution, dripping the mixture onto a glass slide, covering a cover glass, and cooling and solidifying the agar.

As the method for measuring the astaxanthin content of haematococcus pluvialis by using the Raman microscope, in the step (3), the laser confocal Raman microscope is an instrument configuration of a Ranishao confocal laser microscopic Raman instrument:

1) confocal microscope, 5X, 20X, 50X, 100X, total light passing efficiency of Raman spectrometer system is more than 30%, 250 mm focal length, true confocal mode, under 100X objective, spatial resolution is better than 1 μm in horizontal direction and better than 2 μm in longitudinal direction; the spectral repeatability is less than or equal to +/-0.2 cm < -1 >, and the instrument bottom plate is mechanically positioned at three points; research grade come card microscope; 532 nm of semiconductor laser and 50 mW; the Renysha high-power semiconductor laser is 785 nm and larger than 250 mW; the high spectral resolution is less than 0.65 cm < -1 >.

The method for measuring the astaxanthin content of the haematococcus pluvialis by the Raman microscope has the advantages that the astaxanthin accumulation condition in living cells of the haematococcus pluvialis can be monitored in real time, the required sample amount is small, the content of metabolic substances in single cells can be detected, destructive pretreatment on samples is not needed, and the living cells can be directly detected.

Therefore, the method has the characteristics of simple sample pretreatment process, simple instrument operation process, short determination time and the like.

Drawings

FIG. 1 is a schematic flow diagram of the present invention.

Detailed Description

The technical scheme of the invention is further specifically described by the following embodiments and the accompanying drawings.