Method of improving blue algae in-situ detection precision by using anti-fluorescent interference decoupling algorithm

A fluorescence intensity, cyanobacteria technology, applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve problems such as labor-intensive, phycocyanin concentration results are greatly interfered by chlorophyll, and sample processing requirements are complicated and cumbersome.

Active Publication Date: 2017-07-21
威海慧视生物科技有限公司
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Problems solved by technology

However, such methods do not carry out strict screening for specific wavelengths, and the detected phycocyanin concentration results are greatly interfered by chlorophyll: through the fluorescence characteristics of the three pigments of chlorophyll a, chlorophyll b and phycocyanin, namely figure 2 , it can be seen that in the process of detecting phycocyanin by fluorescence method, the fluorescence intensity of phycocyanin will be affected by chlorophyll a and chlorophyll b
In some cases, the content of both pigments in the water body is very large, resulting in a large error in the detection of phycocyanin, and it is impossible to accurately monitor the change trend of cyanobacterial biomass
Therefore, the detection results cannot reflect the real change trend of cyanobacterial biomass
However, flow cytometry or high performance liquid chromatography requires complex and cumbersome sample processing, and the instrument requires certain operating techniques.
More importantly, it cannot meet the requirements of real-time and portable field detection; the microscope counting method not only consumes a lot of manpower, but also has certain technical requirements, and also cannot meet the requirements of real-time and portable detection.

Method used

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  • Method of improving blue algae in-situ detection precision by using anti-fluorescent interference decoupling algorithm
  • Method of improving blue algae in-situ detection precision by using anti-fluorescent interference decoupling algorithm
  • Method of improving blue algae in-situ detection precision by using anti-fluorescent interference decoupling algorithm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Embodiment 1, the fluorescence characteristic analysis of three kinds of pigments

[0154] Take the mother liquor of chlorophyll a, chlorophyll b and phycocyanin, and prepare them as 2mg / L, 1mg / L, 0.5mg / L, 0.25mg / L, 0.125mg / L, 0.075mg / L solutions respectively, and choose 15 mg / L as the dilution solvent % PBS ethanol solution.

[0155] Take 200 μL of the solution in a 96-well plate, adjust the relevant parameters of the fluorescence spectrophotometer, and scan the excitation spectrum and emission spectrum. Then, according to the spectral values ​​of different concentration gradients, the series average value of the fluorescence spectrum of the unit substance is taken, and the obtained results are shown in figure 2 .

[0156] The fluorescence characteristics of the three pigments can be obtained from the excitation and emission spectra of the three pigments:

[0157] Chlorophyll a: The excitation peak is 434nm, and the emission peak is 679nm;

[0158] Chlorophyll b: ...

Embodiment 2

[0161] Example 2, wavelength screening of three kinds of pigment coupling detection and analysis by fluorescence method

[0162] According to Example 1, it can be seen that there is obvious interference between the detection of the three pigments of chlorophyll a, chlorophyll b and phycocyanin. The purpose of this example is to screen different wavelengths of excitation light and emission light to make each other The degree of interference between them is minimal. The operation flow chart of the wavelength screening method is as follows: Figure 4 shown. For a flow chart of the data processing procedure, see Figure 5 .

[0163] The first step is to establish a fluorescence characteristic database for each pigment, see Table 1-Table 3.

[0164] Table 1 Database of fluorescence characteristics of chlorophyll a under different excitation and emission wavelengths

[0165]

[0166] Table 2 Database of fluorescence properties of phycocyanin under different excitation and em...

Embodiment 3

[0190] Example 3. Verification experiment of wavelength screening to realize the separation of three kinds of pigment interference

[0191] According to the establishment method of the detection model proposed by the present invention, the excitation light wavelength and emission light wavelength of the spectrophotometer are respectively adjusted to the wavelength combinations obtained in Example 2 (Table 2). Dilute the three pigments respectively: 2mg / L, 1mg / L, 0.5mg / L, 0.25mg / L, 0.125mg / L, 0.075mg / L, 0.0375mg / L, 0.01875mg / L eight gradient concentrations , to detect the fluorescence intensity under the respective fluorescence conditions. Fit the linear relationship of 8 concentration gradients according to the least square method, and calculate the k under the specific wavelength combination of the three pigments ij with b i . The overall model obtained for the detection of the three pigments is:

[0192]

[0193]

[0194]

[0195] The trimmed result is:

[0196...

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Abstract

The invention discloses a method of improving blue algae in-situ detection precision by using an anti-fluorescent interference decoupling algorithm. The method depends on a fluorescent analysis method, and measurement can be achieved by taking phycocyanin as a major pigment index for detecting blue algae biomass, and using a fluorescent spectrophotometer that is relatively simple to operate in a laboratory. The method can effectively avoid mutual influence of chlorophyll a, chlorophyll b and the phycocyanin; the blue algae biomass in fresh water is monitored accurately; relatively precise alga biomass data is provided for early warning of blue algae water bloom; a theoretical basis is provided for subsequent design of a blue algae biomass detection sensor; the method can be also used for avoiding interference among multiple pigments to the greatest extent when the fluorescent pigments are detected by a fluorescent method; a theoretical basis is provided for scientific and reasonable screening excitation light wavelength and emitting light wavelength in fluorescent analysis; a multivariant correcting feedback piecewise linear detection model is established when an algal pigment concentration in a water body is detected by the fluorescent method; and precise detection of the pigment concentration is achieved.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for improving the in-situ detection accuracy of cyanobacteria by using an anti-fluorescence interference decoupling algorithm. Background technique [0002] Fresh water is a resource that human beings depend on for survival, but algae bloom has become one of the three major aquatic ecological environmental problems facing the world, seriously threatening the sustainable use of human water resources. Related studies have shown that about 81% of algae blooms are caused by cyanobacteria. my country is one of the countries with the most severe cyanobacterial blooms in the world, the most widely distributed and the most types of cyanobacteria. Taihu Lake, Dianchi Lake, Dongting Lake, Chaohu Lake and other inland lakes are the high-frequency areas of cyanobacteria blooms in my country. [0003] The outbreak of algae blooms has four major hazards to humans: first, a large nu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/64
Inventor 蔡中华类成龙周进蔡子文王嵘何永红姜玥璐
Owner 威海慧视生物科技有限公司
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