CN114563362B - A kind of detection method of microalgae content in ship ballast water - Google Patents

Abstract

The invention belongs to the technical field of microalgae content detection of ship ballast water, and particularly relates to an up-conversion nano fluorescent probe which can realize rapid detection of microalgae content through competitive emission. According to the method, a red up-conversion nano fluorescent probe is adopted, under the excitation of near infrared light, the red luminescence of the fluorescent probe is overlapped with the maximum absorption peak height of the chlorophyll a of the ballast water microalgae, a relation curve of the luminescence intensity and the content of the chlorophyll a is established through competitive luminescence measurement, and a relation model of the luminescence intensity and the biomass of the microalgae cells is established according to the corresponding relation of the content of the chlorophyll a and the biomass of the microalgae cells, so that the biomass of the microalgae in a ballast water sample is calculated. The method can realize the rapid detection of the microalgae biomass, has simple and convenient operation and high sensitivity, reduces the photodamage to organisms by adopting safe long-wavelength near-infrared light as an excitation source, does not cause the spontaneous background fluorescence of the microalgae, can improve the detection sensitivity by orders of magnitude, and is suitable for popularization and application.

Description

A detection method for microalgae content in ship's ballast water

technical field

The invention relates to the technical field of detection of microalgae content in ship's ballast water, in particular to the rapid detection of microalgae content by using up-conversion fluorescent probes and competitive emission.

Background technique

With the increasingly globalized economic development, shipping currently undertakes more than 80% of global cargo transportation. In order to control the ship's heel, trim, draft, stability or stress, and ensure the stability and maneuverability of the ship during navigation, it is necessary to load ballast water on the ship. However, the ship’s ballast water carries a large number of aquatic organisms, such as phytoplankton, zooplankton, bacteria, viruses, etc. These alien aquatic organisms will invade the local marine ecosystem with the absorption and discharge of ballast water, causing alien organisms to invade, Red tides and other serious disasters, therefore, are listed as one of the four major hazards to the ocean by the International Maritime Organization (IMO). In particular, marine microalgae, this type of phytoplankton will cause catastrophic damage to the ecosystem in new sea areas due to their strong reproduction and vitality and lack of natural enemies. The invasion of microalgae has caused huge economic and environmental losses worldwide. [Design of rapid detection system for microalgae in ship's ballast water, instrument technology and sensor]. However, there is still a lack of rapid detection methods for microalgal cells in ship's ballast water.

At present, the main detection methods: 1) microscopic counting method, by observing the microalgae cells under a microscope, distinguishing and counting by the size, shape and color of the cells, inferring, but this method requires professionals to detect, manual counting errors 2) Flow cytometry method, using flow cytometry to measure the suspension containing microalgae passing through the laser irradiation area, and measuring the light intensity of the microalgae cells labeled with the dye Signal, to determine the content of microalgae, this method has the advantages of accuracy and high efficiency, but commercial flow cytometers are expensive, bulky, and sample pretreatment is cumbersome, and it is difficult to achieve rapid detection of ballast water tanks; 3) Chlorophyll fluorescence Because chlorophyll a widely exists in all algae, the concentration of chlorophyll a has become the most commonly used indicator for determining the biomass of microalgae. The methods for measuring chlorophyll a concentration mainly include fluorescence spectrophotometry (direct fluorescence spectrophotometry for rapid determination of chlorophyll a in water, Journal of Wuhan University of Technology, 2011, 33, 112-115) and absorption spectrophotometry (a fast and accurate microscopic Algae biomass estimation method, Acta Physiology of Plants, 2021, 57, 216-224): Fluorescence spectrophotometry uses the characteristic of chlorophyll a in marine microalgae to emit red light (680nm) under the excitation of blue light (420nm), and establishes the characteristics of chlorophyll a The correlation between the content and the luminous intensity is used to calibrate the algae biomass, but this method has low sensitivity. Due to the weak fluorescence of chlorophyll, it is not suitable for the content test of low-concentration chlorophyll a. Absorption spectrophotometry uses algae chlorophyll a There are absorption characteristics at ≈649 and ≈665nm wavelengths, and the chlorophyll a content of the sample is calculated by the absorbance value. Further, the patent CN 111896482 A reports the establishment of a model of the color parameters and chlorophyll content of the Chlorella sample, so as to calculate the chlorophyll a content.

Contents of the invention

According to the technical problems set forth above, the object of the present invention is to provide a method for detecting the content of microalgae in ballast water. Utilize the occurrence of up-conversion nano-fluorescence probe at 665nm in the red light region, highly overlap with the maximum absorption peak of chlorophyll a of microalgae to form competitive emission, and realize the detection of microalgae content in ballast water by detecting the luminescence spectrum, so as to quickly judge Whether the living biomass in the ballast water meets the discharge requirements.

In order to achieve the above object, the present invention is achieved through the following technical solutions:

A method for detecting microalgae content in ship's ballast water, comprising the following steps:

Step 1): Take a ballast water sample, filter and centrifuge to obtain a microalgae precipitate, dissolve the microalgae precipitate in a solvent, ultrasonicate and stand still to obtain a chlorophyll solution A, and measure the absorbance value of the chlorophyll solution A, Calculate the content of chlorophyll a;

Step 2): adding the up-conversion nano-fluorescent probe into the same solvent as the chlorophyll solution A for dispersion to form a probe solution B;

Step 3): The chlorophyll solution A of step 1) is diluted with a solvent to prepare chlorophyll solutions with different concentrations, and the chlorophyll solutions of different concentrations are mixed with the probe solution B respectively, and the luminescent spectrum of the mixed solution is measured, and the chlorophyll solution in the chlorophyll solution The absorption of a in the red light region and the emission of the probe solution B in the red light region produce competitive emission, thereby establishing a relationship curve between the content of chlorophyll a and the red luminous intensity;

Step 4): According to the corresponding relationship curve between the content of chlorophyll a and the biomass of microalgae cells, the model of the relationship between the biomass of microalgae cells in the ballast water and the red luminous intensity is obtained.

In the above technical solution, further, in the step 1), measure the absorbance value of the chlorophyll solution A at 649nm and 665nm wavelength, and calculate the chlorophyll a content by the following formula:

C _a =a×A ₆₆₅ -b×A ₆₄₉

In the formula, A ₆₆₅ is the light absorption value of the solution at 665nm, A ₆₄₉ is the absorbance value of the solution at 649nm, a and b are coefficients, and C _a is the content of chlorophyll a.

In the above technical solution, further, the solvent includes dimethyl sulfoxide, acetone, dimethylformamide or chloroform.

In the above technical solution, further, in the step 2), in the probe solution B, the concentration of the up-conversion nano fluorescent probe is 0.05-0.5 mol/L.

In the above technical solution, further, in the step 2), the up-conversion nano fluorescent probe uses near-infrared light with a wavelength of 980 or 1550 nm as the excitation source, and emits red light in the wavelength region of 640-670 nm.

In the above technical solution, further, the up-conversion nano fluorescent probe in the step 2) is based on fluoride or sulfur oxide, the rare earth ion is the luminescent center, and the nanoparticle size is 50-150nm.

In the above technical scheme, further, the up-conversion nanoprobe in the step 2) is prepared with fluoride, oxide, sulfur oxide, molybdate, vanadate, niobate, phosphate, tungstate, Titanate, silicate or tellurate as the matrix, with rare earth ion RE as the luminescent center; the RE is one of Ce, Pr, Nd, Sm, Eu, Tb, Dy, Ho, Er, Tm or Yb species or two or more.

In the above technical solution, further, in the step 4), the same ballast water sample is taken, the number of microalgae in the ballast water is counted by microscopic counting method, and the chlorophyll a content is calculated by the method described in step 1), Establish the corresponding relationship curve between chlorophyll content and microalgae cell biomass.

In the above technical solution, further, the microalgae include Chlorella, Dinoflagellate, Phytophthora, Salina, and Nitzschia.

The beneficial effects of the present invention are:

The method for detecting the content of microalgae in ballast water provided by the present invention is simple and convenient to operate and has high sensitivity. It uses safe long-wavelength near-infrared light (NIR) as an excitation source to reduce photodamage to organisms and will not cause spontaneous microalgae growth. Background fluorescence can increase detection sensitivity by orders of magnitude. At the same time, the inorganic up-conversion luminescence probe is selected, which has good chemical and photostability, no light flicker, and no photobleaching phenomenon when the excitation light is continuously irradiated, which is suitable for the detection of corrosive ballast water environment.

Description of drawings

Fig. 1 is the up-conversion emission spectrum of the up-conversion nano-fluorescent probe of Example 1 of the present invention under 980nm excitation;

Fig. 2 is the red light emission spectrum of the mixed solution of the chlorophyll solution and the up-conversion nano fluorescent probe in Example 1 of the present invention;

Fig. 3 is the relationship curve between the red light luminescence intensity of the up-conversion nano-fluorescence probe of Example 1 of the present invention and the biomass of chlorophyll a;

Fig. 4 is the relational curve of the chlorophyll a content and the biomass of microalgae of the embodiment of the present invention 1;

Fig. 5 is a relationship curve between the red light intensity of the up-conversion nano-fluorescence probe and the biomass of microalgae in ballast water according to Example 1 of the present invention.

Detailed ways

The following examples are used to describe the technical solution of the present invention clearly and in detail, but the examples do not limit the present invention in any form.

Example 1

Step 1): Take 2L of ballast water sample, filter and centrifuge to obtain 45.8mg of microalgae precipitate, add 1ml of dimethyl sulfoxide solvent to the microalgae precipitate, first carry out ultrasonic dispersion, then perform centrifugation and stand still, The obtained supernatant is chlorophyll solution A; measure the light absorption value of chlorophyll solution A at 649nm and 665nm, and calculate the content of chlorophyll a in microalgae solution A by the following formula;

C _a =a×A ₆₆₅ -b×A ₆₄₉

In the formula, C _a is the content of chlorophyll a, A ₆₆₅ is the absorbance value of the solution at 665nm, A ₆₄₉ is the absorbance value of the solution at 649nm, a is 12.47, b is 3.62,

The calculated content of chlorophyll a in the chlorophyll solution A is 20.22 mg/L;

Step 2): Add 0.025mol NaYF ₄ : Yb, Er up-conversion nano fluorescent probe (average particle size is about 50nm) into 0.5ml dimethyl sulfoxide solvent for dispersion, so that the concentration of up-conversion nano fluorescent probe is 0.05 mmol/L, forming probe solution B, the up-conversion emission spectrum of the up-conversion nano fluorescent probe under 980nm excitation is shown in Figure 1;

Step 3): Dilute chlorophyll solution A to obtain chlorophyll solutions with different concentrations (chlorophyll a content: 0.397-6.010mg/L), and then mix them with probe solution B respectively, and obtain The emission wavelength region is located at the red luminous intensity of 640-670nm, as shown in Figure 2; due to the absorption of chlorophyll a at 665nm and NaYF ₄ in the chlorophyll solution: Yb, the emission of Er up-conversion nano fluorescent probe solution B at 665nm overlaps , produce competitive emission, and establish a relationship curve between red luminous intensity and chlorophyll a content, as shown in Figure 3;

Step 4): Count the number of microalgae in the ballast water sample by microscopic counting method, take out 50 μl of the water sample, drop it into a 25×16 type hemocytometer for counting, use a 40× objective lens during the counting process, and select the upper left and lower left during the counting process , the upper right, the lower right, and the five small squares in the middle to count the number of microalgae one by one. Samples were counted 10 times, and the average value was taken to establish a relationship curve between chlorophyll a content and microalgae biomass, as shown in Figure 4.

Step 5): through the relational curve of chlorophyll a content and microalgae biomass, thereby further obtain the relational model of the red light intensity of up-conversion nano fluorescent probe and microalgae biomass, as shown in Figure 5, according to this relational model, The microalgae content of the ballast water sample solution can be calculated by measuring the intensity of the red light.

Take 3 different ballast water samples, and measure the red light luminous intensity of the ballast water microalgae solution and the up-conversion nano-fluorescent probe mixed solution according to the above steps, and bring it into the above relationship curve model to calculate the ballast water sample The biomass of microalgae (calculated value) is compared with the biomass of microalgae measured by traditional microscopic counting method (experimental value), as shown in the table below.

Table 1

Example 2

Step 1): Take 1L of ballast water sample, filter and centrifuge to obtain 25.8mg of microalgae precipitate, add 2ml of acetone solvent to the microalgae precipitate for ultrasonic dispersion, and then perform centrifugation to obtain the supernatant which is chlorophyll Solution A; measure the light absorption value of chlorophyll solution A at 649nm and 665nm, and calculate the content of chlorophyll a in solution A by the following formula:

C _a =a×A ₆₆₅ -b×A ₆₄₉

Here coefficient a=12.21, b=2.59, calculate the content of chlorophyll a in solution A to be 12.32mg/L;

Step 2): Add 0.25mol Y ₂ O ₂ S:Tm,Er up-conversion nano-fluorescence probe (average particle size is about 150nm) into 0.5ml acetone solvent for dispersion, so that the concentration of up-conversion nano-fluorescence probe is 0.5mmol /L to form probe solution B;

Step 3): Dilute chlorophyll solution A to obtain chlorophyll solutions with different concentrations (chlorophyll a content: 0.35-5.23mg/L), and then mix them with probe solution B respectively, and obtain The red luminous intensity in the emission wavelength region of 640-670nm is due to the absorption of chlorophyll a at 665nm in microalgae sample solution A and the emission of Y ₂ O ₂ S:Tm, Er up-conversion nano-fluorescence probe solution B at 665nm, resulting in Competing for emission, establishing a relationship curve between red luminous intensity and chlorophyll a content;

Step 4): according to the corresponding relationship curve of chlorophyll a content and microalgae cell biomass, thus further obtain, the relational model of the red light intensity parameter of up-conversion nano fluorescent probe and microalgae biomass, according to this relational model, can pass Measure the red light intensity to calculate the microalgae content of the ballast water microalgae solution.

Take one type of ballast water sample, measure the red light luminous intensity of the mixed solution of ballast water microalgae solution and up-conversion nano-fluorescent probe according to the above steps respectively, bring it into the above relationship curve model, and calculate the microalgae intensity of the ballast water sample. The algal biomass was 1.302×10 ³ /L.

Claims (7)

1. A method for detecting the microalgae content in ship ballast water is characterized by comprising the following steps:

step 1: taking a pressurized water sample, filtering and centrifuging to obtain microalgae precipitate, dissolving the microalgae precipitate in a solvent, performing ultrasonic treatment and standing treatment to obtain a chlorophyll solution A, measuring the absorbance value of the solution A, and calculating the content of chlorophyll a in the solution A;

and 2, step: adding the up-conversion nano fluorescent probe into the same solvent as the chlorophyll solution A in the step 1 for dispersion to form a probe solution B;

and 3, step 3: diluting the chlorophyll solution A obtained in the step (1) by a solvent to prepare chlorophyll solutions with different concentrations, mixing the chlorophyll solutions with different concentrations with the probe solution B respectively, measuring the luminescence spectrum of the mixed solution, and generating competitive emission through the absorption of chlorophyll a in a red light area in the chlorophyll solution and the emission of the probe solution B in the red light area so as to establish a relation curve between the content of chlorophyll a and the red luminescence intensity;

and 4, step 4: obtaining a relation model between the microalgae cell biomass and the red luminous intensity in the ballast water according to a corresponding relation curve between the chlorophyll a content and the microalgae cell biomass;

the up-conversion nano fluorescent probe is NaYF ₄ Yb, er or Y ₂ O ₂ S is Tm, er; the wavelength of the up-conversion nano fluorescent probe is 980 or1550 The nm near infrared light is used as an excitation source, and the emission wavelength region is red light with the wavelength of 640-670 nm.

2. The detection method according to claim 1, wherein in the step 1, the absorbance values of the chlorophyll solution at the wavelengths of 649nm and 665nm are measured, and the chlorophyll-a content is calculated by the following formula:

in the formula, A ₆₆₅ Absorption value of the solution at 665nm, A ₆₄₉ Absorbance value of the solution at 649nm, a and b are coefficients, C _a I.e. the chlorophyll a content.

3. The detection method according to claim 1, wherein the solvent comprises dimethyl sulfoxide, acetone, dimethylformamide or chloroform.

4. The detection method according to claim 1, wherein in the step 2, the concentration of the up-conversion nano fluorescent probe in the probe solution B is 0.05-0.5mol/L.

5. The detection method according to claim 1, wherein the particle size of the upconversion nanofluorescent probe in the step 2 is 50-150 nm.

6. The detection method according to claim 1, wherein in the step 4, the same water sample of the ballast water is taken, the number of microalgae in the ballast water is counted by using a microscope counting method, the content of chlorophyll a is calculated by using the method in the step 1, and a corresponding relation curve between the content of chlorophyll and the biomass of microalgae cells is established.

7. The method of claim 1, wherein the microalgae comprises chlorella, dinoflagellate, tetraselmis, dunaliella salina, or nitzschia closterium.

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