CN114563362B - Method for detecting microalgae content in ship ballast water - Google Patents
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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
Technical Field
The invention relates to the technical field of microalgae content detection in ship ballast water, in particular to a method for realizing rapid detection of microalgae content by adopting an up-conversion fluorescent probe and through competitive emission.
Background
With the increasing globalization of economic development, ships are currently responsible for over 80% of the global freight transportation. In order to control the heeling, trim, draft, stability or stress of a ship and ensure the stability and maneuverability of the ship during navigation, ballast water needs to be loaded on the ship. However, the ballast water of ships carries a large amount of aquatic organisms such as phytoplankton, zooplankton, bacteria, viruses, etc., and the foreign aquatic organisms invade the local marine ecosystem along with the intake and discharge of the ballast water, causing serious disasters such as invasion of foreign organisms, red tide, etc., and thus, are classified as one of four major hazards to the sea by the International Maritime Organization (IMO). In particular, marine microalgae, which are extremely strong in reproduction and vitality and lack of natural enemies, can cause catastrophic damage to the ecosystem of new sea areas, and the invasion of microalgae causes huge economic and environmental losses worldwide [ design of rapid detection systems for microalgae in ballast water of ships, instruments and sensors ]. However, a rapid detection method for marine ballast water microalgae cells is still lacking at present.
The main detection method at present comprises the following steps: 1) The microscopic counting method is used for observing microalgae cells under a microscope, distinguishing and counting the microalgae cells according to the size, the shape and the color of the microalgae cells and deducing the microalgae cells, but the method needs a professional person for detection, has large manual counting error and long counting time and is difficult to realize rapid detection; 2) The flow cytometry method comprises the steps of measuring a suspension containing microalgae passing through a laser irradiation area by using a flow cytometer, and judging the content of the microalgae by measuring optical signals of dye-labeled microalgae cells, wherein the method has the advantages of accuracy and high efficiency, but the commercial flow cytometer has the disadvantages of high price, large volume and complex sample pretreatment, and is difficult to realize the rapid detection of a ballast water ship base; 3) Chlorophyll a concentration is the most common indicator for measuring microalgae biomass by chlorophyll fluorescence method, because chlorophyll a is widely present in all algae. The method for measuring the concentration of chlorophyll a mainly comprises a fluorescence spectrophotometry (direct fluorescence spectrophotometry for rapidly measuring chlorophyll a in water, university of wuhan theory, 2011, 33, 112-115) and an absorption spectrophotometry (a rapid and accurate microalgae biomass estimation method, plant physiology report, 2021, 57, 216-224): the fluorescence spectrophotometry is characterized in that the chlorophyll a in marine microalgae emits red light (680 nm) under the excitation of blue light (420 nm), and the correlation between the chlorophyll a content and the luminous intensity is established, so that the algae biomass is calibrated, but the method has low sensitivity, and is not suitable for the content test of low-concentration chlorophyll a due to weak fluorescence of chlorophyll; an absorption spectrophotometry method utilizes the characteristic that algae chlorophyll a absorbs at the wavelength of about 649 and about 665nm to calculate the chlorophyll a content of a sample through an absorbance value, and further, a patent CN 111896482A reports a model for establishing color parameters and the chlorophyll content of a chlorella sample so as to calculate the chlorophyll a content.
Disclosure of Invention
In light of the above-mentioned technical problems, an object of the present invention is to provide a method for detecting microalgae content in ballast water. The generation of the up-conversion nano fluorescent probe in a red light region of 665nm is overlapped with the maximum absorption peak height of chlorophyll a of the microalgae to form competitive emission, and the detection of the content of the microalgae in the ballast water is realized by detecting a luminescence spectrum, so that whether the living biomass in the ballast water meets the discharge requirement or not is quickly judged.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a method for detecting the microalgae content in ship ballast water comprises 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 and standing treatment to obtain a chlorophyll solution A, measuring the absorbance value of the chlorophyll solution A, and calculating the content of chlorophyll a;
step 2): adding the upconversion nanometer fluorescent probe into the solvent which is the same as the chlorophyll solution A for dispersion to form a probe solution B;
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 a 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, thereby establishing a relation curve between the content of the chlorophyll a and the red luminous intensity;
step 4): and obtaining a relation model between the microalgae cell biomass and the red luminous intensity in the ballast water according to the corresponding relation curve of the chlorophyll a content and the microalgae cell biomass.
In the above technical solution, further, in the step 1), the absorbance values of the chlorophyll solution a at wavelengths of 649nm and 665nm are measured, and the chlorophyll a content is calculated by the following formula:
C a ＝a×A 665 -b×A 649
in the formula, A 665 Absorption value of the solution at 665nm, A 649 The absorbance value of the solution at 649nm, a and b are coefficients, C a I.e. the chlorophyll a content.
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), the concentration of the upconversion nanometer fluorescent probe in the probe solution B is 0.05-0.5mol/L.
In the above technical solution, further, in the step 2), the upconversion nanometer fluorescent probe emits red light with a wavelength region of 640-670nm by using near-infrared light with a wavelength of 980nm or 1550nm as an excitation source.
In the above technical solution, further, the upconversion nanometer fluorescent probe in step 2) uses fluoride or oxysulfide as a matrix, rare earth ions as a luminescence center, and the size of the nanoparticle is 50 to 150nm.
In the above technical solution, further, the upconversion nanoprobe in step 2) takes fluoride, oxide, oxysulfide, molybdate, vanadate, niobate, phosphate, tungstate, titanate, silicate or tellurate as a substrate, and takes rare earth ion RE as a luminescence center; the RE is one or more of Ce, pr, nd, sm, eu, tb, dy, ho, er, tm or Yb.
In the above technical scheme, further, in the step 4), the same water sample of ballast water is taken, the number of microalgae in the ballast water is counted by using a microscopic counting method, and meanwhile, the chlorophyll a content is calculated by using the method in the step 1), and a corresponding relation curve between the chlorophyll content and the biomass of microalgae cells is established.
In the above technical solution, the microalgae further includes chlorella, dinoflagellate, tetraselmis, dunaliella salina, and nitzschia closterium.
The beneficial effects of the invention are as follows:
the method for detecting the content of the microalgae in the ballast water is simple and convenient to operate and high in sensitivity, adopts safe long-wavelength near infrared light (NIR) as an excitation source, reduces photodamage to organisms, does not cause spontaneous background fluorescence of the microalgae, and can improve the detection sensitivity in an order of magnitude. Meanwhile, an inorganic up-conversion luminescence probe is selected, so that the chemical and light stability is good, no light flicker exists, the photobleaching phenomenon does not exist even if the exciting light is continuously irradiated, and the method is suitable for detecting the corrosive ballast water environment.
Drawings
FIG. 1 is an upconversion emission spectrum of an upconversion nanofluorescent probe according to example 1 of the present invention under excitation at 980 nm;
FIG. 2 is a red light emission spectrum of a mixed solution of chlorophyll solution and upconversion nanofluorescent probe according to example 1 of the present invention;
FIG. 3 is a graph of the relationship between the red light intensity and chlorophyll a biomass of the upconversion nanofluorescent probe in example 1 of the present invention;
FIG. 4 is a plot of chlorophyll a content versus microalgae biomass for example 1 of the present invention;
FIG. 5 is a graph of the relationship between the red light intensity and the ballast water microalgae biomass of the upconversion nano fluorescent probe of example 1 of the present invention.
Detailed Description
The following examples are provided to clearly and specifically describe the technical solutions of the present invention, but the present invention is not limited in any way by the examples.
Example 1
Step 1): taking 2L of a pressurized water sample, filtering, performing centrifugal separation to obtain 45.8mg of microalgae precipitate, adding 1ml of dimethyl sulfoxide solvent into the microalgae precipitate, performing ultrasonic dispersion, performing centrifugal separation, and standing to obtain a supernatant which is a chlorophyll solution A; measuring the absorption light values of the chlorophyll solution A at 649nm and 665nm, and calculating the content of chlorophyll a in the microalgae solution A by the following formula;
C a ＝a×A 665 -b×A 649
in the formula, C a Is the chlorophyll a content, A 665 Is absorbed by the solution at 665nmLight value, A 649 The absorbance of the solution at 649nm, a was 12.47, b was 3.62,
calculating to obtain the chlorophyll a content of 20.22mg/L in the chlorophyll solution A;
step 2): 0.025mol of NaYF 4 Adding 0.5ml of dimethyl sulfoxide solvent into Yb and Er up-conversion nano fluorescent probes (the average particle size is about 50 nm) for dispersing to ensure that the concentration of the up-conversion nano fluorescent probes is 0.05mmol/L to form a probe solution B, wherein the up-conversion emission spectrum of the up-conversion nano fluorescent probes under the excitation of 980nm is shown in figure 1;
step 3): diluting the chlorophyll solution A to obtain chlorophyll solutions with different concentrations (chlorophyll a content: 0.397-6.010 mg/L), mixing with the probe solution B, respectively, and obtaining red luminous intensity with emission wavelength region of 640-670nm under excitation of 980nm near infrared light, as shown in FIG. 2; due to absorption of chlorophyll a at 665nm in chlorophyll solution and NaYF 4 Emitting heights of Yb, er up-conversion nano fluorescent probe solution B are overlapped at 665nm to generate competitive emission, and a relation curve between red luminous intensity and chlorophyll a content is established as shown in figure 3;
step 4): counting the quantity of the microalgae in the water sample of the ballast water by adopting a microscopic counting method, taking out 50 mu l of the water sample, dripping into a 25X 16 type blood cell counting plate for counting, adopting a 40X objective lens in the counting process, selecting five small squares from the upper left, the lower left, the upper right, the lower right and the middle to count the quantity of the microalgae one by one in the counting process, counting by adopting the principle of counting up and counting down and counting left and right when encountering the microalgae pressed on the edge of a grid, repeatedly counting 10 times for each sample, averaging, and establishing a relation curve of the chlorophyll a content and the microalgae biomass, as shown in figure 4.
Step 5): according to the relationship curve of the chlorophyll a content and the microalgae biomass, a relationship model of the red light intensity of the upconversion nanometer fluorescent probe and the microalgae biomass is further obtained, and as shown in fig. 5, according to the relationship model, the microalgae content of the ballast water sample solution can be calculated by measuring the red light intensity.
Taking 3 different ballast water samples, respectively measuring the red light luminous intensity of the ballast water microalgae solution and the up-conversion nano fluorescent probe mixed solution according to the steps, substituting the red light luminous intensity into the relation curve model, calculating the microalgae biomass (calculated value) of the ballast water sample, and comparing the microalgae biomass (calculated value) with the microalgae biomass measured by the traditional micro counting method (experimental value), wherein the table is shown in the specification.
TABLE 1
Example 2
Step 1): taking 1L of a pressurized water sample, filtering, and performing centrifugal separation to obtain 25.8mg of microalgae precipitate, adding 2ml of acetone solvent into the microalgae precipitate, performing ultrasonic dispersion, and performing centrifugal separation to obtain a supernatant, namely a chlorophyll solution A; measuring the absorption light values of the chlorophyll solution A at 649nm and 665nm, and calculating the content of chlorophyll a in the solution A by the following formula:
C a ＝a×A 665 -b×A 649
here, the coefficient a =12.21, b =2.59, and the content of chlorophyll a in the solution a was calculated to be 12.32mg/L;
step 2): 0.25mol of Y 2 O 2 Adding 0.5ml of acetone solvent into Tm and Er up-conversion nano fluorescent probes (the average particle size is about 150 nm) for dispersion, and enabling the concentration of the up-conversion nano fluorescent probes to be 0.5mmol/L to form a probe solution B;
step 3): diluting the chlorophyll solution A to obtain chlorophyll solutions with different concentrations (chlorophyll a content: 0.35-5.23 mg/L), mixing with the probe solution B, respectively, and exciting with 1550nm near infrared light to obtain red light intensity with emission wavelength region of 640-670nm, due to absorption of chlorophyll a in the microalgae sample solution A at 665nm and Y 2 O 2 Tm, er upconversion nanometer fluorescent probe solution B emits at 665nm to generate competitive emission, and establishes red luminous intensity and chlorophylla relation curve between contents;
and step 4): according to the corresponding relation curve of the chlorophyll a content and the microalgae cell biomass, a relation model of the red light intensity parameter of the up-conversion nano fluorescent probe and the microalgae biomass is further obtained, and according to the relation model, the microalgae content of the ballast water microalgae solution can be calculated by measuring the red light intensity.
Taking 1 kind of ballast water samples, respectively measuring the red light luminous intensity of the ballast water microalgae solution and the up-conversion nano fluorescent probe mixed solution according to the steps, substituting the red light luminous intensity into the relation curve model, and calculating the microalgae biomass of the ballast water samples to be 1.302 multiplied by 10 3 And (2) per liter.
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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