CN111707710A - Method for detecting gill potassium ion flow based on non-damage micrometering technology - Google Patents
Method for detecting gill potassium ion flow based on non-damage micrometering technology Download PDFInfo
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- CN111707710A CN111707710A CN202010389921.7A CN202010389921A CN111707710A CN 111707710 A CN111707710 A CN 111707710A CN 202010389921 A CN202010389921 A CN 202010389921A CN 111707710 A CN111707710 A CN 111707710A
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Abstract
The invention discloses a method for detecting potassium ion flow of gills of fish based on a non-damage micrometering technology. The method utilizes a non-damage micrometering technology to detect the discharge condition of potassium ions of the gills of the mosquito-eating fishes, can detect the discharge condition change of the potassium ions of the gills of the fishes before and after the solution of propyl p-hydroxybenzoate pollutants is exposed, and can detect the accelerated discharge of the potassium ions of the gills of the fishes treated by the propyl p-hydroxybenzoate, and the change of the flow velocity of the potassium ions of the gills of the fishes has an indication effect on the pollutants (propyl p-hydroxybenzoate). The invention can detect the discharge speed of the potassium ions of the gills of the fishes, has the advantages of short detection time, high accuracy, simplicity, reliability and the like, can effectively indicate the toxicity and influence of environmental pollutants on the fishes, provides a direct, real-time and rapid detection tool for environmental monitoring and toxicological research, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of environmental toxicology, and particularly relates to a method for evaluating the influence of exposure of a pollutant propylparaben on gills by detecting changes of potassium ion currents of the gills based on a non-damage micrometering technology.
Background
Propyl paraben is a common preservative and is commonly added into cosmetics, nursing products and medicines, and the propyl paraben is widely used in the environment due to the large amount of propyl paraben, so that aquatic organisms such as fishes are exposed to a more dangerous environment.
Mosquito-eating fish (Gambusia affinalis) belongs to the family of killedon tiformes (Cyprinodermames), family of foetidae (Poeciliaceae), genus Gambusia, is small oviparous fish living in freshwater, is native to Central and south America, is introduced into China since 1927, and becomes small freshwater fish throughout south of China. The mosquito-eating fish has small body shape, feeding impurity, rapid growth, short breeding cycle, easy fishing and wide environmental adaptability, is easy to be raised in a laboratory, is an ideal experimental animal for water environment monitoring and toxicity test, and is an effective indicator organism for environmental pollutants.
The non-damage micrometering technology is also called ion-selective microelectrode, the measuring process has the characteristics of no damage, real time, accuracy and the like, and the method is widely applied to a plurality of subjects such as basic biology, physiology, environmental science, material science and the like.
However, the existing target object for detecting the flow rate of ions is mainly plants, and the detection method mainly comprises the steps of detecting the flow direction and the flow rate of heavy metal ions in the plants. At present, the research on the technology for detecting the gill ion flow rate of the small freshwater fishes is rarely reported. Therefore, the method for detecting the potassium ion flow of the fish gills by using the non-damage micrometering technology is provided, and the method is used for judging the influence of the exposure of the propyl p-hydroxybenzoate pollutants on the fish gills, and has the advantages of real-time performance, easy operability, practicability and accuracy.
Disclosure of Invention
The invention aims to provide a method for detecting potassium ion flow of fish gills based on a non-damage micrometering technology, which is used for detecting the influence of the exposed mosquito-eating fish gills to the flow velocity of potassium ions of the fish gills of propyl p-hydroxybenzoate to draw a conclusion that the mosquito-eating fish gills have indication on pollutants (propyl p-hydroxybenzoate).
The invention is realized by adopting the following technical scheme, and the method comprises the following steps:
(1) experiment design: the ion detection experiment of the gill of the mosquito-eating fish is provided with a propyl p-hydroxybenzoate treatment group and a blank control group, wherein each group is provided with five parallel fishes, and each parallel fish is provided with 10 parallel fishes, so that the selected fishes are ensured to be consistent in size and healthy in fish bodies.
(2) Manufacturing a sensor: injecting a potassium ion filling liquid into the tail end of the sensor until the tip of the electrode is filled with 1-2 cm, sucking a corresponding potassium ion exchanger into the front end of the electrode, sleeving the treated electrode into a chlorinated electrode wire socket, and putting the electrode into a correction liquid for correction;
(3) and (3) the fish selected in the step 1 is anesthetized by an anesthetic for 20min and then immediately dissected to take out the gill, and the gill silk of the fish is washed once by using normal saline and then is put into a test buffer solution to be balanced for 10min and then is detected by a sensor.
(4) And processing and analyzing the detection result.
Preferably, the irrigation liquid is 180-200 mM KCl; the length of the inhalation exchanger was 180 μm;
preferably, the test buffer is: CaCl2(0.1mM)、MgCl2·6H2O(0.98mM)、MgSO4(0.81mM), KCl (0.5mM), NaCl (149.4mM), D-galactose (5mM), and sodium pyruvate (5 mM).
Preferably, the Nernst equation of the corrected electrode is calculated to be the ideal value of potassium ion of-23 mV to-56 mV. The flow velocity of the potassium ion flow is 0 to 800pmol cm–2·s–1
The invention is based on the non-damage micrometering technology, realizes the real-time and rapid detection of the gill, has high accuracy and provides an effective means for detecting the gill potassium ion flow.
Drawings
Fig. 1 is a flow chart of a gill potassium ion detection experiment.
Fig. 2 is a diagram of gill detection process.
Fig. 3 is a line graph showing the flow rate change of five parallel potassium ions of the fish gill.
Fig. 4 is a bar graph of flow rate changes of five parallel potassium ions of fish gills.
Detailed Description
The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. The conditions used in the examples may be further adjusted according to the conditions of the particular manufacturer, and the conditions not specified are generally the conditions in routine experiments. If not specified, reference is made to the usual laboratory manuals and to the manufacturer's instructions for the instruments used. Wherein the mosquito-eating fish is purchased from an aquaculture farm.
Example (b):
1.1 preparation of fish gill ion flow velocity detection experiment: the gill potassium ion detection experiment designs a treatment group (4.6mg/l propyl p-hydroxybenzoate) and a blank control group, wherein each group is provided with five parallel fishes, and each parallel fish is provided with 10 parallel fishes, so that the selected fishes are consistent in size and healthy in fish bodies.
1.2 flow rate detection experiment of potassium ions in fish gills
1.2.1 formulation of test buffer: the formula is as follows: CaCl2(0.1mM)、MgCl2·6H2O(0.98mM)、MgSO4(0.81mM), KCl (0.5mM), NaCl (149.4mM), D-galactose (5mM), and sodium pyruvate (5 mM).
1.2.2 making a sensor: the sensor, also known as an ion selective microelectrode, was used in conjunction with a reference electrode to measure the net flux of potassium ions, and the sensor was made under a microscope, as detailed in Table 1. Transferring a microelectrode between two positions close to a target object, controlling the moving distance and frequency of the microelectrode by a hydraulic manipulator driven by a computer, detecting the microelectrode by a non-damage micrometering system at room temperature (26-28 ℃), and placing the target object in a 7.5cm disposable culture dish filled with a test solution for detecting the ion flux.
TABLE 1 ion-selective microelectrodes
1.2.3 calibration of the sensor: firstly, three groups of test solutions with different concentrations are used for calibrating the characteristics of the sensor, and the concentrations of potassium ions in the calibration solution and the test solutions are different. The calibration results were as follows: the voltage of calibration solution 1(1mM potassium ion) is-22.47 mV, the voltage of calibration solution 2(0.25mM potassium ion) is-56.26 mV, the voltage of test solution (0.5mM potassium ion) is-39.89 mV, and the voltage fluctuation of the test solution is within +/-10 mV in the test process, thus meeting the calibration requirement.
1.3 testing the flow rate of potassium ions of the fish gills: firstly, use 0.4 mg.L–1After ethyl m-aminobenzoate mesylate (MS-222) is anesthetized for 20min, immediately dissecting and taking out the gill, washing the gill filaments once with normal saline, putting the gill filaments into a test solution for balancing for 10min, putting a culture dish under a microscope after the balancing is finished, adjusting the culture dish on a display until the gill filaments cells of the gill can be seen clearly, putting a sensor into the culture dish, enabling the sensor to be 1 mu m away from the gill filaments of the gill, starting to detect the flow rate of potassium ions of the gill, and measuring for 10 min.
1.4 data processing
The ion current processing is based on FICK' S diffusion law and is output by non-damage micrometering system data analysis software.
1.5 evaluation of test results:
fig. 3 and 4 show the flow rate of potassium ions in gills of fish before and after propyl p-hydroxybenzoate treatment, each curve in the graph shows the flow rate of potassium ions in gills of one fish, and potassium ions in gills of one fish are generally discharged. The flow velocity range of potassium ions of the gills of the blank group of the fishes is 0 to 200pmol cm–2·s–1The flow velocity range of potassium ions of the propylparaben exposed fish gills is 400-1000 pmol cm–2·s–1The experimental result shows that after the gill is exposed to the propyl hydroxybenzoate, the potassium ion discharge amount in the gill is increased compared with that before exposure, the fluctuation range of the potassium ion flow rate is increased compared with that before exposure, the fluctuation interval range is increased compared with that before exposure, and the difference between the two times of treatment has statistical significance (P is less than 0.05).
The above description is only for the purpose of illustrating the present invention, and is not intended to limit the present invention in any way. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. It is intended that any person skilled in the art can use the above disclosed methods and techniques to make many possible variations and modifications to the disclosed embodiments, or to modify an equivalent embodiment to an equivalent variation without departing from the spirit and scope of the present invention. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Claims (7)
1. A method for detecting potassium ion flow of fish gills based on a non-damage micrometering technology is characterized in that the potassium ion discharge capacity in the fish gills is detected by the non-damage micrometering technology, the potassium ion flow speed change of the fish gills before and after propyl p-hydroxybenzoate exposure is detected to analyze the potassium ion discharge condition of the fish gills, and the propyl p-hydroxybenzoate has an indicating effect.
2. The method of claim 1, wherein the method comprises a gill ion detection experimental design, a sensor manufacturing, a gill anesthesia and immediate dissection of the gill and potassium ion detection of gill filaments, and detection result processing and analysis.
3. The method according to any one of claims 1-2, comprising the steps of:
(1) designing a fish gill ion detection experiment: the propyl hydroxybenzoate exposure group and the blank control group to 4.6mg/l PrP were set, five in each group, 10 fish in each parallel, to ensure the selected fish were healthy and of consistent size;
(2) manufacturing a sensor: injecting a potassium ion filling liquid into the tail end of the sensor until the tip of the electrode is filled with 1-2 cm, sucking a corresponding potassium ion exchanger into the front end of the electrode, sleeving the treated electrode into a chlorinated electrode wire socket, and putting the electrode into a correction liquid for correction;
(3) fish gill dissection: anaesthetizing the fish with an anesthetic for 20min, immediately dissecting and taking out the gill, washing the gill silk of the fish with normal saline once, putting the fish into a test buffer solution for balancing for 10min, and detecting with a corrected electrode;
(4) and processing and analyzing the detection result.
4. The method of claim 3, wherein the irrigating solution is 180-200 mM KCl; the length of the imbibed liquid ion exchanger was 180 μm.
5. The method of claim 3, wherein the test buffer is: 0.1mM CaCl2、0.98mMMgCl2·6H2O、0.81mM MgSO40.5mM KCl, 149.4mM NaCl, 5mM D-galactose and 5mM sodium pyruvate.
6. The method of claim 3, wherein the Nernst equation of the corrected electrode is calculated to be the ideal value of-23 to-56 mV for potassium ions.
7. The method according to claim 3, wherein the flow rate of the potassium ion stream is 0 to 800pmol cm–2·s–1。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115184433A (en) * | 2022-07-16 | 2022-10-14 | 桂林理工大学 | Method for evaluating toxicity of nitrosodiethylamine based on change of fish body calcium ion flow velocity |
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Application publication date: 20200925 |