CN106290759A - Utilize the Na of fish+the method of atpase activity detection water pollution - Google Patents
Utilize the Na of fish+the method of atpase activity detection water pollution Download PDFInfo
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- CN106290759A CN106290759A CN201610546889.2A CN201610546889A CN106290759A CN 106290759 A CN106290759 A CN 106290759A CN 201610546889 A CN201610546889 A CN 201610546889A CN 106290759 A CN106290759 A CN 106290759A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/186—Water using one or more living organisms, e.g. a fish
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- G—PHYSICS
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- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A20/20—Controlling water pollution; Waste water treatment
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Abstract
The invention discloses a kind of Na utilizing fish+The method of ATP (Adenosine Triphosphate) Enzyme assay water pollution.This method is by obtaining the fish in the waters needing detection, and with the Na of described fish+ATP enzyme is as sensitive biological label, it is achieved detect the pollution of water body to be measured.This method utilizes the Na of fish+ATP enzyme is as sensitive biological label, can the pollution level of the heavy metal pollution such as Monitoring of Cadmium sensitively and the extent of injury, sensitive can also detect polycyclic aromatic hydrocarbons contaminated pollution level and the extents of injury such as luxuriant and rich with fragrance (Phe), for water ecological setting monitoring, toxicity diagnosis, regulate and control and prevent and treat to provide scientific method accurately and rapidly.
Description
Technical field
The present invention relates to the use of bio-sensing marker detection water pollution, it is more particularly related to a kind of profit
With the Na of fish+The method of-atpase activity detection water pollution.
Background technology
Along with urbanization and the fast development of industrial or agricultural, water pollution increasingly sharpens.Water pollutant in waters is the most each
Have difference, have for Single Pollution thing, also have multiple pollutant composite pollution.For unknown waters, only whether predict water body
Contaminated, it is the pollution of Single Pollution thing, or the pollution of multiple pollutant, its pollution level is how, reaches biological harm
Which kind of, to degree, could regulate and control and prevent and treat.For time in water body by heavy metal or one or both pollutions of polycyclic aromatic hydrocarbon, with change
Method with qualitative and quantitative analysis to polluting, but can cannot detect its hazardness and the extent of injury.
Summary of the invention
It is an object of the invention to solve at least the above and/or defect, and at least will be described later excellent is provided
Point.
A further object of the invention is just to provide a kind of Na utilizing fish+The side of-atpase activity detection water pollution
Method, this method utilizes the Na of fish+-ATP enzyme, can the pollution of the heavy metal pollution such as Monitoring of Cadmium sensitively as sensitive biological label
Degree and the extent of injury, it is also possible to polycyclic aromatic hydrocarbons contaminated pollution level and the extents of injury such as sensitive detection luxuriant and rich with fragrance (Phe), for water body
ECOLOGICAL ENVIRONMENTAL MONITORING, toxicity diagnose, regulate and control and prevent and treat to provide scientific method accurately and rapidly.
In order to realize according to object of the present invention and other advantages, it is provided that a kind of Na utilizing fish+-ATP enzyme is lived
Property detection degree of water pollution method.This detection method is to obtain the fish in the waters needing detection, and uses described fish
Na+-ATP enzyme is as sensitive biological label.Wherein, Na+-ATP is writing a Chinese character in simplified form of adenosine triphosphate-sodium.
Preferably, the Na of described fish+-atpase activity is negative correlation with the concentration of Heavy Metals in Waters cadmium.
Preferably, the Na of described fish+-atpase activity is negative correlation with the concentration of polycyclic aromatic hydrocarbon in water body.
Preferably, the Na of fish is set up+-atpase activity and the standard curve of pollutant levels relation, then by be detected
The Na of the fish of the of the same race same size in waters+-atpase activity and described standard curve comparison, obtain water body in waters to be detected
The concentration of pollutant.
Preferably, described standard curve is the Na of fish+-atpase activity and the relation curve of heavy metal cadmium concentration, or
The Na of fish+-atpase activity and the relation curve of polycyclic aromatic hydrocarbon concentration.
Preferably, the Na of described fish+-atpase activity measures and comprises the steps:
Step one, sample to be tested process: preparing the homogenate of full fish, be then centrifuged for layering, taking the supernatant is sample to be tested；
Step 2, the mensuration of sample to be tested protein concentration: respectively by the distilled water of equivalent, protein standard substance and sample to be tested
Add in 1,2, No. 3 pipes, and add the Coomassie brilliant blue developer of equivalent respectively to each pipe, mix respectively, stand.Preheat stand-by
Spectrophotometer and with distilled water return to zero after, at 1cm optical path, wavelength 595nm, measure the optical density of solution in 1,2, No. 3 pipes,
It is designated as No. 1 OD value respectively, No. 2 OD values, No. 3 OD values, then substitute into equation below and calculate sample to be tested protein concentration:
Step 3, enzymatic reaction: control tube and measure in pipe each add the reagent one of test kit of equivalent, reagent two,
Reagent four and reagent six, then add sample to be tested, mixing, 37 DEG C of accurate response 10min in described mensuration pipe；
Step 4, termination reaction: in described control tube and mensuration pipe, the reagent seven of the test kit of each addition equivalent terminates
Reaction, then adds the sample to be tested with step 3 equivalent, mixing in described control tube, is centrifuged layering respectively and takes No. 4 supernatants
Liquid and No. 5 supernatant；Reagent seven is reaction terminating agent, adds sample and does not the most continue reaction, therefore control tube is after reagent adding seven
After add sample and be allowed to Fails To Respond, to balance other sample cells, reduce evaluated error.
Step 5, determine phosphorus: equivalent No. 4 supernatant, No. 5 supernatant and phosphorus standard substance application liquid are separately added into 4,5,6
In number pipe, be then respectively adding equivalent determines phosphorus agent mixing, cool to room temperature after 37 DEG C of water-bath 30min, preheats stand-by light splitting light
After degree is counted and returned to zero with distilled water, at 1cm optical path, wavelength 660nm, measure its optical density by pipe, be designated as respectively compareing OD value,
Measure OD value, standard OD value, then substitute into equation below and calculate the Na of fish+-atpase activity:
Preferably, the fish normal saline that in described step one, the homogenate of full fish is full fish and concentration 0.65% is in mass ratio
Configure for 1:9, and described centrifugal layering refrigerated centrifuger, 11028g, 4 DEG C, centrifugal 10min.
Preferably, distilled water, protein standard substance and the sample to be tested of described step 2 moderate is all 0.05ml, albumen
The concentration of standard substance is 0.563g/L, and the amount being separately added into Coomassie brilliant blue developer is 3ml, stands 10min.
Preferably, in described step 3, each addition the reagent 1 μ l of test kit, examination in control tube and mensuration pipe
Agent 2 40 μ l, reagent 4 40 μ l and reagent 6 40 μ l, then add sample to be tested 200 μ that concentration is 1% in described mensuration pipe
L, reaction temperature is 37 DEG C；In described step 4, in control tube and mensuration pipe, each reagent 7 50 μ l adding test kit terminates
Reaction, then adds the sample to be tested 200 μ l that concentration is 1% in described control tube；No. 4 supernatants of described step 5 moderate
Liquid, No. 5 supernatant and phosphorus standard substance application liquid are 200ul, and addition is determined the amount of phosphorus agent and is 2000ul.
Preferably, described fish is that sea water is blue or green.
The present invention at least includes following beneficial effect: the present invention is with the Na of fish in waters+-ATP enzyme is as sensitive biological mark
Note thing, by contrast Na+-atpase activity just can detect whether water body is contaminated, and it is accurate and sensitive not only to detect, and
And intuitively reflect that the pollution in this waters is to the biological extent of injury.The particularly Na of fish+-atpase activity and Heavy Metals in Waters
One or both concentration of cadmium or polycyclic aromatic hydrocarbon is all in negative correlation, so the Na of fish being detected+-atpase activity is the lowest, its
The contaminated degree in waters is the biggest, and expresses one or more pollute by heavy metal cadmium or polycyclic aromatic hydrocarbon.By building
The Na of vertical fish+-atpase activity and the standard curve of heavy metal cadmium concentration relationship, or the Na of fish+-atpase activity is many with phenanthrene etc.
The curve of PAH concentration relationship, can be by the Na of the fish of same size of the same race with Criterion curve in waters to be detected+-ATP
Activity ratio couple, it is thus achieved that polycyclic aromatic hydrocarbon concentration or the comprehensive pollution concentration value such as heavy metal cadmium concentration or phenanthrene in waters, and can be anti-
Mirror current pollution level to biology whether in safety range or be compromised.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, to make those skilled in the art with reference to description
Can implement according to this.
Embodiment 1
This programme utilizes the Na of fish+The method of-atpase activity detection water pollution, obtains in the waters needing detection
Fish, and with the Na of described fish+-ATP enzyme is as sensitive biological label, by detecting the Na of fish+-atpase activity, then comparison
Na with the fish of similar size+-atpase activity and the standard curve of certain pollutant levels relation, it is possible to detect that water body is
No being polluted by certain pollutant, it is accurate and sensitive not only to detect, and can intuitively reflect the pollution of certain pollutant in this waters
Degree and the extent of injury to biology thereof.
Embodiment 2
This programme utilizes the Na of fish+The method of-atpase activity detection degree of water pollution, obtains the waters needing detection
In fish, and with the Na of described fish+-ATP enzyme is as sensitive biological label, according to the Na of fish+-atpase activity and weight in water body
The concentration of cadmium metal is negative correlation, by measuring the Na of fish+-atpase activity, then comparison is with the Na of the fish of similar size+-ATP
Enzymatic activity and the standard curve of cadmium concentration relation, if the Na of detection sample fish+-atpase activity than free of contamination with similar greatly
The Na of little fish+-atpase activity is low, then judge that waters is polluted by cadmium；If than the free of contamination fish with similar size
Na+-atpase activity is the lowest, then judge that the cadmium pollution in waters is the most serious, even can determine whether the journey reaching to endanger aqueous bio
Degree.
Embodiment 3
This programme utilizes the Na of fish+The method of-atpase activity detection degree of water pollution, obtains the waters needing detection
In fish, and with the Na of described fish+-ATP enzyme is as sensitive biological label, according to the Na of fish+-atpase activity is Sino-Philippines with water body
Etc. (Phe) concentration of polycyclic aromatic hydrocarbon is negative correlation, by measuring the Na of fish+-atpase activity, then comparison is with similar size
The Na of fish+-atpase activity and the standard curve of certain polycyclic aromatic hydrocarbon concentration relationship, if the Na of detection sample fish+-atpase activity
Na than the free of contamination fish with similar size+-atpase activity is low, then judge that waters is polluted by certain polycyclic aromatic hydrocarbon；If
Na than the free of contamination fish with similar size+-atpase activity is the lowest, then certain judging waters is polycyclic aromatic hydrocarbons contaminated the most serious,
Even can determine whether the degree reaching to endanger aqueous bio.
Embodiment 4
This programme utilizes the Na of fish+The method of-atpase activity detection water pollution, obtains in the waters needing detection
Fish, and with the Na of described fish+-ATP enzyme is as sensitive biological label.First pass through experiment and set up the Na of fish+-atpase activity with
The standard curve of certain pollutant levels relation, then by the Na of the fish of the of the same race same size in waters to be detected+-atpase activity
With described standard curve comparison, obtain the concentration of certain pollutant in waters to be detected.
The Na of fish is set up by experiment+-atpase activity is as follows with the standard curve of heavy metal cadmium concentration relationship:
The detection sample of this experiment is as a example by sea water green grass or young crops, from the beginning of the prelarva in 10 day age of cultivation, divides 11 groups and carries out, often group
3 parallel is carried out, and experiment terminates each parallel 3 tail fish sample determinations that take, and often group measures 3x3=9 data, will often organize 9 numbers
According to the result obtaining each group of fish after mathematical statistics.1 group of prelarva in 11 groups is stored in-80 DEG C of refrigerators as 0 week sample, to be determined
Compare；It is 0,80,160,240,320,400,480,560,640,720 μ g/L that other 10 groups of prelarvas cultivate respectively at cadmium concentration
Water body in, cultivating condition is all salinity 30 ± 2, and water temperature is 28 ± 2 DEG C, and pH is 8.10, dissolved oxygen amount >=6.10mg/L, Light To Dark Ratio
For 14h:10h, each group experimental implementation is the most identical.Throwing something and feeding every day feedstuff three times, total feeding volume is the 5% of macrura reevesii body weight in wet base of throwing something and feeding.
Examine and record that larva and juvenile is movable every day, ingest, lopsided, dead etc., and tracing detection water body and the change of fish body cadmium, 7
After it, random acquisition fish sample, weighs respectively, be stored in-80 DEG C of refrigerators (failing i.e. to survey because of busy) for molecule, gene, physiology,
Toxicity and biochemical measurement.Mensuration each group fish Ca after mathematical statistics2+0.640,0.604-atpase activity is respectively:,
0.516、0.460、0.375、0.294、0.273、0.242、0.153、0.156μmolPi/mgprot/hour.Sea water green grass or young crops
Na+-atpase activity is negative correlation with the concentration of Heavy Metals in Waters cadmium, then draws standard curve according to experimental result.
The Na of fish is set up by experiment+-atpase activity is as follows with the standard curve of luxuriant and rich with fragrance (Phe) concentration relationship:
The detection sample of this experiment is as a example by sea water green grass or young crops, from the beginning of the prelarva in 10 day age of cultivation, divides 5 groups and carries out, often organize 3
Individual parallel carrying out, experiment terminates each parallel to take 3 sample determinations, and often group measures 3x3=9 data, will often organize 9 data numbers
The result of each group of fish is obtained after reason statistics.5 groups of prelarvas cultivate respectively at the water that luxuriant and rich with fragrance concentration is 0,250,500,750,1000 μ g/L
In body, cultivating condition is all salinity 30 ± 2, and water temperature is 28 ± 2 DEG C, and pH is 8.10, dissolved oxygen amount >=6.10mg/L, and Light To Dark Ratio is
14h:10h, each group experimental implementation is the most identical.Throwing something and feeding three times a day feedstuff, total feeding volume is the 5% of macrura reevesii body weight in wet base of throwing something and feeding.Often
It examines and records that larva and juvenile is movable, ingest, lopsided, dead etc., and tracing detection water body and the luxuriant and rich with fragrance change of fish body, 7 days
Rear random acquisition fish sample, weighs respectively, be stored in-80 DEG C of refrigerators (temporary to be measured) for molecule, gene, physiology, toxicity and
Biochemical measurement.Mensuration each group fish Ca after mathematical statistics2+-atpase activity is respectively: 0.485,0.356,0.318,
0.160、0.088μmolPi/mgprot/hour.The Na that sea water is blue or green+The concentration of-atpase activity and water body Sino-Philippines (Phe) in
Negative correlation, then draws standard curve according to experimental result.
Measure the Na that the sea water in waters to be measured is blue or green+Specifically comprising the following steps that of-atpase activity
Step one, sample to be tested process: take with Criterion curve that to prepare full fish with fish sea water of a size green grass or young crops even
Slurry, is then centrifuged for layering, and taking supernatant is sample to be tested；
Step 2, the mensuration of sample to be tested protein concentration: respectively by the distilled water (blank) of equivalent, protein standard substance
Add in 1,2, No. 3 pipes with sample to be tested, and add the Coomassie brilliant blue developer of equivalent respectively to each pipe, mix respectively, quiet
Put.After preheating stand-by spectrophotometer and returning to zero with distilled water, at 1cm optical path, wavelength 595nm, measure in 1,2, No. 3 pipes molten
The optical density (OD) of liquid, is designated as No. 1 OD value, No. 2 OD values, No. 3 OD values respectively, then substitutes into equation below and calculates sample to be tested
Protein concentration:
Step 3, enzymatic reaction: control tube and measure in pipe each add the reagent one of test kit of equivalent, reagent two,
Reagent four and reagent six, then add sample to be tested in described mensuration pipe, and mixing, 37 DEG C accurately reflect 10min；
Step 4, termination reaction: in described control tube and mensuration pipe, the reagent seven of the test kit of each addition equivalent terminates
Reaction, then adds the sample to be tested with step 3 equivalent, mixing in described control tube, is centrifuged respectively in layering acquirement 4
Clear liquid and No. 5 supernatant；
Step 5, determine phosphorus: equivalent No. 4 supernatant, No. 5 supernatant and phosphorus standard substance application liquid are separately added into 4,5,6
In number pipe, be then respectively adding equivalent determines phosphorus agent, and mixing is cool to room temperature after 37 DEG C of water-bath 30min, preheats stand-by light splitting light
After degree is counted and returned to zero with distilled water, at 1cm optical path, wavelength 660nm, measure its optical density by pipe, be designated as respectively compareing OD value,
Measure OD value, standard OD value, then substitute into equation below and calculate the Na of fish+-atpase activity:
Na blue or green by calculating sea water again+-atpase activity size and standard curve comparison, not only it is known that water body
Whether it is contaminated, and it is known that Cadmium In The Water Body concentration or the size of luxuriant and rich with fragrance concentration or comprehensive pollution big from standard curve
Little, more can know that water body is to the organism extent of injury.
Embodiment 5
Utilize experiment can set up the fish Na of any fingerling of any water body+-atpase activity and heavy metal cadmium concentration relationship
Standard curve, or set up different types of fish Na+-atpase activity and the standard curve of luxuriant and rich with fragrance concentration relationship, then take water to be measured
Fish in territory is cooked sample, as long as sample fish and the fish of Criterion curve are can compare with similar size.
Measure the fish Na in waters to be measured+Specifically comprising the following steps that of-atpase activity
Step one, sample to be tested process: take with Criterion curve with as fish and sizable fish to prepare full fish even
Slurry, the homogenate of full fish is that the fish normal saline of full fish and 0.65% concentration configures for 1:9 in mass ratio, then uses frozen centrifugation
Machine, 11028g, 4 DEG C, centrifugal 10min layering, taking supernatant is sample to be tested；
Step 2, the mensuration of sample to be tested protein concentration: be the protein standard substance of 0.563g/L by distilled water, concentration respectively
0.05ml each with sample to be tested adds in 1,2, No. 3 pipes, and adds Coomassie brilliant blue developer 3ml to each pipe respectively, mixes respectively
Even, stand 10min, be preheating to 35 DEG C, after preheating stand-by spectrophotometer and returning to zero with distilled water, in 1cm optical path, wavelength
At 595nm, measure the optical density of solution in 1,2, No. 3 pipes, be designated as No. 1 OD value, No. 2 OD values, No. 3 OD values respectively, then substitute into
Equation below calculates sample to be tested protein concentration:
Step 3, enzymatic reaction: each addition the reagent 1 μ l of test kit of equivalent, examination in control tube and mensuration pipe
Agent 2 40 μ l, reagent 4 40 μ l and reagent 6 40 μ l, then add sample to be tested, mixing in described mensuration pipe, and 37 DEG C accurately
Reflection 10min；
Step 4, termination reaction: the reagent 7 50 μ l of the test kit of each addition equivalent in described control tube and mensuration pipe
Terminate reaction, in described control tube, then add sample to be tested 200 μ l, mixing, be centrifuged respectively layering obtain No. 4 supernatant and
No. 5 supernatant；
Step 5, determine phosphorus: by each to No. 4 supernatant, No. 5 supernatant and the phosphorus standard substance application liquid that concentration is 0.5umol/ml
200ul is separately added in 4,5, No. 6 pipes, be then respectively adding 2000ul determine phosphorus agent mixing, after 37 DEG C of water-bath 30min, cool extremely
Room temperature, after preheating spectrophotometer and returning to zero with distilled water, at 1cm optical path, wavelength 660nm, measures each pipe optical density, respectively
It is designated as compareing OD value, measures OD value, standard OD value, then substitute into equation below and calculate the Na of fish+-atpase activity:
The Na of fish will be calculated+-atpase activity size and standard curve comparison, not only it is known that whether water body gets dirty
Dye, and it is known that Cadmium In The Water Body concentration or the size of luxuriant and rich with fragrance concentration or the size of comprehensive pollution from standard curve, more can know
Road water body is to the organism extent of injury.
Embodiment 6
Utilize the Na testing the fresh water green grass or young crops setting up 20 ages in days+-atpase activity is bent with the standard of heavy metal cadmium concentration relationship
Line and Na+The standard curve of-atpase activity and Phenanthrene concentration relationship, the fish then taken in waters to be measured cooked sample,
As long as the fish obtaining sample is can compare with similar size with the fish of Criterion curve.
The fresh water green grass or young crops obtaining 20 ages in days in waters to be measured is cooked sample, then measures the Na of this fish in waters to be measured+-
Atpase activity specifically comprises the following steps that
Step one, sample to be tested process: take with Criterion curve with as fish and sizable fish to prepare full fish even
Slurry, the homogenate of full fish is that the fish normal saline of full fish and 0.65% concentration configures for 1:9 in mass ratio, then uses frozen centrifugation
Machine, 11028g, 4 DEG C, centrifugal 10min layering, taking supernatant is sample to be tested；
Step 2, the mensuration of sample to be tested protein concentration: be the protein standard substance of 0.563g/L by distilled water, concentration respectively
0.05ml each with sample to be tested adds in 1,2, No. 3 pipes, and adds Coomassie brilliant blue developer 3ml to each pipe respectively, mixes respectively
Even, stand 10min, be preheating to 35 DEG C, after preheating stand-by spectrophotometer and returning to zero with distilled water, in 1cm optical path, wavelength
At 595nm, measure the optical density of solution in 1,2, No. 3 pipes, be designated as No. 1 OD value, No. 2 OD values, No. 3 OD values respectively, then substitute into
Equation below calculates sample to be tested protein concentration:
Step 3, enzymatic reaction: use the test kit that Nanjing Bioengineering Research Institute produces by control tube and sample to be tested
After enzymatic reaction, it is centrifuged layering respectively and obtains No. 4 supernatant (control tube) and No. 5 supernatant (sample to be tested pipe), wherein enzymatic
Reaction concrete operations such as table 1:
The concrete operations of table 1 enzymatic reaction
Step 4, determine phosphorus: by each to No. 4 supernatant, No. 5 supernatant and the phosphorus standard substance application liquid that concentration is 0.5umol/ml
200ul is separately added in 4,5, No. 6 pipes, be then respectively adding 2000ul determine phosphorus agent mixing, after 37 DEG C of water-bath 30min, cool extremely
Room temperature, after preheating spectrophotometer and returning to zero with distilled water, at 1cm optical path, wavelength 660nm, measures each pipe optical density, respectively
It is designated as compareing OD value, measures OD value, standard OD value, then substitute into equation below and calculate the Na of fish+-atpase activity is 0.510
μm olPi/mgprot/hour:
The Na of fish will be calculated+-atpase activity comparison fish Na respectively+-atpase activity and the mark of heavy metal cadmium concentration relationship
Directrix curve and Na+-atpase activity and the standard curve of Phenanthrene concentration relationship, the cadmium pollution obtaining waters to be measured is dense
Degree is 175 μ g/L, and Phenanthrene concentration is 0；The most both it is known that water body is contaminated, and can from standard curve
To know Cadmium In The Water Body concentration or the size of luxuriant and rich with fragrance concentration, more can know that water body is to the organism extent of injury.
Although embodiment of the present invention are disclosed as above, but it is not restricted in description and embodiment listed
Exemplary applications, it is completely suitable for various applicable the field of the invention, for those skilled in the art, can be the most another
Row revises his use, and therefore under the general concept limited without departing substantially from claim and equivalency range, the present invention is not limited to spy
Fixed details and shown here as with describe embodiment.
Claims (10)
1. the Na utilizing fish+The method of-atpase activity detection water pollution, it is characterised in that obtain the water needing detection
Fish in territory, and with the Na of described fish+-ATP enzyme is as sensitive biological label.
The Na utilizing fish the most according to claim 1+The method of-atpase activity detection water pollution, it is characterised in that institute
State the Na of fish+-atpase activity is negative correlation with the concentration of Heavy Metals in Waters cadmium.
The Na utilizing fish the most according to claim 1+The method of-atpase activity detection water pollution, it is characterised in that institute
State the Na of fish+-atpase activity is negative correlation with the concentration of polycyclic aromatic hydrocarbon in water body.
4. according to the Na utilizing fish described in any one of claim 1-3+The method of-atpase activity detection water pollution, it is special
Levy and be, set up the Na of fish+The standard curve of-atpase activity and pollutant levels relation, then same by waters to be detected
Plant the Na of the fish with size+-atpase activity and described standard curve comparison, obtain water pollution substrate concentration in waters to be detected.
The Na utilizing fish the most according to claim 4+The method of-atpase activity detection water pollution, described standard curve
Na for fish+-atpase activity and the standard curve of heavy metal cadmium concentration relationship, or the Na of fish+-atpase activity and many cyclophanes
The standard curve of hydrocarbon concentration relationship.
The Na utilizing fish the most according to claim 5+The method of-atpase activity detection water pollution, it is characterised in that institute
State the Na of fish+-atpase activity measures and comprises the steps:
Step one, sample to be tested process: preparing the homogenate of full fish, be then centrifuged for layering, taking supernatant is sample to be tested；
Step 2, the mensuration of sample to be tested protein concentration: respectively the distilled water of equivalent, protein standard substance and sample to be tested are added
1, in 2, No. 3 pipes, and respectively to the Coomassie brilliant blue developer of each pipe addition equivalent, mix respectively, stand, preheat spectrophotometric
After counting and returning to zero with distilled water, in 1cm optical path, at wavelength 595nm, measure the optical density of material in 1,2, No. 3 pipes, remember respectively
It is No. 1 OD value, No. 2 OD values, No. 3 OD values, then substitute into equation below and calculate sample to be tested protein concentration:
Step 3, enzymatic reaction: each the addition reagent one of test kit of equivalent, reagent two, reagent in control tube and mensuration pipe
Four and reagent six, in described mensuration pipe, then add sample to be tested, mixing, 37 DEG C of accurate response 10min；
Step 4, termination reaction: in described control tube and mensuration pipe, the reagent seven of each addition equivalent terminates reaction, then in institute
State and control tube adds the sample to be tested with step 3 equivalent, mixing, it is centrifuged layering respectively and obtains No. 4 supernatant and No. 5 supernatants
Liquid；
Step 5, determine phosphorus: equivalent No. 4 supernatant, No. 5 supernatant and phosphorus standard substance application liquid are separately added into 4,5, No. 6 pipes
In, be then respectively adding equivalent determines phosphorus agent mixing, and cool to room temperature after 37 DEG C of water-bath 30min, preheating spectrophotometer is also used
After distilled water zeroing, at 1cm optical path, wavelength 660nm, measure its optical density by pipe, be designated as respectively compareing OD value, measure OD
Value, standard OD value, then substitute into equation below and calculate the Na of fish+-atpase activity:
The Na utilizing fish the most according to claim 6+The method of-atpase activity detection water pollution, it is characterised in that institute
State fish normal saline that the homogenate of full fish in step one is full fish and concentration 0.65% in mass ratio for 1:9 configuration, and described from
Heart layering refrigerated centrifuger, 11028g, 4 DEG C, centrifugal 10min.
The Na utilizing fish the most according to claim 6+The method of-atpase activity detection water pollution, it is characterised in that institute
Stating the distilled water of step 2 moderate, protein standard substance and sample to be tested and be 0.05ml, the concentration of protein standard substance is
0.563g/L, the amount being separately added into Coomassie brilliant blue developer is 3ml, stands 10min.
The Na utilizing fish the most according to claim 6+The method of-atpase activity detection water pollution, it is characterised in that institute
State in step 3, each addition the reagent 1 μ l of test kit, reagent 2 40 μ l, reagent 4 40 μ l in control tube and mensuration pipe
With reagent 6 40 μ l, then adding the sample to be tested 200 μ l that concentration is 1% in described mensuration pipe, reaction temperature is 37 DEG C；Institute
Stating in step 4, in control tube and mensuration pipe, each reagent 7 50 μ l adding test kit terminates reaction, then in described comparison
Pipe adds the sample to be tested 200 μ l that concentration is 1%；No. 4 supernatant of described step 5 moderate, No. 5 supernatant and phosphorus mark
Quasi-product application liquid is 200ul, and addition is determined the amount of phosphorus agent and is 2000ul.
10. according to the Na utilizing fish described in any one of claim 5-9+The method of-atpase activity detection water pollution, it is special
Levying and be, described fish is that sea water is blue or green.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109596549A (en) * | 2018-12-24 | 2019-04-09 | 苏州科铭生物技术有限公司 | A kind of V Activity Assay Kit of chloroplaset complex and method based on micromethod |
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2016
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| CN109596549A (en) * | 2018-12-24 | 2019-04-09 | 苏州科铭生物技术有限公司 | A kind of V Activity Assay Kit of chloroplaset complex and method based on micromethod |
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Application publication date: 20170104 |