CN108845043A - Microcapsule phycotoxin MC-LR Rapid Quantification in a kind of Growth of Microcystis aeruginosa period - Google Patents
Microcapsule phycotoxin MC-LR Rapid Quantification in a kind of Growth of Microcystis aeruginosa period Download PDFInfo
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- 241000192710 Microcystis aeruginosa Species 0.000 title claims abstract description 25
- 238000011002 quantification Methods 0.000 title claims abstract description 19
- 231100000769 Phycotoxin Toxicity 0.000 title claims abstract description 17
- 239000003094 microcapsule Substances 0.000 title claims abstract description 15
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- 239000007788 liquid Substances 0.000 claims abstract description 8
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
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- SRUWWOSWHXIIIA-UKPGNTDSSA-N Cyanoginosin Chemical compound N1C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](C)[C@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C(=C)N(C)C(=O)CC[C@H](C(O)=O)N(C)C(=O)[C@@H](C)[C@@H]1\C=C\C(\C)=C\[C@H](C)[C@@H](O)CC1=CC=CC=C1 SRUWWOSWHXIIIA-UKPGNTDSSA-N 0.000 description 3
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Abstract
The present invention relates to microcapsule phycotoxin MC-LR Rapid Quantifications in a kind of Growth of Microcystis aeruginosa period, MC-LR concentration in Growth of Microcystis aeruginosa period 96h after just inoculation is measured using high performance liquid chromatograph, frustule quantity is calculated using blood counting chamber under the microscope respectively simultaneously, 680nm absorbance measurement is carried out to microcystic aeruginosa with ultraviolet specrophotometer, algae toxin, the direct proportion linear relationship of absorbance and frustule number are established respectively；In subsequent algae toxin toxicological experiment, by the absorbance or frustule quantity of microcystic aeruginosa in measurement growth cycle 96h, same time algae toxin MC-LR concentration can be quantitatively obtained；The present invention is compared with the existing technology, have many advantages, such as that small and nonhazardous is lost in quick, inexpensive, sample, be able to solve low efficiency present in traditional microcystic aeruginosa algae toxin MC-LR quantitative approach, at high cost, sample loss mostly with the technical problems such as operator safety.
Description
[technical field]
The invention belongs to environmental technology field, Microcystin in specifically a kind of Growth of Microcystis aeruginosa period
MC-LR Rapid Quantification.
[background technique]
Cyanobacteria mass propagation causes water hypoxia, water quality to degenerate, and cyanobacterial bloom causes aqueous bio diversity to drastically reduce,
The microcystic aeruginosa that wherein cyanobacteria belongs to is to form one of wawter bloom algae, and it can generate Microcystin, seriously threaten it
His aquatile even health of the mankind.Each pollutant is intricate to the growth effect of cyanobacteria in environment simultaneously, in order to have
Effect control cyanobacterial bloom, is particularly important the monitoring of microcystic aeruginosa.
Currently, chemical analysis detection method, biology measuring technology can be divided into the detection method of microcystin and exempted from
Epidemic disease detection technique：
1, chemical analysis detection method represents method：High performance liquid chromatography mostly uses HPLC-UV to the detection of MC at present
(high performance liquid chromatography-On-line Ultraviolet) method, general analytical procedure are that first toxin is extracted and purified, using positive or
Reverse-phase chromatographic column to toxin carry out Solid phase extraction separation, then by obtained purified toxins carry out ultraviolet detection, by with standard
The appearance time of toxin is compared and carries out Qualitative Identification to toxin type, and is quantified with peak area comparison method to toxin
Analysis, generally ng grades of method detection limit.This method is to detect one of the conventional method of MC content, but this method is needed using standard
Toxin is used to obtain standard curve and absorption peak, and the shortage of normaltoxin limits this method to a certain extent at present
Using.
2, biology measuring technology represents method：Biological test method is taken and is fed or be injected intraperitoneally to mouse to reflect
Determine the toxicity of algae toxin.Its advantage is that it is easy to be intuitive, can relatively coarse judge whether extract is toxic, and there is operation letter
It is single, visual result, it is quick the advantages that；But need to consume more toxin, sensitivity and specificity be not high, can not accurate quantitative analysis,
It can not distinguish the isomers type of toxin, the standing charges height of mouse, heavy workload.Therefore, biological test method is usually only made
For the initial screening method of toxicity detection, and just increasingly replaced other methods.
3, immunoassay technology represents method：ELISA (ELISA) method, substance carries out immune detection in water
When, most common method is competitive heterogeneous enzyme-linked immunization.It is that MC antibody coating is first fixed on enzyme mark that it, which detects program,
On plate, water sample and quantitative enzyme marker are added when use, is eventually adding chromogenic substrate, carries out chromogenic assay.This method is excellent
It is easy to operate for putting, and experimental period is short, and efficiency greatly promotes.But the disadvantage is that toxin cannot be identified well, for isomers
It will appear false positive reaction.
[summary of the invention]
Microcystis aeruginosa in a kind of Growth of Microcystis aeruginosa period is provided present invention aim to solve above-mentioned deficiency
Phycotoxin MC-LR Rapid Quantification, this method have many advantages, such as that small and nonhazardous is lost in quick, low cost, sample, are able to solve
Low efficiency present in traditional microcystic aeruginosa algae toxin MC-LR quantitative approach, the loss of at high cost, sample are mostly and operator
The technical problems such as safety.
Microcapsule phycotoxin MC-LR fast quantification side in a kind of Growth of Microcystis aeruginosa period is designed to achieve the above object
Method includes the following steps：
1) the high performance liquid chromatography measurement of microcystic aeruginosa algae toxin MC-LR：After treatment by algae solution sample, pass through height
Effect liquid phase chromatogram instrument measures the algae toxin MC-LR concentration of sample, wherein the equal genus lucilia Microcystis aeruginosa of algae solution sample, after new inoculation
Under incubator condition of culture, since inoculation grow in 96h different time sections sampling；
2) measurement of microcystic aeruginosa algae solution sample ultraviolet absorptivity：Algae solution sample is moved into cuvette, utilization is ultraviolet
Spectrophotometer, distilled water zeroing, measures the absorbance of counter sample in 680nm；
3) measurement of microcystic aeruginosa algae solution sample frustule quantity：A little algae solution sample is moved into blood counting chamber to count
Room is counted by observing under 40 times of optical microscopy, calculates frustule quantity；
4) interkniting for algae toxin concentration, absorbance and frustule number is established：The algae that step 1) to step 3) is measured
Phycotoxin MC-LR concentration, absorbance and frustule number data are handled, and establish direct proportion linear relationship respectively.
Further, in step 1), the determination condition of algae toxin MC-LR concentration is proportion of mobile phase methanol and water 65:35
Volume ratio, flow velocity 1.0mL/min, column temperature be 40 DEG C, ultraviolet detection wavelength be 238nm, sample volume be 10 μ L, retention time
For 4min.
Further, in step 1), the algae solution sample be microcystic aeruginosa from be inoculated with grow to 0h, 20h, 40h,
The algae solution sample of 60h, 80h, 90h.
Further, in step 4), according to the linear relationship of foundation, in algae toxin toxicological experiment, by measuring verdigris
Microcystis aeruginosa algae solution sample absorbance or frustule count to get same time algae toxin MC-LR concentration out.
Further, it to the sample microcystic aeruginosa for toxicological experiment, cultivates 1-2 weeks, sterilizing is inoculated with again, is cultivated
Liquid is BG-11, is seeded to the conical flask of sterilizing, inoculative proportion 1:5 or 1:6.
The present invention compared with the existing technology, it is fast to provide microcapsule phycotoxin MC-LR in a kind of Growth of Microcystis aeruginosa period
Fast quantitative approach is counted due to directly measuring algae solution absorbance or frustule in continuous mode, to according to MC-LR concentration and inhale
The linear relationship of luminosity or frustule number can fast quantification；The method of the present invention is compared with traditional liquid chromatography, operation
It is more simple, efficient quick；Compared with biological test method, the method for the present invention is tested without the intervention of other animal organisms,
It is easy to operate, environmentally friendly, efficient and convenient, and operator's nonhazardous is acted on；Compared with enzyme-linked immunosorbent assay, the present invention will not
Detected because can not distinguish isomers, cross contamination and measure inaccurate.
[Detailed description of the invention]
Fig. 1 is the curve of absorbance in various time points algae solution sample in 96h of the present invention；
Fig. 2 is the curve of frustule number in various time points algae solution sample in 96h of the present invention；
Fig. 3 is the curve of algae toxin MC-LR and frustule number in algae solution sample in 96h of the present invention；
Fig. 4 is the curve of algae toxin MC-LR and absorbance in algae solution sample in 96h of the present invention；
Fig. 5, Fig. 6 are certain interior two time point algae solution sample of 96h of the present invention blood counting chamber counting chambers under the microscope
Frustule quantity.
[specific embodiment]
Technical principle of the invention is：Microcapsule phycotoxin MC-LR fast quantification side in a kind of Growth of Microcystis aeruginosa period
Method, i.e., using MC-LR concentration in the Growth of Microcystis aeruginosa period 96h after high performance liquid chromatograph measurement just inoculation, same to time-division
Frustule quantity is not calculated using blood counting chamber under the microscope, microcystic aeruginosa is carried out with ultraviolet specrophotometer
680nm absorbance measurement establishes algae toxin, the direct proportion linear relationship of absorbance and frustule number respectively.I.e. in subsequent algae
In toxin toxicological experiment, by the absorbance or frustule quantity of microcystic aeruginosa in measurement growth cycle 96h, it can obtain same
Period algae toxin MC-LR concentration.
The present invention is made combined with specific embodiments below further explained below：
Embodiment 1
Microcapsule phycotoxin MC-LR Rapid Quantification in a kind of Growth of Microcystis aeruginosa period, specific step is as follows：
(1) the high performance liquid chromatography measurement of microcystic aeruginosa algae toxin MC-LR
The algae solution sample of new inoculation microcystic aeruginosa 0,20,40,60,80,90h is taken after treatment by algae solution sample to lead to
High performance liquid chromatograph is crossed, the algae toxin MC-LR concentration of sample is measured.
The method condition is proportion of mobile phase methanol and water 65:35 volume ratio, flow velocity 1.0mL/min, column temperature are
40 DEG C, ultraviolet detection wavelength is 238nm, and sample volume is 10 μ L, and retention time is 4min or so.
(2) measurement of microcystic aeruginosa algae solution sample ultraviolet absorptivity
The algae solution sample for taking new inoculation microcystic aeruginosa 0,20,40,60,80,90h, algae solution sample is moved into cuvette,
Using ultraviolet specrophotometer, distilled water zeroing measures the absorbance of counter sample in 680nm.
(3) measurement of microcystic aeruginosa algae solution sample frustule quantity
The algae solution sample for taking new inoculation microcystic aeruginosa 0,20,40,60,80,90h, moves into blood cell for a little algae solution sample
Tally counting chamber is counted by observing under 40 times of optical microscopy, calculates frustule quantity.
(4) algae toxin concentration is established, absorbance and frustule number interknit
By the algae solution sample of each time point in the Growth of Microcystis aeruginosa period 96h after new inoculation, pass through above-mentioned step
Suddenly the method in (1) (2) (3), measures algae toxin MC-LR concentration, absorbance and frustule number respectively, by MC-LR concentration,
Absorbance and frustule number data are handled, and can establish direct proportion linear relationship respectively.
(5) linear relationship is utilized, in later toxicological experiment, need to only carry out extinction to microcystic aeruginosa algae solution sample
Degree measurement or cell count can quantitatively obtain corresponding algae toxin MC-LR concentration.
Embodiment 2
Microcapsule phycotoxin MC-LR Rapid Quantification in a kind of Growth of Microcystis aeruginosa period, specific step is as follows：
(1) to sample microcystic aeruginosa culture 1-2 weeks for toxicological experiment, sterilizing is inoculated with again, culture solution BG-
11, it is seeded to the conical flask of sterilizing, inoculative proportion is about 1:5 or 1:In 6, the 96h from after being inoculated with, it is intended to fast quantification and measures its sample
Algae toxin content in product.
(2) measurement of microcystic aeruginosa algae solution sample frustule quantity
A little algae solution sample is moved into blood counting chamber by the algae solution sample for taking two different time points in 96h in step (1)
Counting chamber is counted by observing under 40 times of optical microscopy, and Fig. 5, Fig. 6 are two time point algae solution samples blood cell under the microscope
The frustule quantity of tally counting chamber calculates frustule total quantity by formula.
It (3), can be with quantitative scoring using the proportional relationship in embodiment 1, then with frustule total quantity calculated in (2)
Calculating algae toxin concentration in the microcystic aeruginosa algae solution at two time points is respectively 32.721ug/L, 113.716ug/L.
Embodiment 3
Microcapsule phycotoxin MC-LR Rapid Quantification in a kind of Growth of Microcystis aeruginosa period, specific step is as follows：
To sample microcystic aeruginosa culture 1-2 weeks for toxicological experiment, sterilizing was inoculated with again, culture solution BG-11,
It is seeded to the conical flask of sterilizing, inoculative proportion is about 1:5 or 1:In 6, the 96h from after being inoculated with, it is intended to fast quantification and measures its sample
Middle algae toxin content.
(2) measurement of microcystic aeruginosa algae solution sample ultraviolet absorptivity
The algae solution sample for taking two different time points in 96h in step (1) moves into algae solution sample in cuvette, utilizes
Ultraviolet specrophotometer, distilled water zeroing, measures the absorbance 0.175A of two time point counter samples in 680nm respectively,
0.364A。
It, can be with quantitative scoring using the proportional relationship in embodiment 1, then with the frustule sample absorbance read in (2)
Calculating out algae toxin concentration in the microcystic aeruginosa algae solution at two time points is respectively 52.083ug/L, 135.841ug/L.
In conclusion microcapsule phycotoxin MC-LR fast quantification in a kind of Growth of Microcystis aeruginosa period of the present invention
Method measures MC-LR concentration in the Growth of Microcystis aeruginosa period 96h after just inoculation, same time-division using high performance liquid chromatograph
Frustule quantity is not calculated using blood counting chamber under the microscope, microcystic aeruginosa is carried out with ultraviolet specrophotometer
680nm absorbance measurement；By establishing the direct proportion linear relationship of algae toxin, absorbance and frustule number respectively, surveyed with reaching
Determine algae solution sample absorbance or frustule counts, algae toxin MC-LR concentration is quickly obtained by linear relationship.I.e. subsequent
In algae toxin toxicological experiment, by the absorbance or frustule quantity of microcystic aeruginosa in measurement growth cycle 96h, it can quantify
Show that same time algae toxin MC-LR concentration, the measuring method are simple, safe and efficient.
The present invention compared to any other measurement MC-LR method it is all quick, for carry out the culture of laboratory microcystic aeruginosa with
And toxicological experiment research, can save the plenty of time removes complicated MC-LR measurement from.Since the amount of algae after inoculation can be
96h increases in logarithm, therefore the microcystic aeruginosa cultivated, from be inoculated with, the applicable the method for the present invention of algae solution sample within 96h can
Carry out quantitative algae toxin MC-LR, 96h and algae solution sample later are not suitable for this method.
The present invention is simultaneously not limited to the embodiments described above, other any without departing from spiritual essence and principle of the invention
Changes, modifications, substitutions, combinations, simplifications made by lower, should be equivalent substitute mode, are included in protection model of the invention
Within enclosing.
Claims (5)
1. microcapsule phycotoxin MC-LR Rapid Quantification in a kind of Growth of Microcystis aeruginosa period, which is characterized in that including as follows
Step：
1) the high performance liquid chromatography measurement of microcystic aeruginosa algae toxin MC-LR
After treatment by algae solution sample, by high performance liquid chromatograph, the algae toxin MC-LR concentration of sample is measured, wherein algae
The equal genus lucilia Microcystis aeruginosa of liquid sample, after new inoculation under incubator condition of culture, when growing to the difference in 96h from be inoculated with
Between section sample；
2) measurement of microcystic aeruginosa algae solution sample ultraviolet absorptivity
Algae solution sample is moved into cuvette, using ultraviolet specrophotometer, distilled water zeroing measures counter sample in 680nm
Absorbance；
3) measurement of microcystic aeruginosa algae solution sample frustule quantity
A little algae solution sample is moved into blood counting chamber counting chamber, is counted by being observed under 40 times of optical microscopy, calculates algae
Cell quantity；
4) interkniting for algae toxin concentration, absorbance and frustule number is established
Algae toxin MC-LR concentration, absorbance and frustule number data that step 1) to step 3) measures are handled, and
Direct proportion linear relationship is established respectively.
2. microcapsule phycotoxin MC-LR Rapid Quantification in the Growth of Microcystis aeruginosa period as described in claim 1, feature
It is：In step 1), the determination condition of algae toxin MC-LR concentration is proportion of mobile phase methanol and water 65:35 volume ratio, flow velocity
For 1.0mL/min, column temperature is 40 DEG C, and ultraviolet detection wavelength is 238nm, and sample volume is 10 μ L, retention time 4min.
3. microcapsule phycotoxin MC-LR Rapid Quantification in the Growth of Microcystis aeruginosa period as described in claim 1, feature
It is：In step 1), the algae solution sample be microcystic aeruginosa from be inoculated with grow to 0h, 20h, 40h, 60h, 80h, 90h
Algae solution sample.
4. microcapsule phycotoxin MC-LR Rapid Quantification in the Growth of Microcystis aeruginosa period as claimed in claim 1,2 or 3,
It is characterized in that：In step 4), according to the linear relationship of foundation, in algae toxin toxicological experiment, by measuring microcystic aeruginosa
Algae solution sample absorbance or frustule count to get same time algae toxin MC-LR concentration out.
5. microcapsule phycotoxin MC-LR Rapid Quantification in the Growth of Microcystis aeruginosa period as claimed in claim 4, feature
It is：It to the sample microcystic aeruginosa for toxicological experiment, cultivates 1-2 weeks, sterilizing is inoculated with again, and culture solution BG-11 connects
Conical flask of the kind to sterilizing, inoculative proportion 1:5 or 1:6.
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| CN113237817A (en) * | 2021-05-07 | 2021-08-10 | 山东大学 | Depolymerization observation method and application of benthic cyanobacteria algae pad |
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