CN105424671B - A kind of preparation method of the suspending stabilized sample of frond for fluorescence measurement - Google Patents
A kind of preparation method of the suspending stabilized sample of frond for fluorescence measurement Download PDFInfo
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- CN105424671B CN105424671B CN201610014072.0A CN201610014072A CN105424671B CN 105424671 B CN105424671 B CN 105424671B CN 201610014072 A CN201610014072 A CN 201610014072A CN 105424671 B CN105424671 B CN 105424671B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
A kind of preparation method of the suspending stabilized sample of frond for fluorescence measurement, belong to the technical field of algae solution measure, first algae solution sample is prepared using for examination algae solution, then with the chlorophyll in fluorescence spectrometry algae solution sample, centrifugal treating first will be carried out for examination algae solution, then centrifugal treating is obtained into frond and is configured to algae solution sample with suspension stabilizer, wherein the suspension stabilizer is made of agar, xanthans and carragheen.The present invention is by the way that in fluorescence detection, suspension stabilizer is added by the algae solution of detection, and by each parameter of fluoremetry, the attenuation rate of fluorescent value is small, fluctuation is small, shows that the suspension stability of algae solution is high, algae solution stable system, more accurate to the measure of algae solution.
Description
Technical field
The invention belongs to the technical fields of water sample measuring chlorophyll content, are related to a kind of fluorescence spectrometry water sample chlorophyll and contain
The method of amount is more specifically a kind of preparation method of the suspending stabilized sample of the frond for fluorescence measurement, and this method increases
The suspension of algae solution, stability obtain finely dispersed algae solution sample.
Background technology
Water body Determination of Chlorophyll a contents are routine monitoring projects in water quality environmental monitoring, are that body eutrophication refers to
One of mark reacts amount of algae and water quality condition in water body to a certain extent.Chlorophyll-a Content accurately measures particularly important.
Fluorescence spectrophotometry is easy to operate due to high sensitivity, can continuously in real time, monitoring, and realize the reality of water body Determination of Chlorophyll
When in-situ study, be that experimental determination chlorophyll a realizes the most commonly used method to the quantitative determination of phytoplankton.
There is frustules during actual sample chlorophyll measuring to disperse uneven, free settling, suspension stability
The problems such as difference cause fluoroscopic examination Chlorophyll-a Content accuracy of measurement not high, and fluorescent value accurate response algae solution can not be used dense
Degree, this is always a problem of water sample chlorophyll measuring.Find that prior art fluorescence detection directly measures in experimentation
When algae solution sample and actual water sample, frustule sedimentation is apparent, measured value changes over time larger obtained data inaccuracy, can not
Correctly judge aquatic environment, therefore correct measurement aquatic environment is most important.Therefore, the sedimentation of frustule disperseed is reduced
Speed increases the suspension of algae solution, stability obtains emphasis of the finely dispersed algae solution sample as research.
Invention content
When the present invention is solves prior art fluorescence spectrometry water body sample chlorophyll content, since frustule is in water body
The problem of uneven, free settling, suspension system are unstable, and measurement result is inaccurate, error is big provides a kind of for fluorescence survey
The preparation method of the suspending stabilized sample of frond of amount, efficiently solves the above problem.
The present invention for realize its purpose the technical solution adopted is that：
A kind of preparation method of the suspending stabilized sample of frond for fluorescence measurement prepares algae solution using for examination algae solution first
Then sample is measured chlorophyll with fluorescence method, first centrifugal treating will be carried out for examination algae solution, then by centrifugal treating
It obtains frond and is configured to algae solution sample with suspension stabilizer, wherein the suspension stabilizer is by agar, xanthans and carragheen
Composition.
It takes and is placed in a centrifuge for examination algae solution, 15-20min is centrifuged with the rotating speed of 3500-4500r/min, is then outwelled
Clear liquid obtains frond.
Agar in the suspension stabilizer：Xanthans：The content ratio of carragheen is (2-10)：(10-30)：(30-50),
And in sample algae solution agar a concentration of 0.06%.
Agar in the suspension stabilizer：Xanthans：The content ratio of carragheen is 3：7.5：17.5, and in sample algae solution
A concentration of the 0.06% of agar.
Described for examination algae solution is chlorella pyrenoidosa algae solution, as laboratory cultures algae solution sample, centrifugal treating it
Before, first chlorella pyrenoidosa in chlorella pyrenoidosa algae solution is seeded in the conical flask for filling sterilizing BG-11 culture solutions and carried out
Culture.
Described is derived from man-made lake water sample for examination algae solution, is for examination algae solution when being derived from man-made lake water sample as actual water sample
When, it to be immediately available for preparing algae solution sample after sampling or the suspension of 1mL1% magnesium carbonate is added in then every liter of water sample if you need to place
Liquid is kept in dark place with preventing chlorophyllin in 4 DEG C of refrigerators.
The beneficial effects of the invention are as follows：The present invention is creative to add the algae solution of detection by fluorescence detection
Suspension stabilizer is entered, by each parameter of fluoremetry, the attenuation rate of fluorescent value is small, fluctuation is small, shows the suspending stabilized of algae solution
Property high, algae solution stable system, it is more accurate to the measure of algae solution.
By long-term creative research, between inventor has found different suspension stabilizers, even if the viscosity phase of solution
Same or close, liquid suspension stability also differs or even difference is with big, but related notable between suspension and stability, algae
The suspension stability of liquid has significant effect the measure of algae solution, and agar, Huang are selected by the long-term creative research present invention
Virgin rubber, carragheen are suspension stabilizer and auxiliary rheological agents, are mainly used for improving and increase the viscosity of liquid, so as to change
The physical behavior of solution keeps the stability of fluid, has the function of emulsification, homogeneous, stabilization and suspended state is presented.It is and existing
Suspension stability is not considered to the measure of algae solution in technology, is all considering the problems of impurity and absorbance, Yi Jitong substantially
The precision of measuring apparatus is crossed to carry out, the problem of without any algae solution suspension.
Specific embodiment
When the present invention is solves prior art fluorescence spectrometry water body chlorophyll, since frustule is uneven in water body
The problem of even, free settling, suspension system are unstable, and measurement result is inaccurate, error is big provides a kind of for fluorescence measurement
The preparation method of the suspending stabilized sample of frond, efficiently solves the above problem, the present invention is made with reference to specific embodiment into
One step explanation.
First, laboratory apparatus and reagent
Instrument：Illumination box (E-30B0 U.S. Percival)；Visible spectrophotometer (722 type Shanghai spectral instruments
Co., Ltd)；Centrifuge (Z323 type Hermle)；Ultraviolet specrophotometer (the limited public affairs of the U.S. scientific instrument in UV-2600 Shanghai day
Department)；Sepectrophotofluorometer (Shimadzu Corporation of RF-5301 Japan).
Reagent：Hydrochloric acid 1mol.L-1；Culture medium is sterilizing BG-11 culture solutions；10g.L-1Magnesium carbonate suspension.
Suspending agent：Agar, food-grade；Xanthans, food-grade；Carragheen, food-grade.
Sepectrophotofluorometer fluoremetry parameter setting：Excitation wavelength is 436.0nm；Launch wavelength is 672.0nm；Swash
Hair and transmite slit width are 5.0nm；Sensitivity：high；Response time：Auto.
Embodiment 1. is using chlorella pyrenoidosa as trying algae
The chlorella pyrenoidosa (buy from Chinese Academy of Sciences's wildlife matter library --- fresh water algae library) of selection is algae,
Algae is seeded in the conical flask for filling sterilizing BG-11 culture solutions, then conical flask is placed in E-30B0 illumination boxs
Culture, condition of culture are：25 DEG C, illumination condition 2000lx of cultivation temperature, the time is set as 12h daytimes/12h nights, damp condition
75%RH, algae quiescent culture is daily to shake conical flask 2 times while change conical flask position, is taken out simultaneously as confession after ten days
Try algae solution；
25mL is taken to be placed in a centrifuge for examination algae solution, 15min is centrifuged with the rotating speed of 4000r/min, then outwells supernatant,
Frond is obtained, obtained frond and suspension stabilizer, water are then configured to algae solution sample, configured 100mL algae solutions sample is
Example, wherein a concentration of the 0.06% of agar, a concentration of the 0.2% of xanthans, a concentration of the 0.4% of carragheen；Then fluorescence is used
Method is measured algae solution.
2nd, the foundation of chlorella pyrenoidosa algae solution standard curve
It is 436nm in excitation wavelength according to the molecular absorption spectrum of substance and fluorescence spectrum transition principle, chlorophyll a
The fluorescence of 672nm is sent out, fluorescence intensity only has correlativity with the concentration of algae solution when exciting light is stablized, can be in the excitation and transmitting
The concentration of algae solution is measured under wavelength.But there are error is non-negligible, algae solution during measurement smaller due to the too low fluorescent value of algae solution concentration
Fluorescent value, which even declines in slow ascendant trend when concentration quenching has occurred, during excessive concentration causes numerical value inaccurate.Therefore
Its corresponding optimal D (663nm) range and corresponding fluorescence intensity level model are determined according to the standard curve of chlorella pyrenoidosa
It encloses.
It takes the logarithm the chlorella pyrenoidosa algae solution in growth period, is diluted with the BG-11 culture mediums of sterilizing, extension rate is
1-20 measures the extinction of algae sample series with 722 type visible spectrophotometers at the chlorophyll a characteristic absorption wavelength of 663nm
Angle value；The fluorescence intensity of algae sample series is measured under the fluoremetry parameter set with RF5301 sepectrophotofluorometers
Value draws the fluorescence intensity level of chlorella pyrenoidosa with its concentration (being reacted with the absorbance value of chlorophyll a characteristic wave strong point)
Situation of change.
Existed using 722 type spectrophotometers and Shimadzu RF5301 fluorescent spectrophotometer assay various concentration live body algae samples
The relationship of absorbance value and fluorescence intensity at optimal absorption wavelength has shown that the D (663nm) of chlorella pyrenoidosa sample exists
With fluorescence intensity level in good positive correlation, corresponding fluorescence intensity ranging from 40~77 when in the range of 0.3~0.8.
3rd, the characteristic absorption of suspension stabilizer is determined
0.1% agar solution, 0.3% xanthans are scanned at room temperature using the types of UV -2600 ultraviolet-uisible spectrophotometer
Solution and 0.5% carrageenan solutions, the absorbance value in 220~900nm wave-length coverages are determined without characteristic absorption.Therefore fine jade
The addition of fat, xanthans and carragheen is on frond spectrodensitometry result without influence.
4th, the influence that time of repose measures fluorescent value
The purpose of this experiment is：See whether frustule precipitates within a certain period of time, and whether suspension effect is good：Take 25mL albumen
Core chlorella algae solution is settled to 100mL with the BG-11 culture mediums of sterilizing, and the BG-11 culture mediums of sterilizing make as blank reference
With Shimadzu RF5301 sepectrophotofluorometers under the fluoremetry parameter set, initial fluorescent intensity value F is measured0=
89.383, measure the fluorescence intensity of an algae stoste every 1min.Establish the pass between interval time of measurement (T) and fluorescent value (F)
It is curve, establishes the relation table between interval time of measurement and fluorescent value, be shown in Table 1
Relationship between 1 interval time of measurement of table and fluorescent value
As shown in Table 1, fluorescent value is changed greatly with interval time of measurement, and during T=1min, the attenuation rate of fluorescent value reaches △ F/
F0=8.75%, there are large errors for measured value.This is because frustule sedimentation in standing 1min is very fast, algae stoste after 5min
System tends towards stability substantially, therefore it is required that being required when must shake up, measure before determination sample quickly, to reduce because frustule is because heavy
Error caused by drop.
5th, the algae solution sample stability research of suspension stabilizer
3 kinds of suspension stabilizers are chosen, are tested first with single suspension stabilizer, agar powder (0.02-0.10g/
100mL), xanthans (0.10-0.30g/100mL) and carragheen (0.30-0.50g/100mL).According to the algae in algae solution sample
The dosage standard of cell content and related suspension stabilizer chooses various concentration gradient and carries out parallel laboratory test.It primarily determines
After being suitble to the stabilizer of chlorella pyrenoidosa, then refinement experiment is carried out, each stabilizer additive amount and suspension effect are determined, such as table 2-
Shown in table 4.
Research of 5.1 agar to algae solution sample stability
The chlorella pyrenoidosa liquid for taking 5 parts of upgrowth situations identical, every part of 25mL are centrifuged under 4000r/min rotating speeds
15min then takes out the agar solution A that frond uses various concentration respectively1 #、A2 #、A3 #、A4 #、A5 #100mL is settled to, as algae
Liquid sample.Using Shimadzu RF5301 sepectrophotofluorometers a sample is measured every 1min under determining fluoremetry parameter
The fluorescence intensity level of product, and its attenuation rate is sought, and record the time that attenuation rate reaches 5%.
Influence of 2 agar of table to algae solution sample suspension stability
As shown in Table 2, suspension stabilizer is made with agar powder, makes its additive amount in the range of 0.08-0.10g/100mL, it can
Chlorella pyrenoidosa is made to suspend uniform, the stability of algae suspension system is good.
Research of 5.2 xanthans to algae solution sample stability
The chlorella pyrenoidosa liquid for taking 5 parts of upgrowth situations identical, every part of 25mL, in 4000r.min-1It is centrifuged under rotating speed
15min then takes out the xanthan gum solution C that frond uses various concentration respectively1 #、C2 #、C3 #、C4 #、C5 #100mL is settled to, is used
Shimadzu RF5301 sepectrophotofluorometers measure the fluorescence intensity of a sample under determining fluoremetry parameter every 1min
Value, and its attenuation rate is sought, and record the time that attenuation rate reaches 5%.
Influence of 3 xanthans of table to algae solution sample suspension stability
As shown in Table 3, suspension stabilizer is made with xanthans, makes its additive amount that can make albumen for 0.25-0.30g/100mL
Core chlorella suspends uniformly, and algae suspension system stability is good.
Research of 5.3 carragheens to algae solution sample stability
The chlorella pyrenoidosa liquid for taking 5 parts of upgrowth situations identical, every part of 25mL, in 4000r.min-1It is centrifuged under rotating speed
15min then takes out the carrageenan solutions D that frond uses various concentration respectively1 #、D2 #、D3 #、D4 #、D5 #100mL is settled to, is used
Shimadzu RF5301 sepectrophotofluorometers measure the fluorescence intensity of a sample under determining fluoremetry parameter every 1min
Value, and its attenuation rate is sought, and record the time that attenuation rate reaches 5%.
Influence of 4 carragheen of table to algae solution sample suspension stability
As shown in Table 4, suspension stabilizer is made with carragheen, makes its additive amount that can make albumen for 0.45-0.50g/100mL
Core chlorella suspends uniformly, and algae suspension system stability is good, and transparency is good.
The algae solution sample stability research of 5.4 composite suspension stabilizers
On the basis of experiment of single factor, comprehensive study is carried out to three kinds of agar, xanthans, carragheen suspension stabilizers.
Its mobility is combined with xanthans with agar and suspending stabilized ability is strong, but is long placed in and will appear supernatant.Composite suspension agent is configured,
I.e. agar, xanthans, carragheen carry out comprehensive study, optimize algae solution sample stabilising system.
5 composite suspension agent orthogonal test factor of table and level
6 composite suspension agent orthogonal experiments of table
According to table 5,6 orthogonal test of table as a result, by range analysis R1＞ R2＞ R3It is it is found that compound outstanding in trial stretch
To algae solution, the descending factor of suspending stabilized influential effect is followed successively by floating agent：Agar ＞ xanthans ＞ carragheens, formula
A3B2C2For optimum combination, i.e. agar 0.06%, xanthans 0.15%, carragheen 0.35%, at this time the change rate of fluorescent value for-
1.16%.
2. actual water sample of embodiment is for examination algae
The artificial lake water 4L of Hebei University of Science and Technology is acquired as actual water sample, 4 parts is equally divided into, obtains water sample 1, water sample 2, water
Sample 3, water sample 4, are placed in a centrifuge and are centrifuged, and outwell supernatant and obtain frond, and frond and suspension stabilizer, water are matched
It puts, constant volume to 100mL is sufficiently stirred, frustule is made to be evenly distributed in suspension liquid, obtains algae solution sample.It is glimmering with RF5301
The fluorescence intensity level of light spectrophotometric determination algae solution sample, by algae content in its fluorescent value intensity reaction water body, and according to
The change rate of fluorescence intensity level and the relative standard deviation of measured value reflect the suspension stability of algae solution sample solution.
The measure of actual water sample
The actual water sample 0 of suspension stabilizer is not added the water sample 1-4 for adding suspension stabilizer and by fluorescence spectrophotometry
Method is measured, and studies the stability of suspension system, as shown in table 7 below.
Relationship between 7 actual water sample interval time of measurement of table and fluorescent value
It can be obtained from table 7, with the stability of the fluctuation situation reaction suspension system of fluorescent value, in the experiment of 1h time stabilities
Obtain, do not add that the fluctuation of the actual water sample of suspension stabilizer its fluorescent value is larger, and add suspension stabilizer actual water sample its
The change rate of fluorescent value is in ± 3.5%, precision is higher within 1.5%, and the addition of suspension stabilizer can hang frustule
It is floating uniform.
Claims (5)
1. a kind of preparation method of the suspending stabilized sample of frond for fluorescence measurement prepares algae solution sample using for examination algae solution first
Then product are measured chlorophyll with fluorescence method, it is characterised in that：First centrifugal treating will be carried out for examination algae solution, then
Centrifugal treating is obtained into frond and is configured to algae solution sample with suspension stabilizer, wherein the suspension stabilizer is by agar, xanthan
Glue and carragheen form, agar in the suspension stabilizer：Xanthans：The content ratio of carragheen is (2-10)：(10-30)：
(30-50), and in sample algae solution agar a concentration of 0.06%.
2. a kind of preparation method of the suspending stabilized sample of frond for fluorescence measurement according to claim 1, feature
It is：It takes and is placed in a centrifuge for examination algae solution, 15-20min is centrifuged with the rotating speed of 3500-4500r/min, then outwells supernatant
Liquid obtains frond.
3. a kind of preparation method of the suspending stabilized sample of frond for fluorescence measurement according to claim 1, feature
It is：Agar in the suspension stabilizer：Xanthans：The content ratio of carragheen is 3：7.5：17.5, and fine jade in sample algae solution
A concentration of the 0.06% of fat.
4. a kind of preparation method of the suspending stabilized sample of frond for fluorescence measurement according to claim 1, feature
It is：Described for examination algae solution is chlorella pyrenoidosa algae solution, as laboratory cultures algae solution sample, before centrifugal treating,
First chlorella pyrenoidosa in chlorella pyrenoidosa algae solution is seeded in the conical flask for filling sterilizing BG-11 culture solutions and trained
It supports.
5. a kind of preparation method of the suspending stabilized sample of frond for fluorescence measurement according to claim 1, feature
It is：It is described to be derived from man-made lake water sample for examination algae solution, as actual water sample, when it is for examination algae solution to be derived from man-made lake water sample,
It to be immediately available for preparing algae solution sample or if you need to place addition 1mL1% magnesium carbonate suspension in then every liter of water sample after sampling,
And it is kept in dark place in 4 DEG C of refrigerators.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570785A (en) * | 2009-06-10 | 2009-11-04 | 南京大学 | Method for detecting potential inherent toxicity of organic pollutants in water body |
| CN103076297A (en) * | 2012-12-27 | 2013-05-01 | 河北科技大学 | Method for quickly and real-timely measuring water chlorophyll through replacing chlorophyll standard substance with microcystis aeruginosa |
| CN105044065A (en) * | 2015-08-07 | 2015-11-11 | 河北科技大学 | Preparation method for algae solution used for determining chlorophyll content through fluorescence method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570785A (en) * | 2009-06-10 | 2009-11-04 | 南京大学 | Method for detecting potential inherent toxicity of organic pollutants in water body |
| CN103076297A (en) * | 2012-12-27 | 2013-05-01 | 河北科技大学 | Method for quickly and real-timely measuring water chlorophyll through replacing chlorophyll standard substance with microcystis aeruginosa |
| CN105044065A (en) * | 2015-08-07 | 2015-11-11 | 河北科技大学 | Preparation method for algae solution used for determining chlorophyll content through fluorescence method |
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