Background technology
The human lives is among environment, and some composition in the environment may bring harm to people's life and health, therefore necessary environment is detected and monitors.But traditional physico-chemical analysis means can only be carried out single detection to some specific physical and chemical index such as temperature, pH value, dissolved oxygen DO etc., and the various materials in the environment are comprehensive cooperative effects to the influence of human body.Therefore, need the comprehensive effect of these materials be detected, bio-toxicity detects the technology that comes to this.
At present, bio-toxicity detects the method that is adopted has: based on the detection method of bacterial growth inhibition; Based on the detection method of bioenzyme activity, as the toxicity detection method of dehydrogenasa; Detection method based on the Daphnia magna class; Detection method based on algae; Based on detection method of fish etc.
In the middle of above-mentioned detection method, therefore the detection method that suppresses based on bacterial growth has obtained using the most widely owing to have advantages such as easy to detect, quick.
The cardinal principle of the detection method that suppresses based on bacterial growth is: adopt photobacteria, detect as the characteristics of luminescence of deep-sea fluorescent bacteria (Vibrio fischeri).The photobacteria bioluminescence reaction is by the molecular oxygen effect, luciferase catalysis in the cell, flavin mononucleotide (FMN) and the long-chain fat formoxy-of going back ortho states are turned to flavine acid (FMN) and long-chain fatty acid, in these special oxidation reaction process, can produce the 115K/mol free energy, these free energys can make the product of last generation be in excited state, thereby can produce photon.Generally speaking, the photobacteria bioluminescence is because the metabolism of bacterium itself causes, and metabolic influential to bacterium of the various biological toxants in the environment, thereby have influence on the luminous intensity of photobacteria, just can reach the purpose that synthetic biological toxicity in the environment is detected by detection like this luminous intensity.
Because optical detection has noncontact, various advantages such as convenient, so photobacteria growth inhibited detection method becomes the main method of present biological detection.But the photobacteria kind is less, and its living environment is limited, if only with the unique mark of photobacteria as the detection of environmental organism toxicity, can not adapt to the needs of various complex environments.
Summary of the invention
In order to solve above-mentioned deficiency of the prior art, the invention provides a kind of fluorescent characteristic of the common material in the microorganism metabolic processes of utilizing and carry out the new method that environmental organism toxicity detects, satisfied the needs that various environmental organism toxicity detect.
For achieving the above object, the method applied in the present invention is as follows:
A kind of detection method of bio-toxicity may further comprise the steps:
A, selection step
Select microorganism, include fluorescent material in the described microorganism;
B, blend step
In microorganism, add liquid to be measured, and mix; Include sample to be tested in the described liquid to be measured;
C, detection step
The light that light source sends arrives mixed liquor, excites described fluorescent material to send fluorescence;
Measure the fluorescence that described fluorescent material sends;
The variation of analysis of fluorescence obtains the bio-toxicity of sample to be tested.
As preferably, described microorganism is bacterium or fungi.
As preferably, in described step a, in the following way microorganism is mixed with solution:
According to the needs that detect, select bacterial classification;
With described bacterial classification inoculation in nutrient culture media;
When treating that described bacterial classification is cultivated exponential phase, be formulated as certain density bacteria suspension.
As preferably, in described step a, in the following way microorganism is mixed with solution:
According to the needs that detect, select bacterial classification;
With described bacterial classification inoculation in nutrient culture media;
When treating that described bacterial classification is cultivated exponential phase, make freeze dried powder;
Be formulated as certain density bacteria suspension.
Described fluorescent material is lactochrome, go back at least a in ortho states nicotinamide adenine dinucleotide, porphyrin, tryptophane, phenylalanine, the atriphos.
The light that light source sends comprises single or multiple excitation wavelengths, excites one or more fluorescent materials to send fluorescence.
As preferably, described microorganism is bacillus subtilis or saccharomyces cerevisiae.
The fluorescence that the continuous coverage fluorescent material sends.
Ultimate principle of the present invention is: in various cells, generally all contain lactochrome (riboflavin), go back ortho states nicotinamide adenine dinucleotide (NADH), porphyrin (porphyrin), tryptophane (tryptophan), phenylalanine (phenylalanine) and atriphos fluorescent materials such as (ATP), these materials can produce fluorescence under the exciting of extraneous exciting light.Simultaneously, these fluorescent materials all participate in the metabolism of cell again.Therefore, in case the toxicant of influential biology exists in the environment, will have influence on the situations such as concentration, valence state and distribution of these fluorescent materials in bacterium, the nicotinamide adenine dinucleotide (NADH) of for example going back ortho states has fluorescent effect, and the nicotinamide adenine dinucleotide of oxidation state (NAD) does not just have fluorescent effect.Therefore, utilize fluorescence method that concentration, valence state and the distribution of these fluorescent materials in the microorganism are detected, just can reach the testing goal of bio-toxicity.
Compared with prior art, the present invention has following beneficial effect:
Compare with photobacteria bio-toxicity detection method, topmost advantage is exactly a wide spectrum adaptability: the present invention adopts the fluorescent material that contains in the general cell, and the metabolism of pair cell detects, thereby reaches the purpose that detects the sample bio-toxicity.Therefore, the present invention need not to be confined to photogen, but can be according to the needs of actual sample to be tested, selection is used as " sensor " to the most responsive microorganisms such as bacterium of material in the tested sample and carries out optical detection, has so just improved the scope of application and detection sensitivity that bio-toxicity is measured greatly.Therefore, the range of choice that can be used as the microbe species of environmental organism toxicity detection enlarges greatly, helps satisfying the demand of various different occasions.
At present, detection technique of fluorescence is very ripe; Simultaneously,,, optionally metabolic unlike signal thing is detected, reach the purpose of more, the more suitable detection information of extraction so can also pass through the selection of the excitation source of different wave length because fluorescence method is used excitation source.
Embodiment
Below in conjunction with embodiment, the present invention is done further detailed description.
Embodiment 1:
A kind of detection method of bio-toxicity may further comprise the steps:
A, selection step
According to the needs that detect sample to be tested, select bacillus subtilis, contain fluorescent material in the bacillus subtilis, as lactochrome, go back ortho states nicotinamide adenine dinucleotide and atriphos;
Adopt Bacillus subtillis nutrient culture media commonly used to cultivate, wait to cultivate exponential phase, the bacteria suspension that is formulated as debita spissitudo adds in the testing tube;
B, blend step
In testing tube, add liquid to be measured, as contain the solution of sample to be tested mercuric chloride, make it fully to contact with bacterial classification;
C, detection step
Light source sends measuring light, measures light wavelength and comprises 270nm, 292nm and 344nm, and described measuring light arrives mixed liquor;
Wherein, the lactochrome in the optical excitation Bacillus subtillis of 270nm sends the ortho states nicotinamide adenine dinucleotide of going back that atriphos in the optical excitation Bacillus subtillis of fluorescence, 292nm sends in the optical excitation Bacillus subtillis of fluorescence, 344nm and sends fluorescence;
Record fluorescence that lactochrome sends at the 518nm place, record fluorescence that atriphos sends at the 388nm place, record at the 465nm place and go back the fluorescence that the ortho states nicotinamide adenine dinucleotide is sent; Closed described light source 10 minutes;
Open light source afterwards, record once more: the fluorescence that sends at 518nm place lactochrome, the fluorescence that sends at 388nm place atriphos, go back the fluorescence that the ortho states nicotinamide adenine dinucleotide is sent at the 465nm place;
The variation of the fluorescence intensity that twice records relatively, thus the bio-toxicity of sample to be tested obtained.
Embodiment 2:
A kind of detection method of bio-toxicity, as different from Example 1:
Step a is:
According to the needs that detect sample to be tested, select bacillus subtilis, contain fluorescent material in the bacillus subtilis, as lactochrome, go back ortho states nicotinamide adenine dinucleotide and atriphos;
Adopt Bacillus subtillis nutrient culture media commonly used to cultivate, wait to cultivate exponential phase, make freeze dried powder with freeze-drying;
Add solution in freeze dried powder, make bacillus subtilis return to original physiological status, the bacteria suspension that is formulated as debita spissitudo adds in the testing tube.
Embodiment 3:
A kind of detection method of bio-toxicity, as different from Example 2:
Step c is:
Light source sends measuring light, measures light wavelength and comprises 270nm, 292nm and 344nm, and described measuring light arrives mixed liquor;
Wherein, the lactochrome in the optical excitation Bacillus subtillis of 270nm sends the ortho states nicotinamide adenine dinucleotide of going back that atriphos in the optical excitation Bacillus subtillis of fluorescence, 292nm sends in the optical excitation Bacillus subtillis of fluorescence, 344nm and sends fluorescence;
Record fluorescence that lactochrome sends at the 518nm place, record fluorescence that atriphos sends at the 388nm place, record at the 465nm place and go back the fluorescence that the ortho states nicotinamide adenine dinucleotide is sent;
Kept fluorescence measurement 5 minutes, with the dynamic data that obtains reacting always; According to the dynamic change situation of fluorescence, obtain the bio-toxicity of sample to be tested.
Embodiment 4:
A kind of detection method of bio-toxicity may further comprise the steps:
A, selection step
According to the needs that detect sample to be tested, select saccharomyces cerevisiae, contain fluorescent material in the saccharomyces cerevisiae, as lactochrome, go back ortho states nicotinamide adenine dinucleotide and atriphos;
Adopt saccharomyces cerevisiae nutrient culture media commonly used to cultivate, wait to cultivate exponential phase, the bacteria suspension that is formulated as debita spissitudo adds in the testing tube;
B, blend step
In testing tube, add liquid to be measured, as contain the solution of sample to be tested mercuric chloride, make it fully to contact with bacterial classification;
C, detection step
Light source sends measuring light, measures light wavelength and comprises 270nm, 292nm and three excitation wavelengths of 344nm, and described measuring light arrives mixed liquor;
Wherein, the lactochrome in the optical excitation Bacillus subtillis of 270nm sends the ortho states nicotinamide adenine dinucleotide of going back that atriphos in the optical excitation Bacillus subtillis of fluorescence, 292nm sends in the optical excitation Bacillus subtillis of fluorescence, 344nm and sends fluorescence;
Adopt fluorophotometer to measure in the 250-700nm respectively by lactochrome, atriphos, go back the fluorescence that the ortho states nicotinamide adenine dinucleotide is sent; Closed described light source 10 minutes;
Open light source afterwards, adopt fluorophotometer to measure in the 250-700nm respectively by lactochrome, atriphos, go back the fluorescence that the ortho states nicotinamide adenine dinucleotide is sent once more;
The variation of the fluorescence intensity that twice records relatively obtains the bio-toxicity of sample to be tested.
Embodiment 5:
A kind of detection method of bio-toxicity may further comprise the steps:
A, selection step
According to the needs that detect sample to be tested, select bacillus subtilis, contain fluorescent material in the bacillus subtilis, as tryptophane;
Adopt bacillus subtilis nutrient culture media commonly used to cultivate, wait to cultivate exponential phase, the bacteria suspension that is formulated as debita spissitudo adds in the testing tube;
B, blend step
In testing tube, add liquid to be measured, as contain the solution of sample to be tested mercuric chloride, make it fully to contact with bacterial classification;
C, detection step
Light source sends measuring light, and the measurement light wavelength is 280nm, and the tryptophane in the optical excitation Bacillus subtillis of 280nm sends fluorescence, and described measuring light arrives mixed liquor;
Record the fluorescence that tryptophane sends at the 357nm place; Closed described light source 10 minutes;
Open light source afterwards, record the fluorescence that tryptophane sends at the 357nm place once more;
The variation of the fluorescence intensity that twice records relatively obtains the bio-toxicity of sample to be tested.
It is pointed out that above-mentioned embodiment should not be construed as limiting the scope of the invention.Key of the present invention is to utilize the fluorescent characteristic of the common material in the microorganism metabolic processes to carry out the detection of environmental organism toxicity.Under the situation that does not break away from spirit of the present invention, any type of change that the present invention is made all should fall within protection scope of the present invention.