CN107884349A - The assay method of ultra-oxygen anion free radical in a kind of microbial body - Google Patents
The assay method of ultra-oxygen anion free radical in a kind of microbial body Download PDFInfo
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- 239000001301 oxygen Substances 0.000 title claims abstract description 42
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 42
- 230000000813 microbial effect Effects 0.000 title claims abstract description 21
- 238000003556 assay Methods 0.000 title claims abstract description 17
- 238000012360 testing method Methods 0.000 claims abstract description 66
- 239000007853 buffer solution Substances 0.000 claims abstract description 37
- 239000012472 biological sample Substances 0.000 claims abstract description 32
- 230000002906 microbiologic effect Effects 0.000 claims abstract description 32
- 238000002835 absorbance Methods 0.000 claims abstract description 28
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims abstract description 24
- 239000002028 Biomass Substances 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 99
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 44
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims description 36
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 24
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- NULAJYZBOLVQPQ-UHFFFAOYSA-N N-(1-naphthyl)ethylenediamine Chemical compound C1=CC=C2C(NCCN)=CC=CC2=C1 NULAJYZBOLVQPQ-UHFFFAOYSA-N 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 20
- 238000000605 extraction Methods 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 19
- 229940124530 sulfonamide Drugs 0.000 claims description 18
- 150000003456 sulfonamides Chemical class 0.000 claims description 18
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 15
- 238000011088 calibration curve Methods 0.000 claims description 15
- 238000005119 centrifugation Methods 0.000 claims description 14
- 238000002525 ultrasonication Methods 0.000 claims description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 8
- 238000009472 formulation Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 238000002474 experimental method Methods 0.000 claims description 7
- 239000005457 ice water Substances 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 7
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Substances [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 7
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 150000001412 amines Chemical class 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000010802 sludge Substances 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- 230000001186 cumulative effect Effects 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000005070 sampling Methods 0.000 claims description 2
- 150000004985 diamines Chemical class 0.000 claims 2
- 238000000034 method Methods 0.000 abstract description 16
- 244000005700 microbiome Species 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000008363 phosphate buffer Substances 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 9
- 238000000703 high-speed centrifugation Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 6
- -1 hydroxyl radical free radical Chemical class 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000010865 sewage Substances 0.000 description 4
- RAHZWNYVWXNFOC-UHFFFAOYSA-N Sulphur dioxide Chemical group O=S=O RAHZWNYVWXNFOC-UHFFFAOYSA-N 0.000 description 3
- 238000006701 autoxidation reaction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- UCTWMZQNUQWSLP-UHFFFAOYSA-N adrenaline Chemical compound CNCC(O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-UHFFFAOYSA-N 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 238000004939 coking Methods 0.000 description 2
- 239000003344 environmental pollutant Substances 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 2
- 150000005206 1,2-dihydroxybenzenes Chemical class 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229930194542 Keto Natural products 0.000 description 1
- 230000010718 Oxidation Activity Effects 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- ISWQCIVKKSOKNN-UHFFFAOYSA-L Tiron Chemical compound [Na+].[Na+].OC1=CC(S([O-])(=O)=O)=CC(S([O-])(=O)=O)=C1O ISWQCIVKKSOKNN-UHFFFAOYSA-L 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 231100000567 intoxicating Toxicity 0.000 description 1
- 230000002673 intoxicating effect Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- FSVCQIDHPKZJSO-UHFFFAOYSA-L nitro blue tetrazolium dichloride Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 FSVCQIDHPKZJSO-UHFFFAOYSA-L 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229940079877 pyrogallol Drugs 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000013319 spin trapping Methods 0.000 description 1
- 239000002912 waste gas Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- 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
-
- 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
- G01N1/34—Purifying; Cleaning
-
- 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
- G01N1/38—Diluting, dispersing or mixing samples
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- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of assay method of ultra-oxygen anion free radical in microbial body, specific method are as follows：Micro-biological samples are extracted first, formulate standard curve, then test group, control group and blank group are set, the measure of absorbance is carried out to biological specimen respectively, then absorbance of the difference of three groups of absorbance as microorganism in test group is tried to achieve, standard curve and formula is recycled to try to achieve the concentration value of ultra-oxygen anion free radical in microbial body, then by O in formula accounting unit quality biomass body2 ‑.Content, complete the measure of ultra-oxygen anion free radical in microbial body；The present invention is using the carbonate buffer solution that pH is 9.5~10.5, and the phosphate buffer for being 7.8 compared to traditional pH used, pH are 7.6Tris HCl buffer solutions, and system to be measured is to detecting O2 ‑.Sensitivity improve nearly 10 times, and the inventive method selectivity is preferable, simple to operate, take short, easy popularization.
Description
Technical field
The present invention relates to a kind of assay method of ultra-oxygen anion free radical in microbial body, belong to the life of waste water, waste gas
Thing purification treatment technology field.
Background technology
Ultra-oxygen anion free radical (O2 -.) belong to active oxygen group, there is stronger oxidisability.O2 -.In microorganism system
Generally existing, when organism is coerced by pollutant, the generation of activity in vivo oxygen will greatly increase；When it is beyond organism
During anti-oxidative defense ability, the cell component such as lipid, protein, nucleic acid will be in O in biology2 -.Effect is lower to occur irreversible damage
Bad, so as to which interference cell is metabolized or causes cell death, therefore ultra-oxygen anion free radical is also considered as pollutant intoxicating
Important channel and mechanism.In addition, also there are some researches show active oxygen participates in the induction of cell signalling and physiological responses, is thin
A kind of important signaling molecule of born of the same parents.
The active oxygen life-span is extremely short in organism, as the time-to-live of ultra-oxygen anion free radical in aqueous is
1s, the life-span of hydroxyl radical free radical is only 10-6S, activity keto concentration is extremely low in microbial body in addition, causes its qualitative and quantitative detection
It is extremely difficult.Common Measurement Method for Superoxide Anion Radical have NBT (chlorination nitro tetrazolium blue) method, cytochromes method,
Pyrogallol method, adrenaline method and azanol oxidizing process.Cromoci with oxidation activity by ultra-oxygen anion free radical also
After original, form has the ferricytochrome absorbed by force at wavelength 550nm, can be used for the survey of ultra-oxygen anion free radical
It is fixed；But test will be tied if there is other reducing substanceses (such as reproducibility enzyme) in the system of cromoci reducing process
Fruit causes to significantly interfere with.Under superoxide anion radical effect, reduction generates not soluble in water, blue diformazan and replaced NBT
(Diformazan), a length of 560nm of its maximum absorption wave；But diformazan is placed deposited phenomenon occurs for a long time for not soluble in water,
It is dfficult to apply to determine dynamic studies process of the ultra-oxygen anion free radical with time lengthening.Traditional azanol oxidizing process exists low
Content O2 -.Detection difficult, butanol extraction liquid and reaction medium layering unobvious, azanol autoxidation influence test effect etc. no
Foot.Ultra-oxygen anion free radical can also generate a kind of claim with 1,2- dihydroxy benzenes -3,5- sodium disulfonate (Tiron) fast reaction
The spin adduct for " Tiron Semiquinone Radicals ", applying electronic EPR spectrometer (EPR) can be examined at room temperature
Survey；The experiment condition of EPR Spin trapping techniques requirement is stricter, and needs the so expensive analytical and testing instruments of EPR, because
And limit popularizing for the technology.
Ultra-oxygen anion free radical content is relatively low in microbial body, and traditional azanol oxidizing process needs the concentration of oxygen radical
It is of a relatively high；In addition, disturbing factor is more when ultra-oxygen anion free radical is tested, measurement result is sometimes and unstable.At present, also
There is not the method for Concentration of Superoxide Anion Radical in effective measure microbial body, therefore, exploitation operation is relatively simple, surveys
The assay method of ultra-oxygen anion free radical is significant in the high microbial body of examination degree of stability.
The content of the invention
It is an object of the invention to provide a kind of assay method of ultra-oxygen anion free radical in microbial body, this method base
It can be reacted in azanol with the ultra-oxygen anion free radical in microbial body, generate the principle of pink azo-compound, surveyed
Determine Concentration of Superoxide Anion Radical in microbial body, the inventive method is simple to operate, stable testing.
Due to hydroxylamine hydrochloride autoxidation, to add hydroxylamine hydrochloride group as blank group in test, hydrochloric acid hydroxyl can be effectively eliminated
Amine autoxidation and cause measurement result higher.Secondly, to be not added with hydroxylamine hydrochloride group as control group, remaining pigment can effectively be eliminated
And endogenous NO2-Interference.Therefore, in continuous mode, O2-.The extinction of light absorption value-control group of actual A530 values=test group
The light absorption value of value-blank group.
The present invention sets test group, control group and blank group, and carbonic acid is separately added into test group, control group and blank group
Cushioning liquid, and hydroxylamine hydrochloride solution is added into test group and blank group, control group adds the distilled water of same volume, then
Three groups are placed in water bath with thermostatic control simultaneously, supernatant is then added into test group and control group, same volume is added in blank group
Long-pending carbonic acid buffer, and being stood in a water bath after shaking up respectively, then again into test group, control group and blank group respectively according to
It is secondary to add to basic sulfonamide and N-1- naphthyl ethylenediamines, then develop the color in a water bath, extraction is added in the solution after three groups of colour developings
Agent is taken, centrifugal treating is carried out respectively after being sufficiently mixed uniformly, then takes upper strata extraction phase respectively, determines examination at 540nm respectively
The absorbance of group, control group and blank group is tested, the absorbance of measure is asked poor, standard curve side is brought into using this difference as y
Journey, x values are calculated, NO in test group is as corresponded at 540nm2 -Concentration, then by O in formula accounting unit quality biomass body2 -.
Content, that is, complete the measure of ultra-oxygen anion free radical in microbial body.
The assay method of ultra-oxygen anion free radical, concrete operations are as follows in a kind of microbial body：
(1) by activated sludge suspension of the biomass in micro-biological samples more than 800mg/L in 10000~12000r/min
Under the conditions of centrifuge 8~10min, take centrifugation after bottom 1~2g of micro-biological samples, after centrifugation bottom micro-biological samples deficiency
During 1g, repeated sampling adds up to 1~2g, and it is 9.5~10.5 carbonate buffer solutions that pH is then added into micro-biological samples, is added
Measure and add 3.5~4.5mL for every 1g micro-biological samples, then mixed solution is placed in ice-water bath and carries out ultrasonication, finally
Centrifuging and taking supernatant, wherein ultrasonication time are 2~3min, during centrifuging and taking supernatant centrifugal rotational speed be 10000~
120000r/min；
(2) formulation of standard curve：Use NaNO2Prepare NO of 7 groups of concentrations above for 0~30 μm of ol/L2 -Standard liquid, point
200mL is not settled to, then takes the blank cuvette of more than 7 to be separately added into carbonate buffer solution that pH is 9.5~10.5 and dense
Spend and add 0.5 for 50~100mmol/L hydroxylamine hydrochloride solutions, the wherein addition of carbonate buffer solution for every 1g micro-biological samples
~1mL, the addition of hydroxylamine hydrochloride solution are to add 0.1~0.3mL per 1g micro-biological samples, and each group solution then is placed in into 25
10~15min is heated in~35 DEG C of water bath with thermostatic control, the different NO of 0.5mL concentration is then added into each group solution2 -Standard is molten
Liquid, 18~22min is stood after shaking up in 25~35 DEG C of water bath with thermostatic control, then sequentially added into each group solution to basic sulphonyl
Amine aqueous solution and N-1- naphthyl ethylenediamine solution, wherein being by basic sulphur to basic sulfonamide solution and N-1- naphthyl ethylenediamine solution
Acid amides solid and N-1- naphthyl ethylenediamines solid are dissolved in the acetic acid solution that concentration is 10~13mol/L and obtained respectively, to basic sulphur
Amide solution and N-1- naphthyl ethylenediamine solution concentrations are respectively 55~60mmol/L and 5~10mmol/L, and addition is per 1g
Micro-biological samples add 0.8~1mL to basic sulfonamide solution and N-1- naphthyl ethylenediamine solution, are subsequently placed in water-bath and develop the color
20~30min, then extractant is added into each group solution, centrifugal treating is carried out respectively after being sufficiently mixed uniformly, and is taken respectively
Layer extraction phase, the absorbance of each group is determined at 540nm, using the absorbance of measure as y values, by the NO of each group2-Concentration is made
For x values, calibration curve equation is obtained after linear fitting, wherein the volume for adding extractant is each group liquor capacity before addition
0.8~1.2 times, extractant is dichloromethane, chloroform or toluene；
(3) test group, control group and blank group are set, carbonate buffer solution is separately added into in three groups and shakes up, and to
Hydroxylamine hydrochloride solution is added in test group and blank group, control group adds the distilled water of same volume, and 25 are placed in simultaneously by three groups
10~15min is heated in~35 DEG C of water bath with thermostatic control, 0.5mL supernatants, blank group are then added into test group and control group
It is middle to add the carbonic acid buffer of same volume, and 18~22min is stood in 25~35 DEG C of water bath with thermostatic control after shaking up respectively, so
It is sequentially added into again into test group, control group and blank group afterwards molten to basic sulfonamide solution and N-1- naphthyl ethylenediamines
Liquid, then develop the color 20~30min in a water bath, wherein the same step of concentration and addition (2) of each solution；
(4) extractant is added into the solution after three groups of colour developings of step (3), is centrifuged respectively after being sufficiently mixed uniformly
Processing, then takes upper strata extraction phase respectively, and at 540nm determination test group, control group and blank group absorbance, wherein institute
With the same step of extractant (2), the wherein time of centrifugal treating is 3~5min；
(5) absorbance that step (4) determines is asked poor, then brings the calibration curve equation of step (1) into, try to achieve
NO in test group corresponding to 540nm places2 -Concentration, then by O in formula accounting unit quality biomass body2 -.Content, that is, complete micro-
The measure of ultra-oxygen anion free radical in organism, wherein formula areWhereinFor x values,
Unit is μm olL-1, V1For the volume of test group sample to be tested, unit mL, V2After step (2) test group ultrasonication
Centrifuging and taking supernatant cumulative volume, unit mL, ρ are test group sample to be tested sludge solid concentration, unit mgL-1, m1For
The quality of micro-biological samples, unit g, m after test group step (2) centrifugation2After (2) ultrasonication of test group step and centrifuging
The quality of deposit, unit g.
The formulation standard curve and detection sample, the addition of the identical reagent of different test tubes are consistent.
Advantages of the present invention and technique effect：
1st, the present invention uses the carbonate buffer solution that pH is 9.5~10.5, the phosphoric acid for being 7.8 compared to the pH that tradition uses
Buffer solution, pH are 7.6Tris-HCl buffer solutions, and system to be measured is to detecting O2 -.Sensitivity improve nearly 10 times.
2nd, extractant of the invention is toluene, chloroform or dichloromethane, when using dichloromethane as extractant, extraction
Test system liquid phase upper strata is mutually in, and, the absorbance highest of to be measured system obvious with raffinate phase layering.
3rd, use of the invention to basic sulfonamide and N-1- naphthyl ethylenediamines measure ultra-oxygen anion free radical selectivity compared with
It is good, simple to operate, time-consuming short, easy popularization.
Embodiment
The present invention is described in further detail below by embodiment, but the scope of the present invention is not limited in described
Hold.
Embodiment 1：The assay method of ultra-oxygen anion free radical in micro-biological samples body after hydrogen phosphide domestication, specific behaviour
Make as follows：
The suspended 20mL of microorganism after hydrogen phosphide domestication is taken, wherein biomass is about 5000mg/L, in 10000r/min
Under the conditions of centrifuge 8min, take centrifugation bottom of the tube micro-biological samples 1g, add pH be 9.5 carbonate buffer solution 3.5mL, by micro- life
Thing sample is placed in ice-water bath and centrifuges 10min under 10000r/min after ultrasonication 2min, takes supernatant to be used for measuring；
The formulation of calibration curve equation：With NaNO2It is 3,6,9,12,15,18,21 and 24 μm of ol/L to prepare 8 groups of concentration
NO2 -Standard liquid, 8 test tubes are taken, be separately added into the carbonate buffer solution that 0.5mL pH are 9.5, then add 0.1mL concentration
For 70mmol/L hydroxylamine hydrochloride solution, then each group solution is placed in 30 DEG C of water bath with thermostatic control and heats 12min, then to each
The NO that 0.5mL concentration is respectively 3,6,9,12,15,18,21 and 24 μm of ol/L is added in group solution2 -Standard liquid, after shaking up
20min is stood in 30 DEG C of water bath with thermostatic control, then it is 58mmol/L to basic sulphonyl to sequentially add into each group solution 0.8mL concentration
Amine aqueous solution (being dissolved in 10mol/L acetic acid) and the N-1- naphthyl ethylenediamines solution that 0.8mL concentration is 7mmol/L (are dissolved in 10mol/L second
Acid), the 25min that developed the color in 30 DEG C of water-baths is placed in, then adds extractant dichloromethane into each group solution again, added volume is
1 times of above overall solution volume, be sufficiently mixed uniformly after carry out centrifugal treating respectively, under conditions of 10000r/min at a high speed from
Heart 3min, and take upper strata extraction phase respectively, the absorbance of each group is determined at 540nm, using the absorbance of measure as y values,
By the NO of each group2 -Concentration obtains calibration curve equation y=0.0184x+0.1909 (R=as x values after linear fitting
0.9994)；
Test group, control group and blank group are set, carbonate buffer solution is separately added into in three groups and shakes up, and to experiment
Hydroxylamine hydrochloride solution is added in group and blank group, control group adds the distilled water of same volume, and 30 DEG C are placed in simultaneously by three groups
12min is heated in water bath with thermostatic control, 0.5mL supernatants are then added into test group and control group, same volume is added in blank group
Long-pending carbonic acid buffer, and 20min is stood in 30 DEG C of water bath with thermostatic control after shaking up respectively, then again to test group, control group
Be sequentially added into blank group to basic sulfonamide solution and N-1- naphthyl ethylenediamine solution, then develop the color in a water bath
25min, wherein the concentration of added each solution and the same step of addition (2), then add pigment extraction liquid two into three groups of solution
Chloromethanes, the high speed centrifugation 3min under conditions of 10000r/min, three groups of pink upper strata extraction phase is taken respectively and in 540nm
Absorbance is determined, tries to achieve three groups of A530Difference is 0.258, brings calibration curve equation into, tries to achieve x, further according to formulaIt is 0.311 μm of olg that test group concentration of oxygen free radicals, which is calculated,-1。
Embodiment 2：The assay method of ultra-oxygen anion free radical in the micro-biological samples body of municipal sewage plant, specifically
Operation is as follows：
The microorganism suspension 20mL of municipal sewage plant is taken, wherein biomass is about 6800mg/L, in 11000r/
9min is centrifuged under the conditions of min, takes centrifugation bottom of the tube micro-biological samples 1.5g, adds the carbonate buffer solution 6mL that pH is 10.00,
Micro-biological samples are placed in ice-water bath after ultrasonication 2.5min and centrifuge 10min under 11000r/min, take supernatant to be used for
Measuring.
The formulation of calibration curve equation：With NaNO2It is respectively 4,8,12,16,20,24,28 μm of ol/L to prepare 7 groups of concentration
NO2 -Standard liquid, 7 test tubes are taken, be separately added into the carbonate buffer solution that 1.5mL pH are 10.0, then add the dense of 0.45mL
The hydroxylamine hydrochloride solution for 100mmol/L is spent, then each group solution is placed in 35 DEG C of water bath with thermostatic control and heats 10min, then
The NO that 0.5mL concentration is respectively 4,8,12,16,20,24,28 μm of ol/L is added into each group solution2 -Standard liquid, after shaking up
18min is stood in 35 DEG C of water bath with thermostatic control, then it is 55mmol/L to basic sulphonyl to sequentially add into each group solution 1.5mL concentration
Amine aqueous solution (being dissolved in 11mol/L acetic acid) and the N-1- naphthyl ethylenediamines solution that 1.5mL concentration is 5mmol/L (are dissolved in 11mol/L second
Acid), the 20min that developed the color in 35 DEG C of water-baths is placed in, then adds extractant toluene into each group solution again, more than added volume is
0.8 times of overall solution volume, centrifugal treating is carried out respectively after being sufficiently mixed uniformly, and take upper strata extraction phase respectively, at 540nm
The absorbance of each group is determined, using the absorbance of measure as y values, by the NO of each group2 -Concentration is as x values, after linear fitting
Obtain calibration curve equation y=0.0115x+0.1622 (R=0.9997)；
Test group, control group and blank group are set, carbonate buffer solution is separately added into in three groups and shakes up, and to experiment
Hydroxylamine hydrochloride solution is added in group and blank group, control group adds the distilled water of same volume, and 35 DEG C are placed in simultaneously by three groups
10min is heated in water bath with thermostatic control, 0.5mL supernatants are then added into test group and control group, same volume is added in blank group
Long-pending carbonic acid buffer, and 18min is stood in 35 DEG C of water bath with thermostatic control after shaking up respectively, then again to test group, control group
Be sequentially added into blank group to basic sulfonamide solution and N-1- naphthyl ethylenediamine solution, then show in 35 DEG C of water-baths
Color 20min, wherein the concentration of added each solution and the same step of addition (2), then add toluene into three groups of solution,
High speed centrifugation 4min under the conditions of 10000r/min, three groups of pink upper strata extraction phase is taken respectively and determines absorbance in 540nm,
Try to achieve three groups of A530Difference is 0.209, substitutes into standard curve and tries to achieve x again by formulaExamination is calculated
It is 0.102 μm of olg to test group concentration of oxygen free radicals-1。
Embodiment 3：The assay method of ultra-oxygen anion free radical in the micro-biological samples body of Treatment of Coking Effluent factory, specifically
Operation is as follows：
The microorganism suspension 20mL of Treatment of Coking Effluent factory is taken, wherein biomass is about 4500mg/L, in 12000r/
10min is centrifuged under the conditions of min, takes centrifugation bottom of the tube micro-biological samples 2g, adds the carbonate buffer solution 8mL that pH is 10.00, will
Micro-biological samples are placed in ice-water bath and centrifuge 10min under 11000r/min after ultrasonication 2.5min, take supernatant to be used for real
Test is determined；
The formulation of calibration curve equation：With NaNO2Compound concentration is 3,6,9,12,15,18,21 and 24 μm of ol/L NO2 -
Standard liquid, 8 test tubes are taken, be separately added into the carbonate buffer solution that 1mL pH are 10.0, the concentration for then adding 0.3mL is
80mmol/L hydroxylamine hydrochloride solution, then each group solution is placed in 25 DEG C of water bath with thermostatic control and heats 15min, then to each group
The NO that 0.5mL concentration is respectively 3,6,9,12,15,18,21 and 24 μm of ol/L is added in solution2 -Standard liquid, 25 after shaking up
DEG C water bath with thermostatic control in stand 22min, then it is 60mmol/L to basic sulfonamide to sequentially add into each group solution 0.9mL concentration
Solution (being dissolved in 13mol/L acetic acid) and the N-1- naphthyl ethylenediamines solution that 0.9mL concentration is 10mmol/L (are dissolved in 13mol/L second
Acid), the 30min that developed the color in 25 DEG C of water-baths is placed in, then adds extractant dichloromethane into each group solution again, added volume is
1.1 times of above overall solution volume, centrifugal treating is carried out respectively after being sufficiently mixed uniformly, high speed centrifugation under the conditions of 10000r/min
5min, and upper strata extraction phase is taken respectively, the absorbance of each group is determined at 540nm, will using the absorbance of measure as y values
The NO of each group2 -Concentration obtains calibration curve equation y=0.0201x+0.1045 (R=as x values after linear fitting
0.9992)；
Test group, control group and blank group are set, carbonate buffer solution is separately added into in three groups and shakes up, and to experiment
Hydroxylamine hydrochloride solution is added in group and blank group, control group adds the distilled water of same volume, and 25 DEG C are placed in simultaneously by three groups
15min is heated in water bath with thermostatic control, 0.5mL supernatants are then added into test group and control group, same volume is added in blank group
Long-pending carbonic acid buffer, and 22min is stood in 25 DEG C of water bath with thermostatic control after shaking up respectively, then again to test group, control group
Be sequentially added into blank group to basic sulfonamide solution and N-1- naphthyl ethylenediamine solution, then show in 25 DEG C of water-baths
Color 30min, wherein the concentration of added each solution and the same step of addition (2), then add dichloromethane into three groups of solution,
High speed centrifugation 5min under the conditions of 10000r/min, three groups of pink upper strata extraction phase is taken respectively and determines absorbance in 540nm,
Try to achieve three groups of A530Difference is 0.279, substitutes into standard curve and tries to achieve x again by formulaExamination is calculated
It is 0.478 μm of olg to test group concentration of oxygen free radicals-1。
Embodiment 4：The assay method of ultra-oxygen anion free radical in the micro-biological samples body of municipal sewage plant, specifically
Operation is as follows：
The microorganism suspension 20mL of municipal sewage plant is taken, wherein biomass is about 6300mg/L, in 12000r/
10min is centrifuged under the conditions of min, takes centrifugation bottom of the tube micro-biological samples 2g, adds the carbonate buffer solution 9mL that pH is 10.50, will
Micro-biological samples are placed in ice-water bath and centrifuge 10min under 12000r/min after ultrasonication 3min, take supernatant to be used to test
Measure；
The formulation of calibration curve equation：With NaNO2It is 3,6,9,12,15,18,21 and 24 μm of ol/L to prepare 8 groups of concentration
NO2 -Standard liquid, 8 test tubes are taken, be separately added into the carbonate buffer solution that 2mL pH are 9.5, the concentration for then adding 0.6mL is
50mmol/L hydroxylamine hydrochloride solution, then each group solution is placed in 30 DEG C of water bath with thermostatic control and heats 10min, then to each group
The NO that 0.5mL concentration is respectively 3,6,9,12,15,18,21 and 24 μm of ol/L is added in solution2 -Standard liquid, 30 after shaking up
DEG C water bath with thermostatic control in stand 22min, then to sequentially add into each group solution 2mL concentration molten to basic sulfonamide for 58mmol/L
Liquid (being dissolved in 12mol/L acetic acid) and the N-1- naphthyl ethylenediamines solution (being dissolved in 12mol/L acetic acid) that 2mL concentration is 7mmol/L, and
The 30min that develops the color is placed in 30 DEG C of water-baths, then adds extractant chloroform into each group solution again, added volume is molten for more than
1 times of liquid cumulative volume, centrifugal treating is carried out respectively after being sufficiently mixed uniformly, high speed centrifugation 3.2min under the conditions of 10000r/min,
And upper strata extraction phase is taken respectively, the absorbance of each group is determined at 540nm, using the absorbance of measure as y values, by each group
NO2 -Concentration obtains calibration curve equation y=0.0104x+0.1922 (R=0.9997) as x values after linear fitting；
Test group, control group and blank group are set, carbonate buffer solution is separately added into in three groups and shakes up, and to experiment
Hydroxylamine hydrochloride solution is added in group and blank group, control group adds the distilled water of same volume, and 30 DEG C are placed in simultaneously by three groups
10min is heated in water bath with thermostatic control, 0.5mL supernatants are then added into test group and control group, same volume is added in blank group
Long-pending carbonic acid buffer, and 22min is stood in 30 DEG C of water bath with thermostatic control after shaking up respectively, then again to test group, control group
Be sequentially added into blank group to basic sulfonamide solution and N-1- naphthyl ethylenediamine solution, then show in 30 DEG C of water-baths
Color 30min, wherein the concentration of added each solution and the same step of addition (2), then add chloroform into three groups of solution,
High speed centrifugation 3.2min under the conditions of 10000r/min, three groups of pink upper strata extraction phase is taken respectively and determines extinction in 540nm
Degree, tries to achieve three groups of A530Difference is 0.301, substitutes into standard curve and tries to achieve x again by formulaCalculate
It is 0.589 μm of olg to test group concentration of oxygen free radicals-1。
Embodiment 5：The assay method of ultra-oxygen anion free radical, specific behaviour in micro-biological samples body in denitrogenation drop filter tower
Make as follows：
The microorganism suspension 20mL in denitrogenation drop filter tower is taken, wherein biomass is about 8200mg/L, in 12000r/
10min is centrifuged under the conditions of min, takes centrifugation bottom of the tube micro-biological samples 2g, the carbonic acid that pH is 10.00 is added and buffers molten 8.5mL, will
Micro-biological samples are placed in ice-water bath and centrifuge 10min under 12000r/min after ultrasonication 3min, take supernatant to be used to test
Measure.
The formulation of calibration curve equation：With NaNO2It is respectively 3,6,9,12,15,18,21 and 24 μm of ol/ to prepare 8 groups of concentration
L NO2 -Standard liquid, 8 test tubes are taken, be separately added into the carbonate buffer solution that 1mL pH are 10.0, then add the dense of 0.2mL
The hydroxylamine hydrochloride solution for 90mmol/L is spent, then each group solution is placed in 35 DEG C of water bath with thermostatic control and heats 10min, Ran Houxiang
The NO that 0.5mL concentration is respectively 3,6,9,12,15,18,21 and 24 μm of ol/L is added in each group solution2 -Standard liquid, after shaking up
18min is stood in 35 DEG C of water bath with thermostatic control, then it is 58mmol/L to basic sulphonyl to sequentially add into each group solution 2mL concentration
Amine aqueous solution (being dissolved in 10mol/L acetic acid) and the N-1- naphthyl ethylenediamines solution that 2mL concentration is 7mmol/L (are dissolved in 10mol/L second
Acid), the 24min that developed the color in 35 DEG C of water-baths is placed in, then adds extractant dichloromethane into each group solution again, added volume is
1.2 times of above overall solution volume, centrifugal treating is carried out respectively after being sufficiently mixed uniformly, high speed centrifugation under the conditions of 10000r/min
3min, and upper strata extraction phase is taken respectively, the absorbance of each group is determined at 540nm, will using the absorbance of measure as y values
The NO of each group2 -Concentration obtains calibration curve equation y=0.0122x+0.1751 (R=as x values after linear fitting
0.9998)；
Test group, control group and blank group are set, carbonate buffer solution is separately added into in three groups and shakes up, and to experiment
Hydroxylamine hydrochloride solution is added in group and blank group, control group adds the distilled water of same volume, and 35 DEG C are placed in simultaneously by three groups
10min is heated in water bath with thermostatic control, 0.5mL supernatants are then added into test group and control group, same volume is added in blank group
Long-pending carbonic acid buffer, and 18min is stood in 35 DEG C of water bath with thermostatic control after shaking up respectively, then again to test group, control group
Be sequentially added into blank group to basic sulfonamide solution and N-1- naphthyl ethylenediamine solution, then show in 35 DEG C of water-baths
Color 24min, wherein the concentration of added each solution and the same step of addition (2), then add chloroform into three groups of solution,
High speed centrifugation 3min under the conditions of 10000r/min, three groups of pink upper strata extraction phase is taken respectively and determines absorbance in 540nm,
Try to achieve three groups of A530Difference is 0.256, substitutes into standard curve and tries to achieve x again by formulaExamination is calculated
It is 0.325 μm of olg to test group concentration of oxygen free radicals-1。
Claims (5)
1. the assay method of ultra-oxygen anion free radical in a kind of microbial body, it is characterised in that carry out according to the following steps：
(1) by activated sludge suspension of the biomass in micro-biological samples more than 800mg/L in 10000~12000r/min conditions
8~10min of lower centrifugation, 1~2g of micro-biological samples of bottom after centrifugation is taken, the micro-biological samples of bottom are less than 1g after centrifugation
When, repeated sampling adds up to 1~2g, and it is 9.5~10.5 carbonate buffer solutions that pH is then added into micro-biological samples, addition
For every 1g micro-biological samples add 3.5~4.5mL, then mixed solution is placed in ice-water bath and carries out ultrasonication, finally from
The heart takes supernatant；
(2) formulation of standard curve：Use NaNO2Prepare NO of 7 groups of concentrations above for 0~30 μm of ol/L2 -Standard liquid, Ran Houqu
The blank cuvette of more than 7 is separately added into the carbonate buffer solution that pH is 9.5~10.5 and concentration is 50~100mmol/L hydrochloric acid
The addition of hydroxylamine solution, wherein carbonate buffer solution is to add 0.5~1mL per 1g micro-biological samples, hydroxylamine hydrochloride solution
Addition is to add 0.1~0.3mL per 1g micro-biological samples, and then each group solution is placed in 25~35 DEG C of water bath with thermostatic control and added
10~15min of heat, the different NO of 0.5mL concentration is then added into each group solution2 -Standard liquid, at 25~35 DEG C after shaking up
18~22min is stood in water bath with thermostatic control, then is sequentially added into each group solution to basic sulfonamide solution and N-1- naphthyls second two
Amine aqueous solution, wherein being by basic sulfonamide solid and N-1- naphthyl second to basic sulfonamide solution and N-1- naphthyl ethylenediamine solution
Diamines solid is dissolved in the acetic acid solution that concentration is 10~13mol/L and obtained respectively, to basic sulfonamide solution and N-1- naphthyl second
Diamine solution concentration is respectively 55~60mmol/L and 5~10mmol/L, addition be add 0.8 per 1g micro-biological samples~
1mL is subsequently placed in 20~30min of colour developing in water-bath to basic sulfonamide solution and N-1- naphthyl ethylenediamine solution, then to each group
Extractant is added in solution, centrifugal treating is carried out respectively after being sufficiently mixed uniformly, and takes upper strata extraction phase respectively, at 540nm
The absorbance of each group is determined, using the absorbance of measure as y values, by the NO of each group2-Concentration is as x values, after linear fitting
Obtain calibration curve equation；
(3) test group, control group and blank group are set, carbonate buffer solution is separately added into in three groups and shakes up, and to experiment
Hydroxylamine hydrochloride solution is added in group and blank group, control group adds the distilled water of same volume, and 25~35 are placed in simultaneously by three groups
DEG C water bath with thermostatic control in heat 10~15min, 0.5mL supernatants are then added into test group and control group, in blank group plus
Enter the carbonic acid buffer of same volume, and 18~22min, Ran Houzai are stood in 25~35 DEG C of water bath with thermostatic control after shaking up respectively
It is sequentially added into test group, control group and blank group to basic sulfonamide solution and N-1- naphthyl ethylenediamine solution, so
Develop the color 20~30min in a water bath afterwards, wherein the same step of concentration and addition (2) of each solution；
(4) extractant is added into the solution after three groups of colour developings of step (3), is carried out respectively at centrifugation after being sufficiently mixed uniformly
Reason, then takes upper strata extraction phase respectively, and at 540nm determination test group, control group and blank group absorbance, wherein used
The same step of extractant (2)；
(5) absorbance that step (4) determines is asked poor, then brings the calibration curve equation of step (2) into, try to achieve at 540nm
NO in corresponding test group2 -Concentration, then by O in formula accounting unit quality biomass body2 -.Content, that is, complete microbial body
The measure of interior ultra-oxygen anion free radical, wherein formula areWhereinFor 540nm places
NO in corresponding test group2 -Concentration, V1For the volume of test group sample to be tested, V2For after step (2) test group ultrasonication from
The heart takes supernatant cumulative volume, and ρ is test group sample to be tested sludge solid concentration, m1Micro- life after being centrifuged for test group step (2)
The quality of thing sample, m2For the quality of deposit after (2) ultrasonication of test group step and centrifugation.
2. the assay method of ultra-oxygen anion free radical in microbial body according to claim 1, it is characterised in that：Step
(1) the ultrasonication time is 2~3min, and centrifugal rotational speed is 10000~120000r/min during centrifuging and taking supernatant.
3. the assay method of ultra-oxygen anion free radical in microbial body according to claim 1, it is characterised in that：Step
(2) centrifugation time is 3~5min after adding extractant.
4. the assay method of ultra-oxygen anion free radical in microbial body according to claim 1, it is characterised in that：Step
(2) volume that extractant is added in is 0.8~1.2 times of each group liquor capacity before adding.
5. the assay method of ultra-oxygen anion free radical in microbial body according to claim 1, it is characterised in that：Step
(2) extractant is dichloromethane, chloroform or toluene.
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