CN101261242B - Water quality toxicity biosensor microorganism electrode preparation method - Google Patents
Water quality toxicity biosensor microorganism electrode preparation method Download PDFInfo
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- CN101261242B CN101261242B CN2008100363175A CN200810036317A CN101261242B CN 101261242 B CN101261242 B CN 101261242B CN 2008100363175 A CN2008100363175 A CN 2008100363175A CN 200810036317 A CN200810036317 A CN 200810036317A CN 101261242 B CN101261242 B CN 101261242B
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Abstract
The invention relates to a microorganism electrode preparation method of a water quality toxicity biosensor, which is characterized in that: 40 to 100 parts of freezing and drying colon bacillus, 20 to 80 parts of microzyme and 40 to 60 parts of nitrosomonas are taken according to weight and the three germs are blended to get a blended germ; then 0.3 to 1.0g of blended germ is taken and added into 200 to 500 Mu L of 0.85 percent chlorine solution and a sterilized toothpick is used for mixing well the germ , thus blended germ solution is available; a diprosopia tape is stuck to the round of the working area of the carbon electrode; 5 to 10 Mu L of blended germ solution is drawn with a liquid relief gun, which is smeared to the working area of the carbon electrode and is air-cured for 0.5 to 2.0h in dark under room temperature; then a Makrolan film is covered on the air-cured blended solution, thus the microorganism electrode is made by fixing the germ through the diprosipia tape, whichis at last arranged in a fridge of 4 DEG C for 24 to 48h ready for use. The microorganism electrode obtained from the invention improves largely the sensitivity and repetitiveness of the water quality toxicity analysis of the biosensor. The microorganism electrode preparation method of the water quality toxicity biosensor has the advantages of easy techniques and easy operation, can realize the reusage of the biosensor electrode, thus largely reducing the instrument analysis cost.
Description
Technical field
That the present invention relates to is a kind of preparation method who is used for the microorganism electrode of the CellSense biology sensor that water quality toxicity analyzes, adopt the microorganism electrode of this method preparation, both can improve CellSense biology sensor oxicity analysis performance, and can reduce analysis cost again.Belong to the environmental monitoring technology field.
Background technology
Mensuration to wastewater toxicity mainly contains two class methods at present, and a class is a physico-chemical method, and another kind of is biological method.Traditional physico-chemical analysis method can the quantitative test pollutant in the content of principal ingredient, but can not be directly and reflect the combined influence of various noxious materials all sidedly to environment.Biological test can remedy the deficiency of physics and chemistry detection method, and therefore in the water Pollution Study, it has become monitoring and has estimated one of important means of water body environment quality.
In recent years, a kind of CellSense biology sensor oxicity analysis system based on redox mediator (Italian Euroclone company produce) has been subjected to people's favor day by day.Its principle of work is: in the surperficial fixedly toxicity responsive type microorganism of biology sensor carbon working electrode (is reference with Ag/AgCl), it produces electronics down at living things catalysis metabolism state (respiration), transfer function by redox mediator, forming the electric current that is directly proportional with redox active in the cell is detected by system, the power of its toxicity is judged in the variation of the microorganism current signal that the microbial activity inhibiting effect is caused according to toxicant.It can make evaluation to the toxicity of different pollutants (pollution source) by selecting fixedly the certain sensitive indicator microoraganism, has characteristics such as portable, sensitive, quick and on-line analysis.
Being tried microorganism is the key function element of microbiological sensor information capture, and the selection of microorganism directly has influence on the sensitivity and the stability of biology sensor oxicity analysis.At present, in the CellSense Study on Biosensor, existing single bacterial strain of employing such as Escherichia coli (E.coli), pseudomonad (Pseudomonas putida), Fei Shi vibrios (V.fischeri), or hybrid bacterial strain such as active sludge (Activated sludge) are as the research report (Marinella et al.Analytica Chimica 2001, the 426:155-165 that are tried microorganism; Bentley et al.Toxicology Vitro 2001,15:465-479; Luisa et al.Analytica Chimica Acta 2002,456:41-54; Wang et al.Chinese Chemical Letters 2008,19:211-214), but in actual water quality toxicity is analyzed, because the combined pollution system of the multiple often pollutant coexistence of waste water, and different types of microorganism is to the toxicity susceptibility difference of pollutant, therefore, the sensitivity and the relatively poor deficiency of repeatability that have oxicity analysis based on the CellSense biology sensor of single bacterial strain.Simultaneously, on environmental pollution ecological toxicology viewpoint, has the limitation that can not objectively respond the true toxic action of pollutant based on the toxicity assessment of single bacterial strain.And with active sludge as the CellSense biology sensor that is tried microorganism, although have the detection of contamination wide ranges, characteristics such as oxicity analysis real result, but because the flora of different activities mud composition structural difference is bigger, cause the toxicity testing result between the laboratory to be difficult to mutual comparison, above-mentioned existing problems have greatly limited the development of such microbiological sensor.
The microorganism electrode that the CellSense biology sensor is used now, be around in the carbon electrode perform region, to print tackifier in advance, being used to paste polycarbonate membrane directly will be tried microorganism and be covered that fixing method is prepared, the microorganism electrode of this method preparation is a disposable electrode, after finishing a water quality toxicity analysis and abandon, cause analysis cost high, influenced its promotion and application.
Therefore, be to improve CellSense biology sensor oxicity analysis performance and reduce analysis cost, the exploitation that is tried microorganism and fixing aspect remain further research.
Summary of the invention
The objective of the invention is to disclose a kind of preparation method of microorganism electrode of the CellSense of being used for biology sensor.The microorganism electrode that obtains with this method have oxicity analysis highly sensitive, repeat characteristics such as the good and analysis cost of performance is low.
In order to achieve the above object, the present invention is directed to the defective of CellSense biology sensor microorganism electrode, the preparation method who has proposed microorganism electrode is: measure respectively through 40～100 parts of weight of cryodesiccated Escherichia coli (E.coli) earlier, 20～80 parts of weight of saccharomycete (yeast), 40～60 parts of weight of nitrosomonas (Nitrosomonas sp.); Three kinds of bacterium are mixed obtain composite good mixing thalline; Then, get above-mentioned composite good mixing thalline 0.3～1.0g, in thalline, add 200～500 μ L, 0.85% sodium chloride solution, with the sterilization toothpick thalline is stirred evenly and obtain mixed bacteria liquid; Then, around the carbon electrode perform region of the CellSense biology sensor that cleans up, paste double sticky tape, draw 5～10 μ L mixed bacteria liquids with liquid-transfering gun, be applied to the carbon electrode perform region, behind the 0.5～2.0h that in the dark dries in the air under the room temperature condition, polycarbonate membrane is covered on the thalline, paste by double faced adhesive tape the thalline sealing and fixing is made microorganism electrode in the carbon electrode perform region, place behind 4 ℃ of placements of refrigerator, 24～48h the microorganism electrode for preparing stand-by.
Compare with existing CellSense biology sensor, have the following advantages with the CellSense biology sensor of the present invention's preparation based on quantitative Mixed Microbes microorganism electrode based on single bacterial strain and ordinary activated sludge:
1. oxicity analysis is highly sensitive, the present invention adopts by the composite Mixed Microbes conduct of the good bacterial strain of many strains toxicity sensitive property and is tried microorganism, owing to have the responsive complementation of good toxicity between bacterial strain, make this CellSense biology sensor obviously improve to the sensitivity of water body combined pollution system comprehensive toxicity analysis.
2. the repetition performance of oxicity analysis is good, and multiple bacterial strain quantitatively composite overcome the shortcoming that the active sludge flora is complicated and be difficult to quantize, and greatly improved the repetition performance of biology sensor oxicity analysis.
3. oxicity analysis real result more can truly reflect the truth of pollutant toxicity than the toxicity assessment of single bacterium based on the toxicity assessment of many bacterial strains.
4. microorganism electrode preparation method is simple, and the present invention adopts double faced adhesive tape and polycarbonate membrane to carry out the directly fixing of microorganism, and method is simple, is convenient to operation.
5. can realize the repeated use of biological sensor electrode, adopt this fixing means can realize that sensor electrode carries out repeatedly the fixing repeated use of microorganism, greatly reduces the instrumental analysis expense.
Embodiment
Following examples will the invention will be further described.
Embodiment 1
Get 40 parts of 50 parts of cryodesiccated Escherichia coli (E.coli), 20 parts in saccharomycete (yeast) and nitrosomonas (Nitrosomonas sp.), carry out bacterial strain and mix composite; Get the composite good mix bacterium agent of 0.6g and add 250 μ L, 0.85% sodium chloride solution, wet thallus is stirred evenly with the sterilization toothpick; Paste double sticky tape around the perform region with the carbon electrode of the CellSense biology sensor that cleans up, draw 7 μ L mixed bacteria liquids with liquid-transfering gun and be applied to CellSense carbon electrode perform region, 1h in the dark dries in the air under room temperature condition, then polycarbonate membrane is covered on the Mixed Microbes, paste by double faced adhesive tape the thalline sealing and fixing is made microorganism electrode in the carbon electrode perform region, place behind 4 ℃ of placements of refrigerator 48h the microorganism electrode for preparing stand-by.
Adopt the CellSense biology sensor based on the microorganism electrode of quantitative Mixed Microbes of the invention described above, respectively to Cu
2+And Zn
2+Oxicity analysis, (relevant CellSense biology sensor toxicity assay is seen paper: Wang Hong et al.Chinese Chemical Letters 2008,19:211-214).Analysis result shows that the CellSense biology sensor of the microorganism electrode that the present invention makes (quantitatively Mixed Microbes) records Cu
2+And Zn
2+EC
50Value is respectively 4.1mg/L and 48mg/L.Compare based on the CellSense biology sensor of Escherichia coli (E.coli), saccharomycete (yeast), nitrosomonas single bacterial strains such as (Nitrosomonas sp.) and active sludge Mixed Microbes with employing, oxicity analysis sensitivity improves 15～20%, analytical error reduces by 15～30%, and analytical performance obviously improves.
Embodiment 2
Get 50 parts of 80 parts of cryodesiccated Escherichia coli (E.coli), 20 parts in saccharomycete (yeast) and nitrosomonas (Nitrosomonas sp.), carry out bacterial strain and mix composite; Get the composite good mix bacterium agent of 0.5g and add 200 μ L, 0.85% sodium chloride solution, wet thallus is stirred evenly with the sterilization toothpick; Paste double sticky tape around the perform region with the carbon electrode of the CellSense biology sensor that cleans up, draw 5 μ L bacterium liquid with liquid-transfering gun and be applied to CellSense carbon electrode perform region, behind the 0.5h that in the dark dries in the air under the room temperature condition, polycarbonate membrane is covered on the Mixed Microbes, paste by double faced adhesive tape the thalline sealing and fixing is made microorganism electrode in the electrode perform region, place behind 4 ℃ of placements of refrigerator 24h the microorganism electrode for preparing stand-by.
Adopt the CellSense biology sensor of microorganism electrode of the present invention, respectively to 2, the percolate of 4-two chlorophenols and Shanghai refuse landfill carries out bio-toxicity analysis (analytical approach such as example 1).The result shows that quantitative Mixed Microbes CellSense biology sensor of the present invention records 2, the EC of 4-two chlorophenols and percolate
50Value is respectively 62mg/L and 80% (percolate adding volume ratio), than the oxicity analysis sensitivity raising of adopting based on the CellSense biology sensor of Escherichia coli (E.coli), saccharomycete (yeast), nitrosomonas single bacterial strains such as (Nitrosomonas sp.) and active sludge Mixed Microbes 20～30%, analytical error reduces by 25～30%.
Embodiment 3
Get the microorganism electrode of using among the embodiment 2, originally fixing microbial bacteria body and function pure water is removed clean, in superclean bench, sterilize, dry, press the operation steps among the embodiment 2, carry out retightening of microorganism, and be used for 2, the oxicity analysis of 4-two chlorophenols, 20 revision tests analyze 2,4-two chlorophenol EC
50The deviation of value can satisfy the actual analysis needs less than 8%.Not only the preparation method is simple for this biology sensor microorganism electrode, and can realize the repeated use of carbon working electrode, thereby but the lowering apparatus analysis cost reach more than 90%.
Claims (1)
1. the microorganism electrode preparation method of water quality toxicity biosensor, it is characterized in that: measure 40～100 parts of weight of cryodesiccated Escherichia coli earlier, 20～80 parts of weight of saccharomycete, 40～60 parts of weight of nitrosomonas mix three kinds of bacterium and obtain mixing thalline; Get above-mentioned mixing thalline 0.3～1.0g then, in mixing thalline, add 200～500 μ L, 0.85% sodium chloride solution, with the sterilization toothpick thalline is stirred evenly and obtain mixed bacteria liquid; Then, around the perform region of the carbon electrode of the biology sensor that cleans up, paste double sticky tape, draw 5～10 μ L mixed bacteria liquids with liquid-transfering gun, be applied to the perform region of carbon electrode, behind the 0.5～2.0h that in the dark dries in the air under the room temperature condition, polycarbonate membrane is covered on the Mixed Microbes of drying, the thalline sealing and fixing is made microorganism electrode in the carbon electrode perform region by double faced adhesive tape; Being placed in the refrigerator 4 ℃ at last places behind 24～48h stand-by.
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| CN104007154B (en) * | 2013-02-22 | 2018-04-20 | 中国科学院理化技术研究所 | A kind of preparation method and application of the redox hydrosol, the integrated microorganism electrode quickly detected for aqueous bio toxicity |
| CN105986000B (en) * | 2015-02-04 | 2019-08-23 | 中国科学院理化技术研究所 | A kind of method of Mixed Microbes biosensor detection pollutant bio-toxicity |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1051938A (en) * | 1990-12-29 | 1991-06-05 | 复旦大学 | The microbiological sensor of rapid determination biological oxygen demand |
| US5160604A (en) * | 1990-02-05 | 1992-11-03 | Fuji Electric Co., Ltd. | Toxic substance-detecting system with fixed microorganism membrane for water quality-monitoring |
| EP0811838A1 (en) * | 1995-02-22 | 1997-12-10 | Akebono Brake Industry Co., Ltd. | Microbial electrode and microbial sensor |
| CN1293368A (en) * | 2000-12-15 | 2001-05-02 | 清华大学 | Biological sensor of oxygen amount needed biochemically |
-
2008
- 2008-04-21 CN CN2008100363175A patent/CN101261242B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5160604A (en) * | 1990-02-05 | 1992-11-03 | Fuji Electric Co., Ltd. | Toxic substance-detecting system with fixed microorganism membrane for water quality-monitoring |
| CN1051938A (en) * | 1990-12-29 | 1991-06-05 | 复旦大学 | The microbiological sensor of rapid determination biological oxygen demand |
| EP0811838A1 (en) * | 1995-02-22 | 1997-12-10 | Akebono Brake Industry Co., Ltd. | Microbial electrode and microbial sensor |
| CN1293368A (en) * | 2000-12-15 | 2001-05-02 | 清华大学 | Biological sensor of oxygen amount needed biochemically |
Non-Patent Citations (4)
| Title |
|---|
| Hong Wang et al..Toxicity assessment of heavy metals and orgnic compounds using CellSense biosensor with E.coli.《Chinese chemical letters》.2008,第19卷(第2期),211-214. * |
| Marinella et al..Toxicity assessment of organic pollution in wastewaters using a bacterial biosensor.《Analytica Chimica Acta》.2001,第426卷(第2期),155-165. * |
| 孙裕生等.活性污泥微生物膜电极法测定废水中BOD的研究.《河北科技大学学报》.1986,(第1期),96-104. * |
| 麻娟.微生物电极法BOD快速测定仪实验研究.《化工技术与开发》.2008,第37卷(第1期),38-39. * |
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