CN101671718A - Detection method of toxicity of nanomaterials - Google Patents
Detection method of toxicity of nanomaterials Download PDFInfo
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- CN101671718A CN101671718A CN200910180624A CN200910180624A CN101671718A CN 101671718 A CN101671718 A CN 101671718A CN 200910180624 A CN200910180624 A CN 200910180624A CN 200910180624 A CN200910180624 A CN 200910180624A CN 101671718 A CN101671718 A CN 101671718A
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Abstract
The invention relates to a detection method belonging to the technical field of environmental detection, in particular to a method for detecting toxicity of nanomaterials in the environment by adopting Escherichia coli containing green florescent protein plasmids. The method overcomes the interference of a few natural organic matters existing in the environment with the detection of toxicity of the nanomaterials so that the detection of toxicity of the nanomaterials is more convenient and accurate. The specific method selects the Escherichia coli transforming the green florescent protein plasmids as the bacterium for detection and comprises the following steps: 1. using an M9 culture medium to prepare bacterial suspension of the Escherichia coli containing the green florescent protein plasmids; 2. using ultrasonic dispersing nanomaterials to prepare suspension of nano particulate matters; and 3. culturing the nano particulate matters and the bacterial suspension of the Escherichia coliat 37 DEG C, using an enzyme-labeling instrument to detect the change of the florescence intensity of the nano particulate matters in certain period and judging the toxicity of the nano particulate matters to the Escherichia coli according to the difference of the change of the florescence intensity. The invention provides the efficient and stable detection method of toxicity of the nanomaterialsand the method is not likely to be interfered by other factors and is simple and low in cost.
Description
Technical field
The present invention relates to the method that a kind of intestinal bacteria that will contain the egfp grain are applied to nano material toxicity in the testing environment, belong to the environment measuring technical field.
Background technology
Nano material is meant the nano structural material of particle diameter between the 1-100 nanometer, and the material of this yardstick has been showed unique reactive behavior, optical property and electromagnetic property.The source of nanoparticles comprises two kinds, and a kind of is natural source, and another kind is artificial source.Natural source exists very early, the most typically is exactly the aerosol that formed by the dust that volcanic explosion produces in atmosphere, also has the organic colloid that forms down because of biological action in the water body etc.; Artificial source mainly is the nano-scale material that produces by some mankind's activities (as motor vehicle exhaust, the nano material use of coal, natural gas burning and synthetic thereof etc.).In the source of these nanoparticles, with artificial source the most people paid close attention to, and the health that causes thus and environmental problem are even more serious, this paper discusses the toxic effect of artificial source's nanoparticles.
Because of having a unique physical and chemical properties, nano material has obtained than widespread use in scientific research and daily production, life.Adopt the commodity of nanotechnology and nano material extensively to appear on the market at present, it comprises electronic component, optical element, textiles, medical detection and medicine thereof, high-performance sports equipment, makeup, food product pack, water technology, fuel cell, catalyzer, biosensor etc.With titanium dioxide is example, and nano titanium oxide is widely used in self-cleaning glass, solar cell, electrical equipment, foodstuff additive, medicine, makeup and Pollutant Treatment and the pollution reparation, and annual usage quantity number is with ten thousand tons.And, adopt the product of nanotechnology and nano material to get more and more with making along with nanotechnology constantly develops.Someone estimates, and the marketable value of the nano material in the whole world is approximately 10,500,000,000 dollars in the time of 2006, yet this value will reach 1,000,000,000,000 dollars after 2015.This means that the product that more and more contains nano material appears in our daily production and the life.Along with a large amount of uses of these products, a large amount of nano materials will enter into environment by different approaches, as enter into atmospheric environment, water surrounding and edatope.So, have the nano structural material of unique physical chemical attribute and nanoparticles thereof and caused that progressively people are to the environment that may cause and the concern of Health hazard.
The various countries researchist has carried out the toxicity detection to nano material in succession in recent years, and carried out comparatively extensive studies, mainly be the toxicity of judging nano material by the mode of low isocellular Live/Dead such as bacterium, fungi or algae, differentiate by the response to oxidative stress of mammalian cell line (zooblast line, human body cell etc.) in addition.For example, people such as Huang " Langmuir " (Langmuir) magazine (2008 the 24th phase 4140-4144 pages or leaves) go up the toxicity of estimating nanoparticles by the survival rate of bacterium.People such as Long are at " environmental science and technology " (Environmental Science﹠amp; Technology) magazine (2009 the 40th phase 4346-4352 pages or leaves) is gone up the toxicity of estimating nanoparticles by the stress action degree of mammalian cell.More than two kinds of methods exist detect inaccurate, complicated operation, cost an arm and a leg, shortcoming such as repeatable difference.By above analysis as can be known, it is a lot of to detect the problem that the Cytotoxic method of nanoparticles exists at present.
Purpose of the present invention has adopted among the present invention and can stablize fluorescin plasmid that produces fluorescence and the coli somatic that can breed rapidly just in order to solve above-mentioned several problem.On the one hand providing a kind of can judge the toxic method of nanoparticles by fluorescence, adopts intestinal bacteria to simplify the operating method of evaluating apparatus as the host bacterium on the other hand, helps to realize that nanoparticles toxicity detects and analyzes in the environment.
Summary of the invention
The object of the present invention is to provide a kind of by green fluorescent protein can be efficient, the toxic method of nanoparticles in the stable detection environment.This method is to realize the high-throughput and the sensitivity that detect, and the speed of analysis is very fast, and sample dosage is few, and cost is lower.
The technical solution adopted in the present invention is as described below:
A kind of green fluorescent protein that utilizes detects the toxic method of nanoparticles, it is characterized in that: comprise that step is as follows
1) plasmid transforms and the intestinal bacteria cultivation
(a) get a pipe 100 microlitre DH5a competent cells, add egfp grain 1 microlitre, mix, placed 30 minutes in the ice bath;
(b) pipe is put into 42 ℃ of water-bath circulations, thermal shock 90 seconds;
(c) described pipe is transferred in the ice bath 2-5 minute;
(d) add the nonresistant SOB substratum of 700-800 microlitre under the normal temperature in the described pipe behind ice bath, be transferred in the shaking table, shook 1 hour under 37 ℃;
(e) get competent cell 450 microlitres that transformed and join on the substratum of 50 mg/litre kalamycin resistances, wait substratum all to absorb after, cultivated 12-16 hour down at 37 ℃;
(f) identify single bacterium colony by bacterium colony PCR, determine to contain the proteic plasmid that to express green fluorescence;
(g) PCR identify finish errorless after, select this list colony inoculation in the LB substratum that contains kalamycin resistance, cultivated 12-16 hour for 37 ℃;
2) detection of nano material toxicity
(a) will be in the step 1) cultivate the intestinal bacteria that contain the egfp grain that obtain, with 0.9% physiological saline centrifuge washing three times, resuspended with 1 milliliter the M9 substratum that contains kalamycin resistance then;
(b) suspension liquid of prior nanoparticles with ready different sorts of M9 liquid nutrient medium and different concns is joined on the 96 hole black luciferase target orifice plates successively;
(c) the nanoparticles fluorescence intensity by microplate reader detection different sorts and different concns changes;
(d) change by fluorescence intensity in for some time and estimate different sorts and different concns nanoparticles colibacillary toxicity.
As being used for detection method of toxicity of nanomaterials of the present invention, its substratum will be selected the M9 substratum of low fluorescence background for use;
Described nanoparticles is rutile type nano titanic oxide, anatase-type nanometer titanium dioxide, nano cupric oxide or nano zine oxide;
In the method for the present invention, intestinal bacteria to be detected density in orifice plate is 1 * 10
7Individual/cm
2
In the method for the present invention, described nanoparticles in the M9 substratum dispersed particles particle diameter smaller or equal to 100 nanometers, preferred 20 nanometers;
In the method for the present invention, the Infinite M200 that detecting instrument is produced for Tecan company.
Description of drawings
Fig. 1 is the colibacillary excitation wavelength scintigram that contains the egfp grain;
Fig. 2 is the colibacillary emission wavelength scintigram that contains the egfp grain;
Fig. 3 is for adding the nanoparticles (nano-TiO of different concns respectively
2, nano-ZnO, nano-CuO) after, intestinal bacteria fluorescence intensity change curve (p＜0.05);
Wherein, Fig. 3 is a rutile type nano titanic oxide a), Fig. 3 b) be anatase-type nanometer titanium dioxide, Fig. 3 c) be nano cupric oxide, Fig. 3 d) be nano zine oxide.
Embodiment
Be described in detail with reference to the accompanying drawings below in conjunction with embodiment, more deep understanding arranged with purpose, feature and advantage to the inventive method.Following embodiment specific explanations the present invention, scope of the present invention is not subjected to the restriction of embodiment.
Present embodiment relates to the method that nano material toxicity detects, and comprises the steps that wherein said nano material is a rutile type nano titanic oxide.
1. get a pipe 100 microlitre DH5a competent cells, add egfp grain 1 microlitre, mix, ice bath was placed 30 minutes;
2. pipe is put into 42 ℃ of water-baths circulation, thermal shock 90 seconds is transferred in the ice bath 2-5 minute;
3. add the nonresistant SOB substratum of 700-800 microlitre under the normal temperature in the described pipe behind ice bath, be transferred in the shaking table, shook 1 hour under 37 ℃;
4. get competent cell 450 microlitres that transformed and join on the substratum of kalamycin resistance, wait substratum all to absorb after, 37 ℃ of following incubated overnight;
5. select single bacterium colony, and identify by bacterium colony PCR;
6. being seeded in 37 ℃ cultivated 12-16 hour down; Be ready to the black enzyme plate, and prepare the rutile titanium dioxide particulate matter storing solution of 200 mg/litre, ultrasonic dispersing 30 minutes;
7. under 4000 rev/mins of rotating speeds centrifugal 5 minutes, and with 0.9% physiological saline washing three times; Use 1 milliliter M9 liquid nutrient medium resuspended at last;
8. in each detection cell, add the resuspended liquid of intestinal bacteria of 100 microlitres successively, then add rutile type nano titanic oxide, make final concentration be respectively 5,10,20,30,50 mg/litre, allow the volume in final every hole reach 200 microlitres; Each sample be provided with three parallel, and reserve do not add nanoparticles detection cell in contrast;
9. biased sample is put under 150 rev/mins, 37 ℃ of the percussion tables and hatched 30 minutes;
10. utilize the Tecan microplate reader to detect at 488 nanometer exciting lights, the fluorescence intensity under the 520 nanometers emission optical condition changes.
Present embodiment relates to the method that nano material toxicity detects, and comprises the steps that wherein said nano material is an anatase titanium dioxide.Adopt among the embodiment 2 with embodiment 1 in identical nanoparticles toxicity detection board carry out following steps:
1. get a pipe 100 microlitre DH5a competent cells, add egfp grain 1 microlitre, mix, ice bath was placed 30 minutes;
2. pipe is put into 42 ℃ of water-bath circulations, thermal shock 90 seconds; Transfer in the ice bath 2-5 minute;
3. add the nonresistant SOB substratum of 700-800 microlitre under the normal temperature in the described pipe behind ice bath, be transferred in the shaking table, shook 1 hour under 37 ℃;
4. get competent cell 450 microlitres that transformed and join on the substratum of kalamycin resistance, wait substratum all to absorb after, cultivated 12-16 hour down at 37 ℃;
5. select single bacterium colony, and identify by bacterium colony PCR;
6. being seeded in 37 ℃ cultivated 12-16 hour down; Be ready to the black enzyme plate, and prepare the anatase titanium dioxide particulate matter storing solution of 200 mg/litre, ultrasonic dispersing 30 minutes;
7. under 4000 rev/mins of rotating speeds centrifugal 5 minutes, and with 0.9% physiological saline washing three times; Use 1 milliliter M9 liquid nutrient medium resuspended at last;
8. in each detection cell, add the resuspended liquid of intestinal bacteria of 100 microlitres successively, then add anatase titanium dioxide, make final concentration be respectively 5,10,20,30,50 mg/litre, allow the volume in final every hole reach 200 microlitres; Each sample be provided with three parallel, and reserve do not add nanoparticles detection cell in contrast;
9. biased sample is put under 150 rev/mins, 37 ℃ of the percussion tables and hatched 30 minutes;
10. utilize the Tecan microplate reader to detect at 488 nanometer exciting lights, the fluorescence intensity under the 520 nanometers emission optical condition changes.
Present embodiment relates to the method that nano material toxicity detects, and comprises the steps that wherein said nano material is a nano zine oxide.Adopt among the embodiment 3 with embodiment 1 in identical nanoparticles toxicity detection board carry out following steps:
1. get a pipe 100 microlitre DH5a competent cells, add egfp grain 1 microlitre, mix, ice bath was placed 30 minutes;
2. pipe is put into 42 ℃ of water-bath circulations, thermal shock 90 seconds; Transfer in the ice bath 2-5 minute;
3. add the nonresistant SOB substratum of 700-800 microlitre under the normal temperature in the described pipe behind ice bath, be transferred in the shaking table, shook 1 hour under 37 ℃;
4. get competent cell 450 microlitres that transformed and join on the substratum of kalamycin resistance, wait substratum all to absorb after, cultivated 12-16 hour down at 37 ℃;
5. select single bacterium colony, and identify by bacterium colony PCR;
6. being seeded in 37 ℃ cultivated 12-16 hour down; Be ready to the black enzyme plate, and prepare the nano granular of zinc oxide thing storing solution of 100 mg/litre, ultrasonic dispersing 30 minutes;
7. under 4000 rev/mins of rotating speeds centrifugal 5 minutes, and with 0.9% physiological saline washing three times; Use 1 milliliter M9 liquid nutrient medium resuspended at last;
8. in each detection cell, add the resuspended liquid of intestinal bacteria of 100 microlitres successively, then add nano zine oxide, make final concentration be respectively 5,10,20,30,50 mg/litre, allow the volume in final every hole reach 200 microlitres; Each sample be provided with three parallel, and reserve do not add nanoparticles detection cell in contrast;
9. biased sample is put under 150 rev/mins, 37 ℃ of the percussion tables and hatched 30 minutes;
10. utilize the Tecan microplate reader to detect at 488 nanometer exciting lights, the fluorescence intensity under the 520 nanometers emission optical condition changes.
Present embodiment relates to the method that nano material toxicity detects, and comprises the steps that wherein said nano material is a nano cupric oxide.Adopt among the embodiment 4 with embodiment 1 in identical nanoparticles toxicity detection board carry out following steps:
1. get a pipe 100 microlitre DH5a competent cells, add egfp grain 1 microlitre, mix gently, ice bath was placed 30 minutes;
2. pipe is put into 42 ℃ of water-bath circulations, thermal shock 90 seconds; Transfer in the ice bath 2-5 minute;
3. add the nonresistant SOB substratum of 700-800 microlitre under the normal temperature in the described pipe behind ice bath, be transferred in the shaking table, shook 1 hour under 37 ℃;
4. get competent cell 450 microlitres that transformed and join on the substratum of kalamycin resistance, wait substratum all to absorb after, cultivated 12-16 hour down at 37 ℃;
5. select single bacterium colony, and identify by bacterium colony PCR;
6. being seeded in 37 ℃ cultivated 12-16 hour down; Be ready to the black enzyme plate, and prepare the nano cupric oxide particulate matter storing solution of 100 mg/litre, ultrasonic dispersing 30 minutes;
7. under 4000 rev/mins of rotating speeds centrifugal 5 minutes, and with 0.9% physiological saline washing three times; Use 1 milliliter M9 liquid nutrient medium resuspended at last;
8. in each detection cell, add the resuspended liquid of intestinal bacteria of 100 microlitres successively, then add the nano cupric oxide storing solution, make final concentration be respectively 5,10,20,30,50 mg/litre, allow the volume in final every hole reach 200 microlitres;
Each sample be provided with three parallel, and reserve do not add nanoparticles detection cell in contrast;
9. biased sample is put under 150 rev/mins, 37 ℃ of the percussion tables and hatched 30 minutes;
Utilize the Tecan microplate reader to detect at 488 nanometer exciting lights, the fluorescence intensity under the 520 nanometers emission optical condition changes.
Claims (5)
1. one kind is utilized green fluorescent protein to detect the toxic method of nanoparticles, it is characterized in that: comprise the steps 1) plasmid transforms and intestinal bacteria are cultivated
(a) get a pipe 100 microlitre DH5 α competent cells, add egfp grain 1 microlitre, mix, placed 30 minutes in the ice bath;
(b) pipe is put into 42 ℃ of water-bath circulations, thermal shock 90 seconds;
(c) described pipe is transferred in the ice bath 2-5 minute;
(d) add the nonresistant SOB substratum of 700-800 microlitre under the normal temperature in the described pipe behind ice bath, be transferred to then in the shaking table, shook 1 hour under 37 ℃;
(e) get on the substratum of kalamycin resistance that competent cell 450 microlitres that transformed join 50 mg/litre, wait substratum all to absorb after, cultivated 12-16 hour down at 37 ℃;
(f) identify single bacterium colony by bacterium colony PCR, determine to contain the proteic plasmid that to express green fluorescence;
(g) PCR identify finish errorless after, select this list colony inoculation in the LB substratum that contains kalamycin resistance, cultivated 12-16 hour for 37 ℃;
2) detection of nano material toxicity
(a) will be in the step 1) cultivate the intestinal bacteria that contain the egfp grain that obtain, with 0.9% physiological saline centrifuge washing three times, resuspended with 1 milliliter the M9 substratum that contains kalamycin resistance then;
(b) suspension liquid of prior nanoparticles with ready different sorts of M9 liquid nutrient medium and different concns is joined on the 96 hole black luciferase target orifice plates successively;
(c) the nanoparticles fluorescence intensity by microplate reader detection different sorts and different concns changes;
(d) change by fluorescence intensity in 1 hour and estimate different sorts and different concns nanoparticles toxicity.
2. according to the method described in the claim 1, it is characterized in that: intestinal bacteria to be detected density in orifice plate is 1 * 107/cm
2
3. according to the method described in the claim 1, it is characterized in that: described nanoparticles to be detected is rutile type nano titanic oxide, anatase-type nanometer titanium dioxide, nanometer titanium dioxide copper or nanometer titanium dioxide zinc.
4. according to the method described in the claim 1, it is characterized in that: the substratum that is adopted is the M9 substratum of low fluorescence background.
5. according to the method described in the claim 1, it is characterized in that: described nanoparticles in the M9 substratum dispersed particles particle diameter smaller or equal to 100 nanometers, preferred 20 nanometers.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979662A (en) * | 2010-11-17 | 2011-02-23 | 武汉大学 | Bacterial genetic transformation-based method for detecting genetic toxicity of nano material |
| CN102175659A (en) * | 2011-01-20 | 2011-09-07 | 济南市供排水监测中心 | Water quality genotoxicity detection method based on semiconductor opening switch (SOS) effect of recombinant Escherichia coli |
| CN102346147A (en) * | 2011-09-16 | 2012-02-08 | 上海大学 | Method for detecting difference of cell toxicity between atmospheric nano particles and industrial nano particles |
| CN104237534A (en) * | 2014-09-28 | 2014-12-24 | 淮南师范学院 | Method for rapidly detecting bio-availability of nano-material |
| CN113005171A (en) * | 2021-03-19 | 2021-06-22 | 济南国科医工科技发展有限公司 | Preparation method of titanium dioxide nanoparticle culture medium |
| CN114289066A (en) * | 2021-12-29 | 2022-04-08 | 云南大学 | Nano mimic enzyme material, preparation method and application thereof, and method for detecting ovomucoid |
| CN115165821A (en) * | 2022-06-14 | 2022-10-11 | 华南师范大学 | Escherichia coli detection method based on copper oxide nano material |
-
2009
- 2009-10-27 CN CN2009101806245A patent/CN101671718B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979662A (en) * | 2010-11-17 | 2011-02-23 | 武汉大学 | Bacterial genetic transformation-based method for detecting genetic toxicity of nano material |
| CN102175659A (en) * | 2011-01-20 | 2011-09-07 | 济南市供排水监测中心 | Water quality genotoxicity detection method based on semiconductor opening switch (SOS) effect of recombinant Escherichia coli |
| CN102175659B (en) * | 2011-01-20 | 2012-10-31 | 济南市供排水监测中心 | Water quality genotoxicity detection method based on semiconductor opening switch (SOS) effect of recombinant Escherichia coli |
| CN102346147A (en) * | 2011-09-16 | 2012-02-08 | 上海大学 | Method for detecting difference of cell toxicity between atmospheric nano particles and industrial nano particles |
| CN104237534A (en) * | 2014-09-28 | 2014-12-24 | 淮南师范学院 | Method for rapidly detecting bio-availability of nano-material |
| CN104237534B (en) * | 2014-09-28 | 2016-09-07 | 淮南师范学院 | A kind of method of quick detection nano material biological effectiveness |
| CN113005171A (en) * | 2021-03-19 | 2021-06-22 | 济南国科医工科技发展有限公司 | Preparation method of titanium dioxide nanoparticle culture medium |
| CN114289066A (en) * | 2021-12-29 | 2022-04-08 | 云南大学 | Nano mimic enzyme material, preparation method and application thereof, and method for detecting ovomucoid |
| CN114289066B (en) * | 2021-12-29 | 2023-01-20 | 云南大学 | Nano mimic enzyme material, preparation method and application thereof, and method for detecting ovomucoid |
| CN115165821A (en) * | 2022-06-14 | 2022-10-11 | 华南师范大学 | Escherichia coli detection method based on copper oxide nano material |
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