CN109929763A - Microorganism-capturing method and separation method and purposes based on nonimmune effect magnetic material - Google Patents
Microorganism-capturing method and separation method and purposes based on nonimmune effect magnetic material Download PDFInfo
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- CN109929763A CN109929763A CN201910104662.6A CN201910104662A CN109929763A CN 109929763 A CN109929763 A CN 109929763A CN 201910104662 A CN201910104662 A CN 201910104662A CN 109929763 A CN109929763 A CN 109929763A
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- microorganism
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- magnetic bead
- nonimmune
- capture
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Abstract
The present invention provides a kind of microorganism-capturing methods based on nonimmune effect magnetic material, the magnetic bead of the Magnetic particles of common commercialization, amido modified magnetic bead and carboxyl modified is used for the capture of microorganism, there is no the presence of Ag-Ab, the production cost of magnetic microbe capture material is greatly reduced, while making capture material can be with room temperature long-term preservation.Although the not specific recognition of Ag-Ab, the capture of microorganism can be realized by the effect of Electrostatic Absorption and material surface amino or carboxyl.
Description
Technical field
The present invention relates to microorganism fields, more particularly, to the microorganism-capturing method based on nonimmune effect magnetic material
And separation method and purposes
Background technique
During the Testing and appraisal of microorganism, in order to remove complicated substrate, detection limit is reduced, microorganism catches
It is essential for obtaining.General microorganism-capturing material be all built upon Ag-Ab it is immune on the basis of.Can thus it go out
Existing many problems, for example, it is desired to the antigen-antibody to match is found, but the naturally occurring antigen-antibody to match is seldom
, it is unable to satisfy capture of the people to different microorganisms at all；Ag-Ab is sufficiently expensive, although specific can catch
Microorganism is obtained, but the cost of capture material is but greatly improved；Once wrapping up or being connected to anti-on magnetic material surface
The biomaterials such as former or antibody, just influence whether the storage of material, and material needs to be stored in 2~8 DEG C of low temperature environment first,
Preservative substance is also added, the time of storage can also greatly shorten.When material is used for microorganism-capturing, also to carry out a series of
Purification process, otherwise will seriously affect the capture effect of microorganism.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of microorganism of magnetic bead based on nonimmune effect
Capture effect, and the pre-treatment of material is simple, can room temperature store for a long time, antigen-antibody is eliminated, so that the cost of magnetic bead subtracts
It is low, it is economical and practical for the capture of microorganism.
The invention adopts the following technical scheme:,
It is a kind of based on it is nonimmune effect magnetic material microorganism-capturing method, by without any modification Magnetic particles, through ammonia
One of magnetic bead and magnetic bead through carboxyl modified of base modification or it is a variety of be placed directly in microorganism, microorganism is caught in realization
It obtains.
Further, before the magnetic capture microorganism, pre-treatment is first carried out, the pre-treatment is to take appropriate magnetic bead solution
The solvent of magnetic bead itself institute band is removed under the action of an external magnetic field.
Further, magnetic bead is placed in the solution containing microorganism, microorganism is captured.
Further, it after the solution common micro-organisms removal culture medium containing microorganism, cleans and is resuspended in sterile water.
Further, it will be directly added into the solution containing microorganism and carry out after buffer solution for cleaning used in magnetic bead subsequent experimental
Capture.
Further, it after magnetic bead being cleaned three times with the phosphate buffer containing 0.05% polysorbas20, is directly added into containing micro- life
In the solution of object, 25 DEG C, capture 1 hour.
Microorganism described further is escherichia coli.
The present invention also provides a kind of separation method of microorganism, by without any modification Magnetic particles, through amido modified magnetic
One of pearl and magnetic bead through carboxyl modified or it is a variety of be placed directly in microorganism, realize the capture to microorganism, then lead to
Externally-applied magnetic field separation is crossed, realizes the separation of microorganism.
Further, the microorganism is escherichia coli.
The new application that the present invention also provides magnetic beads in microorganism-capturing, by without any modification Magnetic particles, repair through amino
One of the magnetic bead of decorations and magnetic bead through carboxyl modified or it is a variety of be placed directly in microorganism, realize the capture to microorganism.
Beneficial effects of the present invention:
The present invention provides a kind of microorganism-capturing methods based on nonimmune effect magnetic material, by common commercialization
Magnetic particles, amido modified magnetic bead and carboxyl modified magnetic bead be used for the capture of microorganism, the not presence of Ag-Ab,
The production cost of magnetic microbe capture material is greatly reduced, while making capture material can be with room temperature long-term preservation.Although
There is no the specific recognition of Ag-Ab, can but be realized by the effect of Electrostatic Absorption and material surface amino or carboxyl
The capture of microorganism.
And the pre-treatment of material is simple, and biomaterial is not included in magnetic material, storage is convenient, can save for a long time and
It does not need to carry out preservative treatment.
Detailed description of the invention
Fig. 1 is the result figure of embodiment 1.
Fig. 2 is the result figure of embodiment 2.
Fig. 3 is the result figure of embodiment 3.
Specific embodiment
It elaborates below in conjunction with attached drawing to specific embodiments of the present invention.
Capture of 1 Magnetic particles of embodiment to escherichia coli
Specific experiment step 1. takes suitable commercialization Magnetic particles, and the solvent of itself is removed by externally-applied magnetic field, is then used
PBST buffer (PBS buffer solution containing 0.05% polysorbas20) cleans three times；
2. we use the generally acknowledged bovine serum albumin without microorganism-capturing ability in order to highlight the capture effect of Magnetic particles
White (BSA) wraps up Magnetic particles, 25 DEG C, reacts a hour, then carries out Magnetic Isolation, cleaning, obtains having closed
Magnetic bead；
3. cleaning and being resuspended in sterile water after cultured escherichia coli is removed culture medium.It is thin to prepare gradient
Bacterium suspension, gradient are 2 × 103、1×103、500、250CFU·Ml-1；
4. cleaned magnetic bead is added in bacterial suspension, magnetic bead is kept to be in during capturing a hour at 37 DEG C
Suspended state；
5. collecting supernatant and magnetic bead coating respectively and being counted on tryptose soya agar (TSA) culture medium.37 DEG C of items
16h is continuously cultivated under part；
6. experimental result the result is shown in Figure 1, Enrichment of bacteria efficiency is measured using colony counting method.With the coated Magnetic particles of BSA
As control (third column), Magnetic particles have apparent capture (the to escherichia coli under different gradients as the result is shown
One column), it is seen that Magnetic particles have apparent enrichment to escherichia coli.
Capture of the 2 carboxyl modified magnetic bead of embodiment to escherichia coli
1. taking the magnetic bead of suitable commercialization carboxyl modified, the solvent of itself is removed by externally-applied magnetic field, then uses PBST
Buffer (PBS buffer solution containing 0.05% polysorbas20) cleans three times；
2. we use the generally acknowledged BSA without microorganism-capturing ability in order to highlight the capture effect of carboxyl modified magnetic bead
Magnetic particles are wrapped up, 25 DEG C, react a hour, then carried out Magnetic Isolation, cleaning, obtain the magnetic bead closed；
3. cleaning and being resuspended in sterile water after cultured escherichia coli is removed culture medium.It is thin to prepare gradient
Bacterium suspension, gradient are 2 × 103、1×103、500、250CFU·Ml-1；
4. cleaned magnetic bead is added in bacterial suspension, magnetic bead is kept to be in during capturing a hour at 37 DEG C
Suspended state；
5. collecting supernatant and magnetic bead coating respectively and being counted on tryptose soya agar (TSA) culture medium.37 DEG C of items
16h is continuously cultivated under part；
6. experimental result result is shown in that Fig. 2 Enrichment of bacteria efficiency is measured using colony counting method.With the coated carboxyl modified of BSA
Magnetic bead as control (third column), the magnetic bead of carboxyl modified has escherichia coli under different gradients as the result is shown
Apparent capture (first row).It can be seen that carboxyl modified magnetic bead has apparent enrichment to escherichia coli.
Capture of the amido modified magnetic bead of embodiment 3 to escherichia coli
1. the magnetic bead for taking suitable commercialization amido modified, the solvent of itself is removed by externally-applied magnetic field, then uses PBST
Buffer (PBS buffer solution containing 0.05% polysorbas20) cleans three times；
2. we use the generally acknowledged BSA without microorganism-capturing ability in order to highlight the capture effect of amido modified magnetic bead
Magnetic particles are wrapped up, 25 DEG C, react a hour, then carried out Magnetic Isolation, cleaning, obtain the magnetic bead closed；
3. cleaning and being resuspended in sterile water after cultured escherichia coli is removed culture medium.It is thin to prepare gradient
Bacterium suspension, gradient are 2 × 103、1×103、500、250CFU·Ml-1；
4. cleaned magnetic bead is added in bacterial suspension, magnetic bead is kept to be in during capturing a hour at 37 DEG C
Suspended state；
5. collecting supernatant and magnetic bead coating respectively and being counted on tryptose soya agar (TSA) culture medium.37 DEG C of items
16h is continuously cultivated under part；
6. experimental result result is shown in Fig. 3, Enrichment of bacteria efficiency is measured using colony counting method.It is repaired with the coated amino of BSA
The magnetic beads of decorations as control (third column), as the result is shown magnetic bead amido modified under different gradients to escherichia coli all
There is apparent capture (first row).It can be seen that amido modified magnetic bead has apparent enrichment to escherichia coli.
The above is only the preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-described embodiment,
All technical solutions belonged under thinking of the present invention all belong to the scope of protection of the present invention.It should be pointed out that for the art
For those of ordinary skill, several improvements and modifications without departing from the principles of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of microorganism-capturing method based on nonimmune effect magnetic material, it is characterised in that: the catching method is will
The Magnetic particles of no any modification, through one of amido modified magnetic bead and the magnetic bead through carboxyl modified or it is a variety of be placed directly within it is micro-
In biology, the capture to microorganism is realized.
2. the microorganism-capturing method according to claim 1 based on nonimmune effect magnetic material, it is characterised in that: institute
Before stating magnetic capture microorganism, pre-treatment is first carried out, the pre-treatment is to take appropriate magnetic bead solution under the action of an external magnetic field
Remove the solvent of magnetic bead itself institute band.
3. the microorganism-capturing method according to claim 1 based on nonimmune effect magnetic material, it is characterised in that: will
Magnetic bead is placed in the solution containing microorganism, is captured to microorganism.
4. the microorganism-capturing method according to claim 3 based on nonimmune effect magnetic material, it is characterised in that: contain
The solution of microorganism is after common micro-organisms removes culture medium, to clean and be resuspended in sterile water.
5. the microorganism-capturing method according to claim 1 based on nonimmune effect magnetic material, it is characterised in that: will
It is directly added into the solution containing microorganism and is captured after buffer solution for cleaning used in magnetic bead subsequent experimental.
6. the microorganism-capturing method according to claim 1 based on nonimmune effect magnetic material, it is characterised in that: will
After magnetic bead is cleaned three times with the phosphate buffer containing 0.05% polysorbas20, it is directly added into the solution containing microorganism, 25 DEG C, catches
It obtains 1 hour.
7. the microorganism-capturing method according to claim 1 based on nonimmune effect magnetic material, it is characterised in that: institute
Stating microorganism is escherichia coli.
8. a kind of separation method of microorganism, it is characterised in that: by without any modification Magnetic particles, through amido modified magnetic bead and
One of magnetic bead through carboxyl modified or it is a variety of be placed directly in microorganism, capture to microorganism is realized, then by outer
Add magnetic field to separate, realizes the separation of microorganism.
9. the separation method of microorganism according to claim 7, it is characterised in that: the microorganism is escherichia coli
Bacterium.
10. new application of the magnetic bead in microorganism-capturing, it is characterised in that: by without any modification Magnetic particles, through amido modified
Magnetic bead and one of magnetic bead through carboxyl modified or it is a variety of be placed directly in microorganism, realize the capture to microorganism.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110628624A (en) * | 2019-02-01 | 2019-12-31 | 浙江和谱生物科技有限公司 | Magnetic microorganism capturing material and microorganism capturing method |
| CN110819687A (en) * | 2019-10-24 | 2020-02-21 | 广东环凯生物科技有限公司 | Escherichia coli O157 immunomagnetic bead washing liquor |
| WO2021069903A1 (en) | 2019-10-08 | 2021-04-15 | Momentum Bioscience Limited | Microorganism capture from antimicrobial-containing solution |
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| CN102154440A (en) * | 2011-01-01 | 2011-08-17 | 浙江省质量技术监督检测研究院 | Method for removing residual disinfectant left after disinfection by capturing with magnetic beads |
| WO2018131826A1 (en) * | 2017-01-16 | 2018-07-19 | 주식회사 바이오팩트 | Method and composition for isolating plasmid dna using amine-coated magnetic nanoparticle |
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2019
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| CN102154440A (en) * | 2011-01-01 | 2011-08-17 | 浙江省质量技术监督检测研究院 | Method for removing residual disinfectant left after disinfection by capturing with magnetic beads |
| WO2018131826A1 (en) * | 2017-01-16 | 2018-07-19 | 주식회사 바이오팩트 | Method and composition for isolating plasmid dna using amine-coated magnetic nanoparticle |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628624A (en) * | 2019-02-01 | 2019-12-31 | 浙江和谱生物科技有限公司 | Magnetic microorganism capturing material and microorganism capturing method |
| WO2021069903A1 (en) | 2019-10-08 | 2021-04-15 | Momentum Bioscience Limited | Microorganism capture from antimicrobial-containing solution |
| EP4283300A2 (en) | 2019-10-08 | 2023-11-29 | Momentum Bioscience Limited | Microorganism capture from antimicrobial-containing solution |
| CN110819687A (en) * | 2019-10-24 | 2020-02-21 | 广东环凯生物科技有限公司 | Escherichia coli O157 immunomagnetic bead washing liquor |
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