CN101906409A - Preparation method of live magnetic microbe - Google Patents
Preparation method of live magnetic microbe Download PDFInfo
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- CN101906409A CN101906409A CN2009100624288A CN200910062428A CN101906409A CN 101906409 A CN101906409 A CN 101906409A CN 2009100624288 A CN2009100624288 A CN 2009100624288A CN 200910062428 A CN200910062428 A CN 200910062428A CN 101906409 A CN101906409 A CN 101906409A
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Abstract
The invention provides a preparation method for obtaining a live magnetic microbe by efficiently introducing magnetic nano particles to the surface of a microbe. The preparation method comprises the following steps of: after activation culture of the microbe to be magnetized, modifying the surfaces of microbial cells by using appropriate polycation and polyanion dilute solution; then adding CaCl2 solution, aqueous solution of the magnetic nano particle, and Na2HPO4 and NaOH into the microbe; depositing the generated calcium phosphate on the surfaces of the microbial cells; and doping the magnetic nano particles into a deposition layer of the calcium phosphate and adhering the magnetic nano particles to the surfaces of the microbial cells, wherein the prepared magnetic microbe can keep the activity. The preparation method has strong operability, has high universality for different types of microbes, can ensure that a general microbial live cell additionally obtains magnetotaxis, and can be used for the process control of microbial microbe bio-manufacturing to further assemble a micro-nano new material and a new device.
Description
Technical field
The invention belongs to microorganism and biological manufacturing field, be specifically related to microorganism magnetization technology method.
Background technology
The biological manufacturing is life science, Materials science and biotechnology to be dissolved into make in the subject, is the new branch of science that micro-nano manufacturing technology and life science intersection produces.It comprises bionical manufacturing, biomass and organism manufacturing, uses the principle and the method for modern manufacturing science and life science, by controlled three-dimensional processing and the assembling of cell or microorganism, makes novel material, device and biosystem.Biology is fabricated to the minute manufacturing technology provides a class brand-new manufacturing means, has expanded the border and the category in traditional manufacturing field.
The biological essence of making be with biological as the main body of finishing manufacturing processed, constitute various microstructures in the controlled self-assembly self-assembly of micro-nano-scale by biology.Mainly comprise biomacromolecules such as DNA, protein, polysaccharide are handled in order; To microorganism directional induction or ordered arrangement, utilize its natural structure and functional development new function material; And, finish life entity moulding and manufacturing with metabolic feature by the controlled assembling of cell.
Microorganism is a life entity the most ancient on the earth, comprise bacterium, fungi, virus etc., of a great variety, form and biological function are different, its individual size from tens nanometers to micron order, have extremely strong vitality and adaptability, can cultivate in enormous quantities at lower cost, have great application potential at micro-nano manufacture field.In addition; utilize microorganism to carry out the friendly process process that materials processing is less energy-consumption, saving soil and natural resources, protection environment; therefore also orientable cultivation new variety are natural can be used for nanometer, micron and stride " elementary cell " that yardstick is processed at many levels.Is the machine of molecule assembling with the microorganism, it is carried out two-dimensional digital location and patterned arrangement or three-dimensional little manipulation, can design thus and formulate out novel material with specific function with the course control method for use of micro-nano-scale.Its key is orientation movement and the ordered arrangement with suitable method controlling microbial colony, and then can utilize its natural biological to learn function and finish processes such as self-assembly, orderly assembling.
The micro-nano manufacture method that can be used in biological process control at present mainly contains mechanical effect spraying technique, direct writing technology, electrospinning silk technology etc.And this wherein most methods can not be applied to control to micro-organisms living cell, mainly be because its control condition is gentle inadequately, can not guarantee the complete of the survival of microorganism individuality in whole control process and biological function.
The contriver has proposed the new ideas of live magnetic microorganism, is meant the artificial constructed living microorganism that can carry out the magnetotaxis motion under externally-applied magnetic field.It will can be used for setting up a kind ofly comes the biological process control method of controlling microbial with magnetic field, and this still belongs to blank in the biology field of making.The magnetic control method compared to other course control method for use have mild condition, workable, fine degree is high, can carry out the control of orientation or localization, can dynamically control and easily with advantages such as other course control method for use is used in combination.
Magnetotactic bacteria is natural magnetic microbe.The United States Patent (USP) of patent No. US2006073540-A1 " Controlling of micro-object (s) involves modifying the orientation of magneticfield to modify the displacement path of magnetotactic bacterium " thus once related to a kind of by changing the method that magnetic field orientating is controlled micron-sized object, a kind of system based on magnetotactic bacteria has been set up in this invention, thereby promptly by making this system have magnetic response on micron-sized object and magnetotactic bacteria coupling, and then utilization magnetic field control.But the magnetotactic bacteria biomass is little, must under exacting terms, cultivate, therefore and different biological manufacturing processedes require differently to microbial function and form, need set up simple and easy to do and different strain is had the method for broad applicability, come artificial constructed magnetic microbe.Applying date 2002.10.11, it is the hollow metal particle and preparation method thereof of template with the microorganism cells that the Chinese patent application file of application number 02130836.5 " being hollow metal particle of template and preparation method thereof with the microorganism cells " once related to a kind of, it is to utilize the microorganism cells of different geometric shapes to be template, by chemical plating method at its surface deposition metal level or alloy layer, be prepared into light hollow metallic conduction or magnetic particle, but this method will make microorganism cause death, and what it was prepared can only be metal particle, and can not further be applied to assemble biomaterial in the biology field of making, biological device.The Japanese Patent of patent No. JP2005349254-A " Moving a minuteobject (e.g.a microorganism) bound to or containing magnetic microparticles; comprises moving a magnetic probe to move the object on a substrate " once related to a kind of utilize needle guiding in conjunction with or contain the method that the small items of magnetic particle carries out permutatation, this invention can be used for studying the behavior of cell or the interaction between cell and microorganism etc., but this invention does not relate on the microorganism surface or the inner preparation method who imports magnetic particle.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of live magnetic microbe.
Realize that technical scheme of the present invention is:
This preparation method of live magnetic microbe provided by the invention may further comprise the steps:
(1) activation culture microorganism to be magnetized according to a conventional method.
(2), polycation and polyanion alternately are adsorbed on activated cultured microorganism cell surface, and to make the microorganism cells outermost layer be polyanion for the electronegative microorganism of cell surface; For the positively charged microorganism of cell surface, then polyanion and polycation alternately are adsorbed on activated cultured microorganism cell surface, and to make the microorganism cells outermost layer be polyanion, obtaining the cell outermost layer is the microbial bacteria suspension of polyanion, and used polycation and polyanion are for waiting to magnetize polycation and the polyanion that microbial metabolism function and cellularstructure do not damage to selected.
(3) in being the microbial bacteria suspension of polyanion, the cell outermost layer adds CaCl
2The solution and the magnetic nanoparticle aqueous solution then, maintain the pH of microbial bacteria suspension with NaOH solution again and slowly drip Na under the weakly acidic condition
2HPO
4Solution, the calcium phosphate precipitation that reaction is generated is deposited on the microorganism cells surface uniformly, and magnetic nanoparticle also is incorporated in the settled layer of calcium phosphate equably.
For the unlikely suction of microorganism cells is risen brokenly, the whole operations that relate to should be carried out under 0～4 ℃ of condition in above-mentioned steps (2) and (3); Employed whole reagent comprise deionized water, said polycation solution, polyanion solution, CaCl in above-mentioned steps (2) and (3)
2Solution, the magnetic nanoparticle aqueous solution, Na
2HPO
4Solution and NaOH solution should add NaCl when preparation, select suitable concentration according to handled microorganism cells, press to keep Premeabilisation of cells; Employed whole reagent must pass through autoclave sterilization before use in above-mentioned steps (2) and (3).
The described activation culture of above-mentioned steps (1) method of microorganism to be magnetized is: choose microbial strains to be magnetized, being seeded to liquid nutrient medium activation culture to the cell concn in the microbial culture medium according to a conventional method is 10
6～10
8ML
-1Microorganism to be magnetized can be fungi or bacterium, and its form can be spherical, shaft-like or spirrillum, and its surface can be to have positive charge, also can be electronegative.Activation culture waits to magnetize the ordinary method of microorganism can be referring to Zhou Deqing chief editor's " microbiology experiment study course ", Higher Education Publishing House's second edition in 2006.
Above-mentioned steps (2) is described alternately to be adsorbed on the electronegative microorganism surface of activated cultivation with polycation and polyanion, and to make the microorganism cells outermost layer be that the concrete grammar of polyanion may further comprise the steps:
A) clean the microorganism cells surface: the microbial culture medium of getting after the activation culture of certain volume is transferred in the centrifuge tube, usually the microbial culture medium of getting after 5～10mL activation culture is transferred in the centrifuge tube, centrifugal with suitable centrifugal rotational speed and centrifugation time, centrifugal rotational speed and centrifugation time are determined according to selected microbial strains, abandon supernatant liquor, add with the isopyknic deionized water of microbial culture medium after the activation culture of getting, shake up and make microorganism cells form bacteria suspension, centrifugal with in this step, abandon supernatant liquor, add isopyknic deionized water, the process that shakes up repeats 1～3 time;
B) absorption of cell surface polycation: bacteria suspension is centrifugal, abandon add behind the supernatant with step a) in to add the isopyknic mass percent of deionized water be 0.01～0.5% polycation dilute solution, left standstill after shaking up 10～30 minutes;
C) remove the polycation that does not adsorb in the bacteria suspension: the bacteria suspension that step b) was handled is centrifugal, abandon supernatant, institute adds the isopyknic deionized water of deionized water in adding and the step a), shake up, with in this step centrifugal, abandon supernatant, add deionized water, the process that shakes up repeats 1～3 time;
D) absorption of cell surface polyanion: the bacteria suspension that step c) was handled is centrifugal, abandon supernatant, add with step a) in to add the isopyknic mass percent of deionized water be 0.01～0.5% polyanion dilute solution, left standstill after shaking up 10～30 minutes;
E) remove the polyanion that does not adsorb in the bacteria suspension: the bacteria suspension that step d) was handled is centrifugal, abandon supernatant, institute adds the isopyknic deionized water of deionized water in adding and the step a), shake up, with in this step centrifugal, abandon supernatant, add deionized water, the process that shakes up repeats 1～3 time;
F) polycation and polyanion are in the alternately absorption on microorganism cells surface: with step b), c), d), e) repeat successively 1～4 time, obtaining the cell outermost layer is the microbial bacteria suspension of polyanion.
Above-mentioned steps (2) is described alternately to be adsorbed on the positively charged microorganism surface of cell surface with polyanion and polycation, and to make the microorganism cells outermost layer be that the concrete grammar of polyanion may further comprise the steps:
A ') clean the microorganism cells surface: the microbial culture medium of getting after the activation culture of certain volume is transferred in the centrifuge tube, usually the microbial culture medium of getting after the 5-10mL activation culture is transferred in the centrifuge tube, centrifugal with suitable centrifugal rotational speed and centrifugation time, centrifugal rotational speed and centrifugation time are determined according to selected microbial strains, abandon supernatant liquor, add with the isopyknic deionized water of microbial culture medium after the activation culture of getting, shake up and make microorganism cells form bacteria suspension, centrifugal with in this step, abandon supernatant liquor, add isopyknic deionized water, the process that shakes up repeats 1～3 time;
B ') absorption of cell surface polyanion: bacteria suspension is centrifugal, abandon behind the supernatant add and step a ') in to add the isopyknic mass percent of deionized water be 0.01～0.5% polyanion dilute solution, left standstill after shaking up 10～30 minutes;
C ') removes the polyanion that does not adsorb in the bacteria suspension: the bacteria suspension that step b) was handled is centrifugal, abandon supernatant, add and step a ') in institute add the isopyknic deionized water of deionized water, shake up, with in this step centrifugal, abandon supernatant, add deionized water, the process that shakes up carries out 1～3 time repeatedly;
D ') absorption of cell surface polycation: with step c ') bacteria suspension of handling is centrifugal, abandon supernatant, add and step a ') in to add the isopyknic mass percent of deionized water be 0.01～0.5% polycation dilute solution, left standstill after shaking up 10～30 minutes;
E ') remove in the bacteria suspension the not polycation of absorption: with steps d ') bacteria suspension handled is centrifugal, abandon supernatant, add and step a ') in institute add the isopyknic deionized water of deionized water, shake up, with in this step centrifugal, abandon supernatant, add deionized water, the process that shakes up carries out 1～3 time repeatedly;
F ') with above-mentioned steps b '), c '), d '), e ') repeat successively 1～4 time, and when repeating the last time, only carry out step b ' successively), c '), making the cell outermost layer is polyanion, obtaining the cell outermost layer is the microbial bacteria suspension of polyanion.
Above-mentioned steps (2) is described, and to wait to magnetize the polycation that microbial metabolism function and cellularstructure do not damage can be diallyl dimethyl ammoniumchloride, polypropylene ammonium chloride or cationic polyacrylamide to selected, and described to wait to magnetize the polyanion that microbial metabolism function and cellularstructure do not damage can be sodium polyacrylate, sodium polystyrene sulfonate, anionic polyacrylamide or sodium alginate to selected.
Above-mentioned steps (3) is described to add CaCl in the cell outermost layer is the microbial bacteria suspension of polyanion
2The solution and the magnetic nanoparticle aqueous solution then, make the pH of microbial bacteria suspension maintain the slow Na of dropping under the weakly acidic condition with NaOH solution
2HPO
4Solution, the calcium phosphate precipitation that reaction is generated is deposited on the microorganism cells surface uniformly, and the concrete grammar that magnetic nanoparticle also is incorporated in the settled layer of calcium phosphate equably may further comprise the steps:
G) CaCl of adding question response
2: the bacteria suspension after will handling through above-mentioned steps (2) will be centrifugal, abandon supernatant, add with aforementioned cleaning microorganism cells surface step in to add the isopyknic concentration of deionized water be the CaCl of 0.005～0.05mol/L
2Solution shakes up;
H) add magnetic nanoparticle: adding concentration in the bacteria suspension that step g) was handled is the aqueous solution of the magnetic nanoparticle of 0.01～0.1g/L, and its volume is added CaCl by step g)
21/20～1/5 of solution shakes up.Magnetic nanoparticle in the used magnetic nano particle aqueous solution can be Fe
3O
4Or γ-Fe
2O
3Median size is less than 50nm, but preparation method's reference of the aqueous solution of magnetic nanoparticle " New technique for synthesizing iron ferrite magnetic nanosized particles; Langmuir; 1997; 13 (15): 3927-3933 " or " Synthesis and surface engineering ofiron oxide nanoparticles for biomedical applications; Biomaterials; 2005,26 (18): 3995-4021 ".
I) to through step h) slowly dropping and CaCl that step g) adds in the bacteria suspension that contains magnetic nanoparticle handled
2The concentration that liquor capacity is identical is the Na of 0.005～0.05mol/L
2HPO
4Solution does not stop to shake up, and is the NaOH solution adjusting pH of 0.005～0.05mol/L with concentration simultaneously, makes Na
2HPO
4PH maintains 6.7～6.8 in the whole dropping process of solution;
J) Na
2HPO
4After all dropwising with NaOH solution, continue to shake up 10～60min;
K) remove excessive and unreacted reagent: with step j) bacteria suspension after handling is centrifugal, abandon supernatant, institute adds the isopyknic deionized water of deionized water in adding and the aforementioned cleaning microorganism cells surface step, shake up, with in this step centrifugal, abandon supernatant, add deionized water, the process that shakes up carries out 1～3 time repeatedly, promptly makes magnetic microbe.
Based on the inventive method, the invention provides a kind of live magnetic microorganism of artificial preparation, there is the magnetic nanoparticle that adheres to manual method on the surface of this microorganism cells, and the magnetic nanoparticle that adheres to is the Fe of median size less than 50nm
3O
4Or γ-Fe
2O
3
Experimental data
1. the observation of the magnetic yeast magnetotaxis that embodiment 1 is made
The magnetic yeast sample concentration of preparation is that the tetracycline hydrochloride of 0.05g/L is to surperficial Ca
2+Ion carries out fluorescent dye, and carries out Fluirescence observation with opticmicroscope (Olympus IX71) under 360～370nm UV-light.Prepare the control group yeast in addition,, but in preparation process, do not add magnetic nanoparticle γ-Fe according to the preparation of embodiment 1 method
2O
3, carry out observing after the fluorescent dye with tetracycline hydrochloride equally.
Magnetotaxis with small magnetic needle test yeast cell.After the result showed the adding small magnetic needle, the magnetic yeast was assembled near small magnetic needle in a large number, presents bright fluorescent effect (Fig. 1 c), illustrates that this yeast has good magnetotaxis; The control group yeast then can not be gathered in (Fig. 2 c) around the small magnetic needle.Two groups of experimental results can illustrate that magnetic nanoparticle successfully is equipped on the yeast surface, and make yeast have magnetotaxis.
2. the proof of the magnetic yeast viability that embodiment 1 is made
To carry out the magnetic resolution purifying with small magnet by the magnetic yeast that embodiment 1 makes, be stored in the NaCl solution of 7g/L under 4 ℃ of conditions.After depositing a week, the hydrochloric acid soln of using 0.01mol/L is seeded to liquid nutrient medium with magnetic yeast suspension adjust pH to 6, and 30 ℃ of constant temperature shaking tables were cultivated 48 hours.The result proves that yeast can well grow, and it is consistent with the yeast of common inoculation to observe its morphological specificity under opticmicroscope.
3. the observation of the magnetic yeast surface microstructure that embodiment 1 is made
Observe the saccharomycetic surface topography of magnetic with scanning electronic microscope (QUANTA200), sample is handled through lyophilize.Observe magnetic yeast cell tangent plane pattern with transmission electron microscope (FEI Tecnai G2 20 TWIN), adopt glutaraldehyde---carry out ultrathin section(ing) after osmic acid double fixed method is fixing, observe with acetic acid uranium and lead citrate dyeing back.Fig. 3 has demonstrated the saccharomycetic whole pattern of magnetic that scanning electron microscopic observation arrives, and there is the continuous folding relief fabric on the surface.The natural smooth corrugationless of yeast cell wall surface (Fig. 4 b).There is significantly " sawtooth " shape structure on yeast surface after handling through embodiment 1, and (Fig. 4 a), and cell walls outermost layer segment thickness obviously increases, and this part-structure distributes continuous, and is comparatively even.Firm attachment is in yeast cell surface can to infer magnetic nanoparticle thus, and this also is to make cell that the reason of magnetotaxis be arranged.
The invention discloses a kind of at the efficient magnetic nanoparticle that imports in the microorganism surface of living, acquisition preparation method of live magnetic microbe.The inventive method is workable, and the different microorganisms bacterial classification is had good broad applicability, and the microorganism of preparation has magnetotaxis, and can keep survival.
The live magnetic microorganism is the new ideas that proposed by the present invention, and the live magnetic microorganism is meant the artificial constructed living microorganism that can carry out the magnetotaxis motion under externally-applied magnetic field.Though can not be hereditary by the magnetic microbe magnetic that the present invention makes, the division of cell increment simultaneously is restricted, but can keep active, this is what further explore based on micro-organisms living cell, and the biology that can bring into play whole biological function of microorganism and advantage is made the magnetic control process and laid a good foundation.Combination by biotechnology and micro-nano technology, with magnetic microbe is micro-nano robot, method ixed control and ordered arrangement living microorganism in micro-nano size range with magnetic field control, bring out its distinctive biological function, carry out biological manufacturing processedes such as controlled self-assembly, be expected to design thus and formulate a series of novel specific function materials and device.
Description of drawings
Fig. 1 is for testing the observations of the magnetic yeast magnetotaxis that under opticmicroscope embodiment 1 is made described in 1, when not adding needle, under opticmicroscope, observe the magnetic yeast, figure a), b) be respectively the fluorescence result and the common result of shooting the same visual field under; After adding needle, under opticmicroscope, observe the magnetic yeast, figure c), d) be respectively the fluorescence result and the common result that take under the same visual field, after the result shows the adding small magnetic needle, the magnetic yeast is assembled near small magnetic needle in a large number, present bright fluorescent effect, illustrate that this yeast has good magnetotaxis;
Fig. 2 is the observations of control experiment described in the test 1, when not adding needle, observes the control group yeast under opticmicroscope, figure a), b) be respectively the fluorescence result and the common result of shooting the same visual field under; After adding needle, under opticmicroscope, observe the control group yeast, figure c), d) be respectively the fluorescence result and the common result that take under the same visual field, the result shows that the control group yeast can not be gathered in around the small magnetic needle, contrast with Fig. 1 result, two groups of experimental results can illustrate that magnetic nanoparticle successfully is equipped on the yeast surface, and make yeast have magnetotaxis;
The magnetic saccharomycetic sem photograph of Fig. 3 for being made by embodiment 1 described in the test 3 demonstrated the saccharomycetic whole pattern of magnetic that scanning electron microscopic observation arrives, and there is the continuous folding relief fabric on the surface;
The magnetic saccharomycetic transmission electron microscope picture (a) of Fig. 4 for making by embodiment 1 described in the test 3, transmission electron microscope picture (b) with control group unartificial yeast bacterium, demonstrate the natural smooth corrugationless of yeast cell wall surface among the figure, through the yeast surface after embodiment 1 processing significantly " sawtooth " shape structure is arranged, and cell walls outermost layer segment thickness obviously increases, this part-structure distributes continuously, comparatively even, firm attachment is in yeast cell surface can to infer magnetic nanoparticle thus, and this also is to make yeast that the reason of magnetotaxis be arranged.
Embodiment
The present invention is further elaborated below in conjunction with specific embodiment.
Whole the operating under 0～4 ℃ of condition that relates in following examples 1 described step (2), (3) and embodiment 2 described steps (2), (3) carried out.
(1) chooses and wait to magnetize microorganism yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), be seeded to the back 30 ℃ of constant temperature shaking tables of YPD liquid nutrient medium (peptone 2%, yeast powder 1%, glucose 2%) and cultivate 48h.
(2) microorganism cells surface Modification: with the deionized water compound concentration is the NaCl solution of 7g/L, prepares other solution that will use in all preparation process, autoclave sterilization with this solution again; The yeast culture liquid of getting after 10mL cultivates is transferred in the centrifuge tube, the centrifugal 6min of 5000rpm (all centrifugal these conditions that adopt among the embodiment 1) abandons supernatant liquor, adds 10mLNaCl solution, shake up into bacteria suspension, with centrifugal, abandon supernatant, add NaCl solution, the process that shakes up carries out 2 times repeatedly; The yeast suspension is centrifugal, abandon that to add the 10mL mass percent behind the supernatant be 0.1% diallyl dimethyl ammoniumchloride solution, left standstill after shaking up 20 minutes; Bacteria suspension is centrifugal, abandon supernatant, add 10mLNaCl solution, shake up; Bacteria suspension is centrifugal, abandon supernatant, adding 10mL mass percent is 0.1% sodium polyacrylate solution, leaves standstill after shaking up 20 minutes; Bacteria suspension is centrifugal, abandon supernatant, add 10mLNaCl solution, shake up; Above-mentioned steps is centrifugal, abandon supernatant, add diallyl dimethyl ammoniumchloride solution, shake up, leave standstill, centrifugal, abandon supernatant, add NaCl solution, shake up, centrifugal, abandon supernatant, add sodium polyacrylate solution, shake up, leave standstill, centrifugal, abandon supernatant, add NaCl solution, shake up, repeat successively to carry out again 1 time.
(3) magnetic nanoparticle adhering in yeast cell surface: the bacteria suspension after step (2) processing is centrifugal, abandon supernatant, adding 10mL concentration is the CaCl of 0.01mol/L
2Solution shakes up; To wherein adding magnetic nanoparticle γ-Fe that concentration is 0.1g/L
2O
3Aqueous solution 1mL, shake up the γ-Fe in the used magnetic nano particle aqueous solution
2O
3Median size is 20nm～30nm; Slowly dripping 10mL concentration in the bacteria suspension that contains magnetic nanoparticle is the Na of 0.01mol/L
2HPO
4Solution does not stop to shake up, and is the NaOH solution adjusting pH of 0.01mol/L with concentration simultaneously, makes Na
2HPO
4PH maintains 6.8 in the whole dropping process of solution; Na
2HPO
4After all dropwising with NaOH solution, continue to shake up 60min; Bacteria suspension after handling is centrifugal, abandon supernatant, add 10mLNaCl solution, shake up, with centrifugal, abandon supernatant, add NaCl solution, the process that shakes up carries out 2 times again, promptly makes the magnetic yeast.
Embodiment 2
(1) chooses microorganism Escherichia coli to be magnetized (Escherichia coli), be seeded to the back 37 ℃ of constant temperature shaking tables of LB liquid nutrient medium (NaCl 1% for peptone 1%, yeast powder 0.5%, and pH 7.4) and cultivate 12h.
(2) microorganism cells surface Modification: with the deionized water compound concentration is the NaCl solution of 5g/L, prepares other solution that will use in all preparation process, autoclave sterilization with this solution again; The intestinal bacteria nutrient solution of getting after 10mL cultivates is transferred in the centrifuge tube, the centrifugal 10min of 6000rpm (all centrifugal these conditions that all adopt among the embodiment 2), abandon supernatant liquor, add 10mLNaCl solution, shake up into bacteria suspension, with centrifugal, abandon supernatant, add NaCl solution, the process that shakes up carries out 1 time repeatedly; The intestinal bacteria bacteria suspension is centrifugal, abandon that to add the 10mL mass percent behind the supernatant be 0.05% polypropylene ammonium chloride solution, left standstill after shaking up 10 minutes; Bacteria suspension is centrifugal, abandon supernatant, add 10mLNaCl solution, shake up; Bacteria suspension is centrifugal, abandon supernatant, adding 10mL mass percent is 0.05% sodium polystyrene sulfonate solution, leaves standstill after shaking up 10 minutes; Bacteria suspension is centrifugal, abandon supernatant, add 10mLNaCl solution, shake up; Above-mentioned steps is centrifugal, abandon supernatant, add the polypropylene ammonium chloride solution, shake up, leave standstill, centrifugal, abandon supernatant, add NaCl solution, shake up, centrifugal, abandon supernatant, add sodium polystyrene sulfonate solution, shake up, leave standstill, centrifugal, abandon supernatant, add NaCl solution, shake up, repeat successively to carry out again 2 times.
(3) magnetic nanoparticle adhering on the Bacillus coli cells surface: the bacteria suspension after step (2) processing is centrifugal, abandon supernatant, adding 10mL concentration is the CaCl of 0.005mol/L
2Solution shakes up; To wherein adding the magnetic nanoparticle Fe that concentration is 0.01g/L
3O
4Aqueous solution 0.5mL, shake up the Fe in the used magnetic nano particle aqueous solution
3O
4Median size is 10nm～20nm; Slowly dripping 10mL concentration in the bacteria suspension that contains magnetic nanoparticle is the Na of 0.005mol/L
2HPO
4Solution does not stop to shake up, and is the NaOH solution adjusting pH of 0.005mol/L with concentration simultaneously, makes Na
2HPO
4PH maintains 6.75 in the whole dropping process of solution; Na
2HPO
4After all dropwising with NaOH solution, continue to shake up 30min; Bacteria suspension after handling is centrifugal, abandon supernatant, add 10mLNaCl solution, shake up, with centrifugal, abandon supernatant, add NaCl solution, the process that shakes up carries out 1 time again, promptly makes the magnetic intestinal bacteria.
The observation of the magnetic yeast magnetotaxis that 3 couples of embodiment 1 of embodiment make
The magnetic yeast sample concentration of preparation is that the tetracycline hydrochloride of 0.05g/L is to surperficial Ca
2+Ion carries out fluorescent dye, and carries out Fluirescence observation with opticmicroscope (Olympus IX71) under 360～370nm UV-light.Prepare the control group yeast in addition,, but in preparation process, do not add magnetic nanoparticle γ-Fe according to the preparation of embodiment 1 method
2O
3, carry out observing after the fluorescent dye with tetracycline hydrochloride equally.
Magnetotaxis with small magnetic needle test yeast cell.After the result showed the adding small magnetic needle, the magnetic yeast was assembled near small magnetic needle in a large number, presents bright fluorescent effect, sees Fig. 1 c, illustrates that this yeast has good magnetotaxis; The control group yeast then can not be gathered in around the small magnetic needle, sees Fig. 2 c.Two groups of experimental results can illustrate that magnetic nanoparticle successfully is equipped on the yeast surface, and make yeast have magnetotaxis.
The proof of the magnetic yeast viability that 4 couples of embodiment 1 of embodiment make
To carry out the magnetic resolution purifying with small magnet by the magnetic yeast that embodiment 1 makes, be stored in the NaCl solution of 7g/L under 4 ℃ of conditions.After depositing a week, the hydrochloric acid soln of using 0.01mol/L is seeded to liquid nutrient medium with magnetic yeast suspension adjust pH to 6, and 30 ℃ of constant temperature shaking tables were cultivated 48 hours.The result proves that yeast can well grow, and it is consistent with the yeast of common inoculation to observe its morphological specificity under opticmicroscope.
The observation of the magnetic yeast surface microstructure that 5 couples of embodiment 1 of embodiment make
Observe the saccharomycetic surface topography of magnetic with scanning electronic microscope (QUANTA200), sample is handled through lyophilize.Observe magnetic yeast cell tangent plane pattern with transmission electron microscope (FEI Tecnai G2 20 TWIN), adopt glutaraldehyde---carry out ultrathin section(ing) after osmic acid double fixed method is fixing, observe with acetic acid uranium and lead citrate dyeing back.
Fig. 3 has demonstrated the saccharomycetic whole pattern of magnetic that scanning electron microscopic observation arrives, and there is the continuous folding relief fabric on the surface.The natural smooth corrugationless of yeast cell wall surface is seen Fig. 4 b.There is significantly " sawtooth " shape structure on yeast surface after handling through embodiment 1, sees Fig. 4 a, and cell walls outermost layer segment thickness obviously increases, and this part-structure distributes continuous, and is comparatively even.Firm attachment is in yeast cell surface can to infer magnetic nanoparticle thus, and this also is to make cell that the reason of magnetotaxis be arranged.
Claims (15)
1. preparation method of live magnetic microbe may further comprise the steps:
(1) activation culture microorganism to be magnetized according to a conventional method;
(2), polycation and polyanion alternately are adsorbed on activated cultured microorganism cell surface, and to make the microorganism cells outermost layer be polyanion for the electronegative microorganism of cell surface; For the positively charged microorganism of cell surface, then polyanion and polycation alternately are adsorbed on activated cultured microorganism cell surface, and to make the microorganism cells outermost layer be polyanion, obtaining the cell outermost layer is the microbial bacteria suspension of polyanion, and used polycation and polyanion are for waiting to magnetize polycation and the polyanion that microbial metabolism function and cellularstructure do not damage to selected.
(3) in being the microbial bacteria suspension of polyanion, the cell outermost layer adds CaCl
2The solution and the magnetic nanoparticle aqueous solution then, maintain the pH of microbial bacteria suspension with NaOH solution again and slowly drip Na under the weakly acidic condition
2HPO
4Solution, the calcium phosphate precipitation that reaction is generated is deposited on the microorganism cells surface uniformly, and magnetic nanoparticle also is incorporated in the settled layer of calcium phosphate equably.
2. preparation method of live magnetic microbe according to claim 1 is characterized in that, described microorganism to be magnetized can be fungi or bacterium.
3. preparation method of live magnetic microbe according to claim 1 is characterized in that, described microorganism to be magnetized is that the surface has microorganism positive charge or negative charge.
4. preparation method of live magnetic microbe according to claim 1 is characterized in that, described microorganism to be magnetized, and its form can be spherical, shaft-like or spirrillum.
5. preparation method of live magnetic microbe according to claim 1, it is characterized in that, described activation culture method of microorganism to be magnetized is: choose microbial strains to be magnetized, being seeded to liquid nutrient medium activation culture to the cell concn in the microbial culture medium according to a conventional method is 10
6～10
8ML
-1
6. preparation method of live magnetic microbe according to claim 1, it is characterized in that, described polycation and polyanion alternately are adsorbed on the electronegative microorganism surface of activated cultivation, and to make the microorganism cells outermost layer be that the concrete grammar of polyanion may further comprise the steps:
A) clean the microorganism cells surface: the microbial culture medium of getting after the activation culture of certain volume is transferred in the centrifuge tube, centrifugal with suitable centrifugal rotational speed and centrifugation time, abandon supernatant liquor, add with the isopyknic deionized water of microbial culture medium after the activation culture of getting, shake up and make microorganism cells form bacteria suspension, with in this step centrifugal, abandon supernatant liquor, add isopyknic deionized water, the process that shakes up repeats 1～3 time;
B) absorption of cell surface polycation: bacteria suspension is centrifugal, abandon add behind the supernatant with step a) in to add the isopyknic mass percent of deionized water be 0.01～0.5% polycation dilute solution, left standstill after shaking up 10～30 minutes;
C) remove the polycation that does not adsorb in the bacteria suspension: the bacteria suspension that step b) was handled is centrifugal, abandon supernatant, institute adds the isopyknic deionized water of deionized water in adding and the step a), shake up, with in this step centrifugal, abandon supernatant, add deionized water, the process that shakes up repeats 1～3 time;
D) absorption of cell surface polyanion: the bacteria suspension that step c) was handled is centrifugal, abandon supernatant, add with step a) in to add the isopyknic mass percent of deionized water be 0.01～0.5% polyanion dilute solution, left standstill after shaking up 10～30 minutes;
E) remove the polyanion that does not adsorb in the bacteria suspension: the bacteria suspension that step d) was handled is centrifugal, abandon supernatant, institute adds the isopyknic deionized water of deionized water in adding and the step a), shake up, with in this step centrifugal, abandon supernatant, add deionized water, the process that shakes up repeats 1～3 time;
F) polycation and polyanion are in the alternately absorption on microorganism cells surface: with step b), c), d), e) repeat successively 1～4 time, obtaining the cell outermost layer is the microbial bacteria suspension of polyanion.
7. preparation method of live magnetic microbe according to claim 1, it is characterized in that, describedly will wait to magnetize polyanion and the polycation that microbial metabolism function and cellularstructure do not damage and alternately be adsorbed on the positively charged microorganism surface of cell surface, and to make the microorganism cells outermost layer be that the concrete grammar of polyanion may further comprise the steps selected:
A ') clean the microorganism cells surface: the microbial culture medium of getting after the activation culture of certain volume is transferred in the centrifuge tube, centrifugal with suitable centrifugal rotational speed and centrifugation time, abandon supernatant liquor, add with the isopyknic deionized water of microbial culture medium after the activation culture of getting, shake up and make microorganism cells form bacteria suspension, with in this step centrifugal, abandon supernatant liquor, add isopyknic deionized water, the process that shakes up repeats 1～3 time;
B ') absorption of cell surface polyanion: bacteria suspension is centrifugal, abandon add behind the supernatant with step a) in to add the isopyknic mass percent of deionized water be 0.01～0.5% polyanion dilute solution, left standstill after shaking up 10～30 minutes;
C ') removes the polyanion that does not adsorb in the bacteria suspension: the bacteria suspension that step b) was handled is centrifugal, abandon supernatant, institute adds the isopyknic deionized water of deionized water in adding and the step a), shake up, with in this step centrifugal, abandon supernatant, add deionized water, the process that shakes up carries out 1～3 time repeatedly;
D ') absorption of cell surface polycation: the bacteria suspension that step c) was handled is centrifugal, abandon supernatant, add and step a ') in to add the isopyknic mass percent of deionized water be 0.01～0.5% polycation dilute solution, left standstill after shaking up 10～30 minutes;
E ') removes the polycation that does not adsorb in the bacteria suspension: the bacteria suspension that step d) was handled is centrifugal, abandon supernatant, add and step a ') in institute add the isopyknic deionized water of deionized water, shake up, with in this step centrifugal, abandon supernatant, add deionized water, the process that shakes up carries out 1～3 time repeatedly;
F ') with above-mentioned steps b '), c '), d '), e ') repeat successively 1～4 time, and when repeating the last time, only advance successively to go step b '), c '), making the cell outermost layer is polyanion, obtaining the cell outermost layer is the microbial bacteria suspension of polyanion.
8. preparation method of live magnetic microbe according to claim 1, it is characterized in that described to wait to magnetize the polycation that microbial metabolism function and cellularstructure do not damage be diallyl dimethyl ammoniumchloride, polypropylene ammonium chloride or cationic polyacrylamide to selected; It is described that to wait to magnetize the polyanion that microbial metabolism function and cellularstructure do not damage be sodium polyacrylate, sodium polystyrene sulfonate, anionic polyacrylamide or sodium alginate to selected.
9. preparation method of live magnetic microbe according to claim 1 is characterized in that, describedly adds CaCl in the cell outermost layer is the microbial bacteria suspension of polyanion
2The solution and the magnetic nanoparticle aqueous solution then, make the pH of microbial bacteria suspension maintain the slow Na of dropping under the weakly acidic condition with NaOH solution
2HPO
4Solution, the calcium phosphate precipitation that reaction is generated is deposited on the microorganism cells surface uniformly, and the concrete grammar that magnetic nanoparticle also is incorporated in the settled layer of calcium phosphate equably may further comprise the steps:
G) CaCl of adding question response
2: the bacteria suspension after will handling through the described step of claim 1 (2) will be centrifugal, abandon supernatant, add with the described step a) of claim 6 or with the described step a ' of claim 7) in to add the isopyknic concentration of deionized water be the CaCl of 0.005～0.05mol/L
2Solution shakes up;
H) add magnetic nanoparticle: adding concentration in the bacteria suspension that step g) was handled is the aqueous solution of the magnetic nanoparticle of 0.01～0.1g/L, and its volume is added CaCl by step g)
21/20～1/5 of solution shakes up;
I) to through step h) slowly dropping and CaCl that step g) adds in the bacteria suspension that contains magnetic nanoparticle handled
2The concentration that liquor capacity is identical is the Na of 0.005～0.05mol/L
2HPO
4Solution does not stop to shake up, and is the NaOH solution adjusting pH of 0.005～0.05mol/L with concentration simultaneously, makes Na
2HPO
4PH maintains 6.7～6.8 in the whole dropping process of solution;
J) Na
2HPO
4After all dropwising with NaOH solution, continue to shake up 10～60min;
K) remove excessive and unreacted reagent: with step j) bacteria suspension after handling is centrifugal, abandon supernatant, add with the described step a) of claim 6 or with the described step a ' of claim 7) in institute add the isopyknic deionized water of deionized water, shake up, with in this step centrifugal, abandon supernatant, add deionized water, the process that shakes up carries out 1～3 time repeatedly.
10. according to each described preparation method of live magnetic microbe in the claim 1 to 9, it is characterized in that the magnetic nanoparticle in the described magnetic nanoparticle aqueous solution is Fe
3O
4Or γ-Fe
2O
3, its median size is less than 50nm.
11. preparation method of live magnetic microbe according to claim 1 is characterized in that, described step (2) and step (3) are to carry out under 0～4 ℃ of condition.
12., it is characterized in that described deionized water, said polycation solution, polyanion solution, CaCl according to each described preparation method of live magnetic microbe in the claim 1 to 11
2Solution, the magnetic nanoparticle aqueous solution, Na
2HPO
4Added NaCl in solution and the NaOH solution, to keep microorganism cells osmotic pressure, the add-on of NaCl is determined according to the required osmotic pressure of handled microorganism cells.
13., it is characterized in that described deionized water, said polycation solution, polyanion solution, CaCl according to each described preparation method of live magnetic microbe in the claim 1 to 12
2Solution, the magnetic nanoparticle aqueous solution, Na
2HPO
4Solution and NaOH solution pass through autoclave sterilization before use.
14. the live magnetic microorganism of artificial preparation is characterized in that there is the magnetic nanoparticle that adheres to manual method on the surface of this microorganism cells.
15. the magnetic microbe according to the described artificial preparation of claim 14 is characterized in that, the magnetic nanoparticle that this microorganism cells surface is adhered to manual method is the Fe of median size less than 50nm
3O
4Or γ-Fe
2O
3
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105727599A (en) * | 2016-02-23 | 2016-07-06 | 同济大学 | Method for adopting magnetic nanometer particles for modifying demulsifying bacteria to reinforce lactescence demulsification |
| CN106512482A (en) * | 2016-10-13 | 2017-03-22 | 同济大学 | Method for enhancing demulsifying property of demulsifying bacteria |
| CN113337498A (en) * | 2021-05-27 | 2021-09-03 | 青岛农业大学 | Magnetic microsphere immobilized protease, instant plant protein with high nitrogen solubility index prepared from magnetic microsphere immobilized protease and preparation method of instant plant protein |
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2009
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105727599A (en) * | 2016-02-23 | 2016-07-06 | 同济大学 | Method for adopting magnetic nanometer particles for modifying demulsifying bacteria to reinforce lactescence demulsification |
| CN105727599B (en) * | 2016-02-23 | 2017-11-07 | 同济大学 | The method that demulsifying bacteria is used to strengthen emulsion breakdown is modified using magnetic nano-particle |
| CN106512482A (en) * | 2016-10-13 | 2017-03-22 | 同济大学 | Method for enhancing demulsifying property of demulsifying bacteria |
| CN106512482B (en) * | 2016-10-13 | 2019-08-06 | 同济大学 | A method of enhancing demulsifying bacteria demulsification performance |
| CN113337498A (en) * | 2021-05-27 | 2021-09-03 | 青岛农业大学 | Magnetic microsphere immobilized protease, instant plant protein with high nitrogen solubility index prepared from magnetic microsphere immobilized protease and preparation method of instant plant protein |
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