CN107085102A - A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe Nb Cu B nano-probes - Google Patents
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe Nb Cu B nano-probes Download PDFInfo
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Abstract
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe Nb Cu B nano-probes, belongs to tcm development applied technical field.The present invention dependent on set up based on prepares Fe Nb Cu B nano-probes be used for screen suppression anaerobic bacteria Chinese medicine preparation method.The characteristics of paramagnetic Fe Nb Cu B nano-probes prepared can be specifically bound to object bacteria is coated with using antibody, the influence using Fe Nb Cu B paramagnetic properties to the NMR relaxation time detects whether contain object bacteria in sample.Paramagnetic Fe Nb Cu B nano-probes, NMR relaxation time showing outlet sexual intercourse within the specific limits, that is nanometer Fe Nb Cu B contents are big, the nuclear magnetic resonance spin Lattice Relaxation Time and transverse relaxation time value of sample are smaller, detection object bacteria can be quantified in certain limit, so that the fungistatic effect of indirect assessment Chinese medicine preparation.This method can be used for the recipe determination for having the Chinese medicine of inhibitory action to anaerobic bacteria, accelerate the development rate of tcm product.
Description
Technical field
The present invention relates to a kind of screening technique of Chinese medicine preparation.
Background technology
Antibiotic simultaneously, will also result in human body infringement, influence liver, renal function, gastrointestinal reaction etc. in sterilization.Abuse is anti-
Raw element may cause the generation of some bacterial resistance phenomenons, and the treatment to infection can become very difficult.At present, almost without one
Plant antibacterials and resistance phenomenon is not present.Chinese medicine and formula of the exploitation with antibacterial action have positive effect.
Many Chinese medicines and magistral medicines have antibacterial and anti-inflammation functions, and some medium-height grass the effective elements of the medicines and molecular structure etc. are also
Through studying clear whole or in part.The main component berberine of such as coptis and golden cypress stop dysentery(Berberine), radix scutellariae antibacterial
Main component baicalein etc..A host of facts prove, the medicine that ancient Chinese working people is accumulated by long-term practice
Legacy is extremely abundant, extremely valuable.We should value the great treasure-house of this motherland's medicine and pharmacology, make great efforts to excavate, be subject to
Improve, can therefrom develop the Chinese medicine with antibacterial and anti-inflammation functions, can replace or part substitution antibiotic.
Anaerobic bacteria be a class under anaerobic than the bacterium grown in aerobic environment, and can not be with the presence of air
In the case of the bacterium that grows.This kind of bacterium lacks complete metabolism enzyme system, and its energetic supersession is carried out in the way of anaerobic fermentation.
It can cause the infection of human body different parts, including appendicitis, cholecystitis, tympanitis, mouth infection, endocarditis, osteomyelitis,
A variety of diseases such as peritonitis.It is usually in sterilizing in Chinese medicine new formulation procedures of traditional exploitation for certain anaerobic bacteria
Traditional Chinese medicinal components are added after inoculation of medium anaerobic bacteria, are cultivated in special anaerobic culture box.Sampled again after a period of time into
Row line culture, calculates clump count, so that it is determined that whether certain Chinese medicine compositing formula has bacteriostasis.Line culture is also required to detesting
Cultivated in oxygen incubator, and cultivation cycle at least takes several days, therefore whole cycle is long, efficiency is low.This method is asked for above-mentioned
Inscribe a kind of rapid screening method of Chinese medicine preparation of invention.
The general principle of this method:Monoclonal antibody or antigen molecule are by Covalent bonding together, and this combination will not change
Monoclonal antibody, the immunological characteristic of antigen and bioactivity, specific monoclonal antibody only can be with specific antigen knots
Close.And quaternary Fe-Nb-Cu-B materials have ferromagnetism, its element of Fe outer layer has multiple unpaired electrons, with high saturation magnetic
Change intensity, particle diameter it is small to a certain extent when, it may appear that paramagnetic and superparamagnetic characteristic, Fe-Nb-Cu-B nano materials can be to surrounding
The influence of nuclear magnetic resonance spin-Lattice Relaxation Time of hydrone is very big, the effect with amplification nuclear magnetic resonance relaxation signals.
The NMR relaxation time that very micro Fe-Nb-Cu-B nano materials will result in water declines to a great extent.Therefore it can pass through
Fe-Nb-Cu-B nano-probes are built, detect whether there is object bacteria from the angle of magnetic resonance, so that indirect assessment Chinese medicine
The bacteriostasis of composition.
Key step:
1)Prepared by Fe-Nb-Cu-B nano-probes.Commercially paramagnetic Fe-Nb-Cu-B nano materials, can also pass through
Other method prepares nano level Fe-Nb-Cu-B.Using silane coupler, its formula is:Y(CH2)nSiX3.Herein, n is 0-
3;X is hydrolyzable group;Y is organo-functional group.X is typically chloro, methoxyl group, ethyoxyl, acetoxyl group etc., these groups
Silanol (Si (OH) is generated during hydrolysis3), and combined with inorganic substances, form siloxanes.Y be vinyl, amino, epoxy radicals,
Methacryloxy, sulfydryl.These reactive groups can react and combine with organic substance.Therefore, by using silane coupled
Agent, can erect " molecular bridge " between inorganic substances and the interface of organic substance, and the material of two kinds of property great disparities is connected to one
Play a part of to improve the performance of composite and increase adhesive strength.Surface modification can be obtained by silane coupler reaction
The Fe-Nb-Cu-B nano materials of amination or carboxylated.Further by coupled antibody, surface-functionalized, formation spy can be achieved
Specific immunological probe, closes unnecessary avtive spot, can separate excessive antibody after centrifugation, you can to obtain resistance
The Fe-Nb-Cu-B nano-probes of modification.
2)The sample cell made using polystyrene material carries out Chinese medicine screening.Polystyrene material and ELISA Plate class
Seemingly, detecting probe surface antibody can be combined on the surface of polystyrene material due to suction-operated, still, and the antibody of detecting probe surface is inhaled
The speed of polystyrene material surface is attached to compared with the speed of detecting probe surface antibody binding object bacteria, the former is slow-footed more,
Both are competitive relations.Therefore, the culture medium of the anaerobic bacteria of sterilizing, vaccination target anaerobic bacteria are added in polystyrene sample pipe
The Chinese medicine extract for needing to screen is added afterwards, after closing anaerobic culture for a period of time, adds step 1)Prepared Fe-Nb-Cu-
B nano-probes, are sufficiently mixed concussion reaction for a period of time, if there is target bacteria growing in sample cell, Fe-Nb-Cu-B nanometers of spies
The antibody of wire surface can be combined with object bacteria first, so that Fe-Nb-Cu-B nano-probes are stayed in a liquid.If sample cell
In there is no object bacteria, then the antibody on Fe-Nb-Cu-B nano-probes surface can be adsorbed gradually in polystyrene sample pipe surface, from
And make there is no Fe-Nb-Cu-B nano-probes in liquid.Preceding to have addressed, micro Fe-Nb-Cu-B nano materials will result in water
The NMR relaxation time decline to a great extent.Using the sterile medium liquid without vaccination target bacterium as blank, what is measured is outstanding
The relaxation time of turbid liquid compared to significantly reducing, then illustrates to contain probe in liquid, so that have object bacteria in indirect proof sample,
The amount of probe and the drop-out value in relaxation time are proportional, and the amount of object bacteria can be gone out with indirect quantification by the quantitative probe of mark-on.Have
Object bacteria exist, then illustrate Traditional Chinese medicine bacteriostatic effect it is unobvious, it is on the contrary then illustrate have bacteriostasis.
The major advantage of this method is exactly quick, sensitivity height.Object bacteria can be realized and continuously existed to a certain extent
Line is monitored.Time of even several days 2-3 days relative to anaerobic bacteria culture, magnetic resonance detection only needs a few minutes.The method is not required to
Anaerobic culture box is wanted, can cultivate the sample seal of tube after need to being only inoculated with anaerobic, will be quantitative with syringe during detection
Probe injection can carry out follow-up magnetic resonance detection.Therefore, the screening of extensive Chinese medicine and formula can be made of this method.
The content of the invention
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe-Nb-Cu-B nano-probes.This method is a kind of objective
The method of object bacteria, can do the quick screening of extensive Chinese medicine and formula, so as to a certain degree in effective detection sample
On significantly reduction be directed to certain anaerobic bacteria effective tcm development time.
A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe-Nb-Cu-B nano-probes.Utilize NMR pair
The response sensibility of paramagnetic meterial, proposes NMR relaxation Parameters variation and Fe-Nb-Cu-B nano-particle paramagnetic immunological probes
The correlation metric of content.This method is used for the detection of object bacteria in sample dependent on paramagnetic nano Fe-Nb-Cu-B nano-probes
To assess the fungistatic effect of Chinese medicine preparation.Using the paramagnetic Fe-Nb-Cu-B nano-probes for being coupled monoclonal antibody specific,
Specific anaerobic bacteria in sample can be detected.Because NMR is very quick to Fe-Nb-Cu-B nano-particles
Sense, there is micro paramagnetic Fe-Nb-Cu-B nano-particles, then the spin-lattice relaxation time of water and/spin-spin relaxation just
It can be remarkably decreased.The immunological probe content in sample is quantitatively detected by blank control and mark-on, so as to indirect quantification
The anaerobic bacteria clump count gone out in sample.The anaerobism bacterial content detected and probe content linear correlation, degree of fitting are preferable.It is final with
Corresponding relation between paramagnetic Fe-Nb-Cu-B nano-probes and anaerobic bacteria is tie, determines the anaerobic bacteria clump count in sample.From
And determine whether the bacteriostasis of Chinese medicine is effective indirectly.Time of even several days 2-3 days relative to anaerobic bacteria culture, nuclear magnetic resonance
Detection only needs a few minutes.Therefore, the screening of extensive Chinese medicine and formula can be made of this method.
The present invention is achieved in that step is as follows:
1)Target bacteria antibody is coated with the preparation of paramagnetic nano Fe-Nb-Cu-B nano-probes.
2)The culture medium of the anaerobic bacteria of sterilizing, vaccination target anaerobic bacteria are added in polystyrene sample pipe.Adding needs
The Chinese medicine extract of screening, after culture a period of time, after culture a period of time, adds step 1)Prepared Fe-Nb-Cu-B receives
Rice probe, is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, first can be with Fe-Nb-Cu-B nanometers of spies
The antibody binding of wire surface, so that Fe-Nb-Cu-B nano-probes are stayed in a liquid.If not there is object bacteria in sample cell,
Then the antibody on Fe-Nb-Cu-B nano-probes surface can gradually be adsorbed and separated in polystyrene sample pipe surface with liquid.This
When, the measure of NMR relaxation time is carried out to sample cell.Using the sterile medium liquid without vaccination target bacterium as sky
In vain, the relaxation time of the suspension measured, then explanation contained probe, so as to have mesh in indirect proof sample compared to significantly reducing
Bacterium is marked, the amount of probe and the decline in relaxation time are proportional, indirect quantification goes out the amount of object bacteria by mark-on quantitative probe.
The Fe-Nb-Cu-B nano-probes are the nanoscale Fe-Nb-Cu-B materials with paramagnetic properties, and nanometer particle size is small
In 300 nanometers.
The change in final relaxation time of the detection evaluation method based on nuclear magnetic resonance technique of described object bacteria, during relaxation
Between include spin-lattice relaxation time and spin spin relaxation time.
Beneficial effects of the present invention:The present invention, which provides a kind of objectively quick screening, the Chinese medicine of inhibitory action to anaerobic bacteria
The method of formula, it is characterized in that using the corresponding relation between paramagnetic Fe-Nb-Cu-B nano-probes and anaerobic bacteria as tie, determining sample
Anaerobic bacteria clump count in product.So as to determine whether the bacteriostasis of Chinese medicine is effective indirectly.Relative to anaerobic bacteria culture 2-3 days very
To the time of several days, magnetic resonance detection only needed a few minutes.Therefore, the screening of extensive Chinese medicine and formula can be made of this method.
Embodiment
Example 1
Screening is to Propiobacterium(Anaerobic bacteria)There is the Chinese medicine preparation of suppression or therapeutic action.
1)It is prepared by nanometer Fe-Nb-Cu-B immunological probes:
Fe-Nb-Cu-B nano-particles can commercially, can also bibliography with either physically or chemically synthesizing.
Silicon dioxide coated nanometer Fe-Nb-Cu-B:47.5g sodium metasilicate is taken, is dissolved in deionized water in beaker, salt is used
Acid for adjusting pH value is 12-13.5.0g nanometer Fes-Nb-Cu-B is taken to be added in this beaker, mechanical agitation (using glass bar) 5min.
By mixed liquor ultrasound 30min, stirring in good time.85 C are warming up to, salt acid for adjusting pH value 6-7, generation precipitation is added dropwise.Side magnetic point
Precipitation is washed with deionized from side, washs 3-4 times.Then, precipitation is scattered in 250mL methanol.Above procedure repeats three
It is secondary, it is ensured that silicon is attached on Fe-Nb-Cu-B.
Amino containing silane Fe-Nb-Cu-B nano materials:Obtained silicon dioxide coated nanometer Fe-Nb-Cu-B is added
Into 25mL methanol, 1mLH is used2O and methanol dilution are to 150mL.Then the mixing of 150mL glycerine is added.Ultrasonic 30min, is transferred to
Have in the 500mL three-necked flasks of agitating device.Add 10mL amino silicane coupling agents(AEAPS), quickly stirred under 80-90 C
Mix after 3h, be transferred out of product.Product is washed with deionized 3 times, and methanol washs 2 times (using Buchner funnel suction filtration).Vacuum drying.
It should be noted that suction filtration is due to there is glycerine, so slow, supernatant liquid can be sucked with suction pipe in good time after a period of time by taking out,
Suction filtration process about needs 6-8h.Obtained Aminosilylation Fe-Nb-Cu-B nano materials are finally dried in vacuo 12h.
Antibody modification:Weigh a certain amount of amido modified Fe-Nb-Cu-B nano-particles, add 2 times of quality EDC and
NHS, adds 0.01M each 1mL of PBS.It is placed on blending instrument, 15 turns of rotating speed, reacts 45min.)By soak time to sample enter
Row 10000r/min is centrifuged, time 5min, and the PBS for then adding 0.01M carries out washing centrifugation, is repeated 3 times.By what is centrifuged
Sample respectively adds excessive Propiobacterium antibody, and 1h is coupled on blending instrument.The 10%BSA confining liquids added after the completion of coupling
45min.After closing terminates, rotating speed 8000r/min centrifuges 5min, is centrifuged, is repeated 3 times with 0.01M PBS, then will
Unnecessary antibody, BSA are washed away, and are eventually adding PBS and are resuspended.Preparation resistance modification Fe-Nb-Cu-B nano-particles be
Immunological probe, be stored in 4 DEG C it is stand-by.
2)The culture medium sterilized in right amount is each added in a collection of polystyrene sample pipe, acne bar is inoculated with oxygen free condition
Bacterium, is separately added into after the Chinese medicine extract sealing for needing to screen, after culture a period of time, adds step 1)Prepared Fe-Nb-
Cu-B nano-probes, are sufficiently mixed concussion reaction, if there is object bacteria in sample cell, first can be with Fe-Nb-Cu-B nano-probes
The antibody binding on surface, so that Fe-Nb-Cu-B nano-probes are stayed in a liquid;If not there is object bacteria in sample cell,
The antibody on Fe-Nb-Cu-B nano-probes surface can be adsorbed gradually in polystyrene sample pipe surface.After reaction 1 hour, to sample
Pipe carries out the measure of NMR relaxation time(Shanghai knob steps NMR20 NMRs).There is no vaccination target bacterium with sterile
Medium liquid be blank, the relaxation time spin-lattice relaxation time and spin spin relaxation time of the suspension measured
Compared to significantly reducing, then explanation contains probe, illustrates there is object bacteria in sample cell, so that the bacteriostasis of side light Chinese medicine
Substantially, it is not on the contrary then anti-.The amount of probe and the decline of spin-lattice relaxation time and spin spin relaxation time are proportional, lead to
Cross the quantitative probe of mark-on and indirect quantification goes out the amount of object bacteria.Illustrate that the Chinese medicine added does not have inhibitory action with the presence of object bacteria.
It is on the contrary then anti-.
Example 2
Screening is to clostridium(Anaerobic bacteria)There is the Chinese medicine preparation of suppression or therapeutic action.
1)It is prepared by nanometer Fe-Nb-Cu-B immunological probes:
Fe-Nb-Cu-B nano-particles bibliography physics and can also commercially can be chemically synthesized.
Silicon dioxide coated nanometer Fe-Nb-Cu-B:47.5g sodium metasilicate is taken, is dissolved in deionized water in beaker, salt is used
Acid for adjusting pH value is 12-13.5.0g nanometer Fes-Nb-Cu-B is taken to be added in this beaker, mechanical agitation (using glass bar) 5min.
By mixed liquor ultrasound 30min, stirring in good time.85 C are warming up to, salt acid for adjusting pH value 6-7, generation precipitation is added dropwise.Side magnetic point
Precipitation is washed with deionized from side, washs 3-4 times.Then, precipitation is scattered in 250mL methanol.Above procedure repeats three
It is secondary, it is ensured that silicon is attached on Fe-Nb-Cu-B.
Amino containing silane Fe-Nb-Cu-B nano materials:Obtained silicon dioxide coated nanometer Fe-Nb-Cu-B is added
Into 25mL methanol, 1mLH is used2O and methanol dilution are to 150mL.Then the mixing of 150mL glycerine is added.Ultrasonic 30min, is transferred to
Have in the 500mL three-necked flasks of agitating device.Add 10mL amino silicane coupling agents(AEAPS), quickly stirred under 80-90 C
Mix after 3h, be transferred out of product.Product is washed with deionized 3 times, and methanol washs 2 times (using Buchner funnel suction filtration).Vacuum drying.
It should be noted that suction filtration is due to there is glycerine, so slow, supernatant liquid can be sucked with suction pipe in good time after a period of time by taking out,
Suction filtration process about needs 6-8h.Obtained Aminosilylation Fe-Nb-Cu-B nano materials are finally dried in vacuo 12h.
Antibody modification:Weigh a certain amount of amido modified Fe-Nb-Cu-B nano-particles, add 2 times of quality EDC and
NHS, adds 0.01M each 1mL of PBS.It is placed on blending instrument, 15 turns of rotating speed, reacts 45min.)By soak time to sample enter
Row 10000r/min is centrifuged, time 5min, and the PBS for then adding 0.01M carries out washing centrifugation, is repeated 3 times.By what is centrifuged
Sample respectively adds excessive clostridium antibody, and 1h is coupled on blending instrument.The 10%BSA confining liquids added after the completion of coupling
45min.After closing terminates, rotating speed 8000r/min centrifuges 5min, is centrifuged, is repeated 3 times with 0.01M PBS, then will
Unnecessary antibody, BSA are washed away, and are eventually adding PBS and are resuspended.Preparation resistance modification Fe-Nb-Cu-B nano-particles be
Immunological probe, be stored in 4 DEG C it is stand-by.
2)The culture medium sterilized in right amount is each added in a collection of polystyrene sample pipe, fusiform bud is inoculated with oxygen free condition
Spore bacillus, is separately added into after the Chinese medicine extract sealing for needing to screen, after culture a period of time, adds step 1)Prepared
Fe-Nb-Cu-B nano-probes, are sufficiently mixed concussion reaction, if there is object bacteria in sample cell, can be received first with Fe-Nb-Cu-B
The antibody binding of rice detecting probe surface, so that Fe-Nb-Cu-B nano-probes are stayed in a liquid.If not there is mesh in sample cell
Bacterium is marked, then the antibody on Fe-Nb-Cu-B nano-probes surface can be adsorbed gradually in polystyrene sample pipe surface.Reaction 1 hour
Afterwards, the measure of NMR relaxation time is carried out to sample cell(Shanghai knob steps NMR20 NMRs).Do not connect with sterile
The medium liquid for planting object bacteria is blank, the spin-lattice relaxation time and spin spin relaxation time of the suspension measured
Compared to significantly reducing, then explanation contains probe, illustrates there is object bacteria in sample cell, so that the bacteriostasis of side light Chinese medicine
Substantially, it is not on the contrary then anti-.The amount of probe and the decline of spin-lattice relaxation time and spin spin relaxation time are proportional, lead to
Cross the quantitative probe of mark-on and indirect quantification goes out the amount of object bacteria.Illustrate that the Chinese medicine added does not have inhibitory action with the presence of object bacteria.
It is on the contrary then anti-.
Claims (3)
1. a kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe-Nb-Cu-B nano-probes, its characterization step is as follows:
1)Target bacteria antibody is coated with the preparation of paramagnetic Fe-Nb-Cu-B nano-probes;
2)The culture medium of the anaerobic bacteria of sterilizing is added in polystyrene sample pipe, vaccination target anaerobic bacteria, addition needs screening
Chinese medicine preparation extract solution, after closed culture a period of time, step 1 is quantitatively adding with syringe)Prepared Fe-Nb-Cu-B
Nano-probe, is sufficiently mixed concussion reaction for a period of time, if there is object bacteria in sample cell, Fe-Nb-Cu-B nano-probes surface
Antibody can be combined first with object bacteria so that Fe-Nb-Cu-B nano-probes are stayed in a liquid;If not having in sample cell
Object bacteria, then the antibody on Fe-Nb-Cu-B nano-probes surface can gradually adsorb and leave culture in polystyrene sample pipe surface
Liquid;Now, the measure of NMR relaxation time is carried out to sample cell;With the sterile medium liquid without vaccination target bacterium
For blank, the relaxation time of the suspension measured, then explanation contained probe, so that in indirect proof sample compared to significantly reducing
There is object bacteria, the amount of probe and the decline in relaxation time are proportional, indirect quantification goes out object bacteria by mark-on quantitative probe
Amount;There is no object bacteria to illustrate that the traditional Chinese medicine ingredients added have a fungistatic effect in sample cell, it is on the contrary then anti-.
2. the method according to claim 1 that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe-Nb-Cu-B nano-probes, its
It is characterized in that the nanometer Fe-Nb-Cu-B nano-probes are the nanometer Fe-Nb-Cu-B materials with paramagnetic properties, nanometer particle size is small
In 300 nanometers.
3. the method according to claim 1 that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe-Nb-Cu-B nano-probes, its
It is characterized in the change in final relaxation time of the detection evaluation method based on nuclear magnetic resonance technique of described object bacteria.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110726841A (en) * | 2019-10-21 | 2020-01-24 | 华中农业大学 | Method for detecting veterinary drug residues based on enzymatic click reaction signal amplification magnetic relaxation time immunosensor |
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2017
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Non-Patent Citations (2)
Title |
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申静等: "基于NMR纳米探针的生物传感器用于快速检测沙门氏菌", 《食品工业科技》 * |
顾立刚: "《医学免疫学与微生物学》", 31 August 2004, 中国中医药出版社 * |
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CN110726841A (en) * | 2019-10-21 | 2020-01-24 | 华中农业大学 | Method for detecting veterinary drug residues based on enzymatic click reaction signal amplification magnetic relaxation time immunosensor |
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