CN103217449A - NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Ni alloy probe indirect enrichment - Google Patents
NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Ni alloy probe indirect enrichment Download PDFInfo
- Publication number
- CN103217449A CN103217449A CN2013100976353A CN201310097635A CN103217449A CN 103217449 A CN103217449 A CN 103217449A CN 2013100976353 A CN2013100976353 A CN 2013100976353A CN 201310097635 A CN201310097635 A CN 201310097635A CN 103217449 A CN103217449 A CN 103217449A
- Authority
- CN
- China
- Prior art keywords
- probe
- alloy
- antibody
- nano
- paramagnetic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention relates to a NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Ni alloy probe indirect enrichment. The invention belongs to the technical field of pathogen rapid detection in food safety. The method is dependent on an established nuclear magnetic resonance (NMR) detection method which can be used in detecting pathogens in food liquid samples. Target bacteria are captured by using a first antibody; the target bacteria are enriched and separated by using paramagnetic nano-Fe-Ni alloy probes coated by the antibody of the first antibody, which is a second antibody; and with the influence of the nano-Fe-Ni alloy paramagnetic properties upon the relaxation time of the NMR attenuated signals, the existence of the target pathogen in the sample is detected. Specific correspondences comprise: the paramagnetic nano-Fe-Ni alloy probes show a linear relationship under a certain condition, wherein the larger the nano-Fe-Ni alloy content is, the smaller the sample T2/T1 value is. Therefore, target bacteria can be quantitatively detected within a certain range. The method can be used in rapid detection of harmful pathogens in food samples, and can be used in rapid screening of large batches of samples to be detected.
Description
Technical field
The present invention relates to the fast detecting side of a kind of pathogenic bacteria, relate in particular to a kind of NMR food-borne pathogens method for quick based on the indirect enrichment of paramagnetic nano Fe-Ni alloy probe.
Background technology
Ultimate principle: monoclonal antibody or antigen molecule and enzyme molecule are by covalent bonds, and this combination can not change the immunological characteristic and the biologically active of monoclonal antibody, antigen and enzyme, and specific monoclonal antibody only can combine with specific antigen.The Fe-Ni alloy material has ferromagnetism, when particle diameter little of to a certain degree the paramagnetic characteristic occurring.Do not have magnetic when promptly not having externally-applied magnetic field, and when externally-applied magnetic field is arranged, show certain magnetic, can be used for magnetic and separate.Simultaneously, paramagnet is very remarkable to the influence of NMR signal, and the paramagnet of trace will make NMR signal show variation.Therefore can make up paramagnetic specificity Fe-Ni alloy nano probe biology sensor, do detection from the angle of magnetic resonance.
The principle steps that it is main: 1. add the 1st antibody that detects object bacteria in sample, if having object bacteria in the sample, then the combination by antibody antigen forms 1 anti-compound, and resisted the signal amplification this moment 1.2. buy paramagnetic nano Fe-Ni alloy material from the market, also can prepare nano level Fe-Ni alloy by additive method.Use silane coupling agent, its general formula is: Y (CH
2) nSiX
3Herein, n is 0-3; X is hydrolyzable group; Y is an organo-functional group.X is chloro, methoxyl, ethoxy, acetoxyl group etc. normally, generates silanol (Si (OH) during these group hydrolysis
3), and combine with dead matter, form siloxane.Y is vinyl, amino, epoxy radicals, methacryloxy, sulfydryl.These reactive groups can combine with the organic substance reaction.Therefore,, can between the interface of dead matter and organic substance, erect " molecule bridge ", the material of two kinds of character great disparities is linked together improve the effect of performance of composites and increase bonding strength by using silane coupling agent.Can realize surface-functionalizedly by modified antibodies, form the specific immunity probe, seal unnecessary avtive spot again.Because nanometer Fe-Ni alloy probe has the paramagnetic characteristic, therefore, can separate the antibody that does not have on the connection by externally-applied magnetic field.What nanometer Fe-Ni alloy material was modified is the antibody of the 1st antibody, and promptly 2 is anti-.3. adopt certain method to be fixed on surface of solid phase carriers object bacteria specificity the 1st antibody, and unnecessary avtive spot sealing is standby.4. go on foot the sample of handling the 1st, add the paramagnetic nano Fe-Ni alloy probe that the 2nd step made, fully mix concussion reaction a period of time, grasp the after-applied externally-applied magnetic field of object bacteria, because nanometer Fe-Ni alloy has the paramagnetic characteristic, the Fe-Ni alloy probe can gather magnetic field on one side, siphons away supernatant and then can isolate probe.If object bacteria is arranged in the sample to be checked, then at first can be when the 1st step and the 1 anti-1 anti-compound that forms, 1 anti-compound can be again with 2 of detecting probe surface resist compound, by externally-applied magnetic field by enrichment, separation.After separating, washing, magnetic adds the paramagnetic nano Fe-Ni alloy probe suspension that a small amount of aseptic deionized water then forms object bacteria.At this moment, the excess probe of catching object bacteria and not catching object bacteria still mixes.5. with the above-mentioned probe that mixes, be added to the surface of solid phase carriers in the 3rd step, the probe that has then grasped object bacteria will combine with surface of solid phase carriers monoclonal antibody generation specificity, form double antibodies sandwich, can will the probe wash-out of combination not take place with aseptic washed with de-ionized water.6. adopt eluant, eluent that the specific nano immunological probe of the combination on the immobilization carrier is washed again, the method for separating with magnetic washes ion, solvent.If this part probe exists, grasped the probe of object bacteria exactly.Because the Fe-Ni alloy has the paramagnetic characteristic, very responsive for the resonance instrument, for other molecules, the Fe-Ni alloy of trace can significantly reduce the spin-lattice relaxation parameter T1 and the spin spin relaxation time T2 of aseptic deionized water, and aseptic deionized water is under certain even field intensity, and T1/T2 fixes.Eluent is placed nuclear magnetic resonance analyser, contrast with aseptic deionized water control group.The explanation that the reduction of T1/T2 value significantly takes place has probe to exist, thereby has pathogenic bacteria to detect in the side light food samples.Probe content and T1/T2 value reduce proportional.By mark-on, can the detection by quantitative object bacteria.The Fe-Ni alloy probe is the means of separation and concentration in this method, and the paramagnetic characteristic that has of Fe-Ni alloy simultaneously can be used as the probe of detection by quantitative again.The major advantage of this method is exactly quick, highly sensitive.With respect to microbe culture 2-3 days even several days the time of pathogenic bacteria.The method depends primarily on the The pretreatment time, and magnetic resonance detection only needs a few minutes.All food standards, pathogenic bacteria all must not detect.Therefore, can do the positive-selecting of extensive sample to be checked with this method, to a certain extent can detection by quantitative.At present, also there is not this method of bibliographical information both at home and abroad.
Summary of the invention
A kind of NMR food-borne pathogens method for quick based on the indirect enrichment of paramagnetic nano Fe-Ni alloy probe is used for various food samples is estimated.This method is a kind of objective method that effectively detects harmful pathogenic bacteria in the food, thereby has reduced the screening time of the harmful pathogenic bacteria of food samples to a certain extent greatly.
A kind of NMR food-borne pathogens method for quick based on the indirect enrichment of paramagnetic nano Fe-Ni alloy probe, utilize the response susceptibility of nuclear magnetic resonance analyser, propose the correlativity index of variation of NMR (Nuclear Magnetic Resonance) relaxation parameter and Fe-Ni alloy nano particle paramagnetic immunological probe content paramagnetic material.Different pathogenic bacteria detect the lower limit difference.
This method depends on harmful pathogenic bacteria specificity paramagnetic nano Fe-Ni alloy probe enrichment, the separation in the food samples of can be used for of foundation, and the angle from the NMR (Nuclear Magnetic Resonance) relaxation signal parameter changes detects the harmful pathogenic bacteria in the sample.The paramagnetic nano Fe-Ni alloy probe of monoclonal antibody specific that adopted coupling can carry out enrichment with the specificity pathogenic bacteria in the sample.Because nuclear magnetic resonance analyser SPIN-LATTICE RELAXATION efficient and spin-spin relaxation efficient are very responsive to the Fe-Ni alloy nano particle, promptly in deionized water, the paramagnetic Fe-Ni alloy nano particle that has trace, then the spin-lattice relaxation time of water (T1) and/spin-spin relaxation (T2) will significantly descend.Under certain condition, the paramagnetic characteristic of paramagnetic immunological probe reduces the relaxation decay signal T2/T1 generation linearity of nuclear magnetic resonance.Go out immunological probe content in the sample by detection by quantitative, thereby can quantitatively go out the harmful pathogenic bacteria content in the food samples indirectly.Detected pathogenic bacteria content and probe content linear dependence, degree of fitting is better.Final is tie with the corresponding relation between paramagnetic immunological probe and pathogenic bacteria, determines the pathogenic bacteria clump count in the food samples.And all food standards, pathogenic bacteria all must not detect.Therefore, this method can be done the positive-selecting of extensive sample to be checked, to a certain extent can detection by quantitative.
The present invention is achieved in that step is as follows:
1) add the 1st antibody that detects object bacteria in sample, if having object bacteria in the sample, then the combination by antibody antigen forms 1 anti-compound.
2) antibody of the 1st antibody of detection object bacteria, promptly 2 anti-bags are by the preparation of paramagnetic nano Fe-Ni alloy probe;
3) with object bacteria specificity the 1st antibody fixing standby on solid phase carrier.
4) enrichment aimed strain, and separate: the sample of handling in the 1st step, add the paramagnetic nano Fe-Ni alloy probe that the 2nd step made, fully mix concussion reaction a period of time, grasp the after-applied externally-applied magnetic field of object bacteria, because the paramagnetic characteristic of nanometer Fe-Ni alloy, then the Fe-Ni alloy probe just is pooled to magnetic field on one side, siphons away supernatant and then can isolate probe.If object bacteria is arranged in the sample to be checked, then at first can be when the 1st step and the 1 anti-1 anti-compound that forms, 1 anti-compound can be again with 2 of detecting probe surface resist compound, by externally-applied magnetic field by enrichment, separation.After separating, washing, magnetic adds the paramagnetic nano Fe-Ni alloy probe suspension that a small amount of aseptic deionized water then forms object bacteria.At this moment, the excess probe of catching object bacteria and not catching object bacteria still mixes.
5) the probe suspension with enrichment is added on the solid phase carrier of having fixed monoclonal antibody of the 3rd step making, if exist object bacteria then to form double antibodies sandwich; With aseptic washed with de-ionized water, the probe that does not then grab object bacteria is just washed off, if there is not object bacteria, then all probes are all washed off.
6) afterwards, washing with the probe of eluant, eluent with the double antibodies sandwich on the fixed head, fall ion and solvent with the method for externally-applied magnetic field separate probe with aseptic washed with de-ionized water, is exactly the probe of catching object bacteria if also there is probe.This part probe, the suspension that adds aseptic deionized water formation probe, carrying out the relaxation time of nuclear magnetic resonance measures, with aseptic deionized water is blank, the relaxation time T1/T2 of the suspension that records compares aseptic deionized water remarkable reduction, and then explanation contains probe, thereby in the indirect proof sample target pathogenic bacteria is arranged, the amount of probe and the decline of T1/T2 are proportional, can quantitatively go out the amount of object bacteria by quantitative probe to a certain extent indirectly.
Described NMR probe is the nanoscale Fe-Ni alloy material with paramagnetic characteristic, and nanometer particle size is less than 1000 nanometers.
Described object bacteria finally detect the variation of evaluation method based on the relaxation time characterisitic parameter of nuclear magnetic resonance technique.
Described relaxation time characteristic is meant spin-lattice relaxation time (T1) and spin spin relaxation time (T2).
Described relaxation time characteristic, T1 adopts 180 °-τ-90 ° of pulse method to measure, and T2 adopts cpmg sequence row method to measure.
Described 1 is anti-for detecting the specific antibody of object bacteria, preferably monoclonal antibody.2 anti-be the antibody of the 1st antibody.
Beneficial effect of the present invention: the invention provides a kind of objective fast detecting and go out the method for the harmful pathogenic bacteria in the food, it is characterized in that depending on the magnetic resonance detection method that can be used for detecting the indirect enrichment of paramagnetic nano Fe-Ni alloy probe of foundation.This method can objectively detect harmful pathogenic bacteria in the food effectively, confirms that than the biological culture of pathogenic bacteria this method has the advantage of fast detecting, can be used for the rapid screening of extensive sample.
Embodiment
Example 1
Measure it in the check food samples and whether contain harmful pathogenic bacteria---Listeria monocytogenes.
1. nanometer Fe-Ni alloy immunological probe preparation: the 1st antibody adopts the anti-IgG monoclonal antibody of rabbit of Listeria monocytogenes, and 2 anti-ly are the goat anti-rabbit igg of Listeria monocytogenes.The Fe-Ni alloy nano particle can be bought from the market.Such as Deco island, Beijing gold Science and Technology Co., Ltd. or the super prestige nano material in Shanghai company, 20nm, ratio 1:1, purity 99.5% separately.
Silicon dioxide coated nanometer Fe-Ni alloy: getting the 47.5g sodium silicate, be dissolved in the beaker with deionized water, is 12-13 with the salt acid for adjusting pH value.Get 5.0g nanometer Fe-Ni alloy and join in this beaker, mechanical raking (using glass bar) 5min.With the ultrasonic 30min of mixed liquor, stir in good time.Be warmed up to 85 ℃, dropwise add salt acid for adjusting pH value 6-7, generate precipitation.Limit magnetic separates the limit and precipitates with deionized water wash, washs 3-4 time.Then, precipitation is scattered in the 250mL methyl alcohol.Above process triplicate guarantees that silicon is attached on the Fe-Ni alloy.
Amino containing silane Fe-Ni alloy nano-material: the silicon dioxide coated nanometer Fe-Ni alloy that will make joins in the 25mL methyl alcohol, uses 1mLH
2O and methyl alcohol are diluted to 150mL.Adding 150mL glycerine then mixes.Ultrasonic 30min transfers in the 500mL there-necked flask of stirring apparatus.Add 10mL amino silicane coupling agent (AEAPS), behind 80-90 ℃ of quick down stirring 3h, migrate out product.Product deionized water wash 3 times, methanol wash 2 times (using the Buchner funnel suction filtration).Vacuum drying.It should be noted that suction filtration because glycerine is arranged, thus slow, to take out can inhale with suction pipe after a period of time in good time and remove supernatant liquid, the suction filtration process needs 6-8h approximately.At last with the amino containing silane Fe-Ni alloy nano-material vacuum drying 12h that obtains.
Antibody modification: get a certain amount of amination Fe-Ni alloy nano particle, add the goat anti-rabbit igg antibody of excessive Listeria monocytogenes, 26 ℃ hatch, wash after, add excessive 1% bovine serum albumin (BSA), 22 ℃, the active room of confining surface, washing, resuspension.Because of nanometer Fe-Ni alloy has the paramagnetic characteristic, externally-applied magnetic field separates, and nanometer Fe-Ni alloy will be pooled to magnetic field on one side, siphons away supernatant, and washing is then with unnecessary antibody, BSA flush away.The preparation the paramagnetic nano immunological probe be kept at 4 ℃ stand-by.
2. monoclonal antibody is fixed: can adopt conventional ELISA Plate fixing means, also can adopt following method.With clean cover glass 5 * 5mm
2Square, coating machine spray one deck Cr (2 –, 4 nm) earlier are fixing golden in order to help.Be used in surface sputtering again and spray one deck nm of gold, adopt 200 microlitres, 2 mmol disulfide group-succinimide-propionic esters (DSP) that nm of gold is modified (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) again.Add the Listeria monocytogenes first antibody, the anti-IgG monoclonal antibody of rabbit is about to 100
L100
The g/mL monoclonal antibody be fixed on the glass plate by e and 37 ℃ hatch 45 min.Add 1% bovine serum albumin (BSA), 22 ℃, 1 hour, remaining avtive spot on the plate is sealed and drying.
3. food samples is carried out pre-service, adopt the FDA enrichment in case of necessity, sample is filtered, increases pre-service such as bacterium activation, obtain sample to be checked.The 1st antibody, the anti-IgG of the rabbit of Listeria monocytogenes will be added in the sample to be checked.If have Listeria monocytogenes in the sample to be checked, will with the 1 anti-1 anti-compound that forms.Fully shake after the goat anti-rabbit igg probe of the 1st the 2nd antibody Listeria monocytogenes that makes of step added.Last magnetic force frame separate probe adds the suspension that a small amount of aseptic deionized water obtains probe.At this moment,, then pass through the interaction of the 2nd antibody and the 1st antibody, thereby catch this compound, reached the purpose of object bacteria enrichment if the target Listeria monocytogenes is arranged in the sample to be checked.At this moment, combine 1 anti-compound of object bacteria and do not have excessive 1 of combining target bacterium to resist and all can close, still mix with 2 resistive connections of detecting probe surface.The probe suspension of this enrichment is added on the prepared 1 anti-fixed head of the 2nd step, then combines the probe of Listeria monocytogenes in the probe suspension and can be further combine, form the double antibodies sandwich structure with monoclonal antibody generation specificity on the fixed head.This moment is with aseptic washed with de-ionized water, just can be with less than the probe flush away in conjunction with Listeria monocytogenes.The probe that just only combines Listeria monocytogenes that on fixed head, is left.
4. with eluent (methyl alcohol etc.) probe that combines Listeria monocytogenes on the fixed head is eluted.Last magnetic force frame, separate probe is also cleaned 1-2 time, with the ion flush away.The solution that obtains is with the T1 and the T2 of nuclear magnetic resonance analyser (NMR20, Niu Mai company) mensuration solution.With aseptic deionized water is blank, and the T1/T2 that solution records compares with blank, and there were significant differences, and illustrating has probe to exist in the solution, thereby in the explanation sample Listeria monocytogenes is arranged.The amount of probe and the drop-out value of T1/T2 are proportional.The bright probe of speaking more more that descends is many more, thereby the side light Listeria monocytogenes is many more, by the mark-on checking can the detection by quantitative sample in the number of object bacteria.All food standards all must not detect Listeria monocytogenes, and the method can fast detecting go out whether contain Listeria monocytogenes in the sample.
Embodiment
Example 2
Measure food samples and whether contain harmful pathogenic bacteria---Escherichia coli O 157: H7.
1. nanometer Fe-Ni alloy immunological probe preparation: the 1 anti-anti-IgG monoclonal antibody of rabbit that adopts O157:H7,2 anti-ly are the goat anti-rabbit igg of O157:H7, can be that how anti-monoclonal antibody also can be.The Fe-Ni alloy nano particle can be bought from the market.Such as Deco island, Beijing gold Science and Technology Co., Ltd. or the super prestige nano material in Shanghai company, 20nm, ratio 1:1, purity 99.5% separately.
Silicon dioxide coated nanometer Fe-Ni alloy: getting the 47.5g sodium silicate, be dissolved in the beaker with deionized water, is 12-13 with the salt acid for adjusting pH value.Get 5.0g nanometer Fe-Ni alloy and join in this beaker, mechanical raking (using glass bar) 5min.With the ultrasonic 30min of mixed liquor, stir in good time.Be warmed up to 85 ℃, dropwise add salt acid for adjusting pH value 6-7, generate precipitation.Limit magnetic separates the limit and precipitates with deionized water wash, washs 3-4 time.Then, precipitation is scattered in the 250mL methyl alcohol.Above process triplicate guarantees that silicon is attached on the Fe-Ni alloy.
Amino containing silane Fe-Ni alloy nano-material: the silicon dioxide coated nanometer Fe-Ni alloy that will make joins in the 25mL methyl alcohol, uses 1mLH
2O and methyl alcohol are diluted to 150mL.Adding 150mL glycerine then mixes.Ultrasonic 30min transfers in the 500mL there-necked flask of stirring apparatus.Add 10mL amino silicane coupling agent (AEAPS), behind 80-90 ℃ of quick down stirring 3h, migrate out product.Product deionized water wash 3 times, methanol wash 2 times (using the Buchner funnel suction filtration).Vacuum drying.It should be noted that suction filtration because glycerine is arranged, thus slow, to take out can inhale with suction pipe after a period of time in good time and remove supernatant liquid, the suction filtration process needs 6-8h approximately.At last with the amino containing silane Fe-Ni alloy nano-material vacuum drying 12h that obtains.
Antibody modification: get a certain amount of amination Fe-Ni alloy nano particle, add excessive 2 anti-, i.e. the goat anti-rabbit igg antibody of Escherichia coli O 157: H7,26 ℃ hatch, wash after, add excessive 1% bovine serum albumin (BSA), 22 ℃, the active room of confining surface, washing, resuspension.Because of nanometer Fe-Ni alloy has the paramagnetic characteristic, externally-applied magnetic field separates, and nanometer Fe-Ni alloy will be pooled to magnetic field on one side, siphons away supernatant, and washing is then with unnecessary antibody, BSA flush away.The preparation the paramagnetic nano immunological probe be kept at 4 ℃ stand-by.
2. monoclonal antibody is fixed: can adopt conventional ELISA Plate fixing means, also can adopt following method.With clean cover glass 5 * 5mm
2Square, coating machine spray one deck Cr (2 –, 4 nm) earlier are fixing golden in order to help.Be used in surface sputtering again and spray one deck nm of gold, adopt 200 microlitres, 2 mmol disulfide group-succinimide-propionic esters (DSP) that nm of gold is modified (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) again.Add the 1st antibody, promptly the anti-IgG monoclonal antibody of rabbit is about to 100
L100
The g/mL monoclonal antibody be fixed on the glass plate by e and 37 ℃ hatch 45 min.Adding bovine serum albumin seals remaining avtive spot on the plate and drying.
3. food samples is carried out pre-service, adopt the FDA enrichment in case of necessity, sample is filtered, increases pre-service such as bacterium activation, obtain sample to be checked.The 1st antibody, the anti-IgG of the rabbit of O157:H7 will be added in the sample to be checked.If have O157:H7 in the sample to be checked, will with the 1 anti-1 anti-compound that forms.With the 2nd antibody that the 1st step made, the goat anti-rabbit igg probe of O157:H7 fully shakes after adding.Last magnetic force frame separate probe adds the suspension that a small amount of aseptic deionized water obtains probe.At this moment,, then pass through the interaction of the 2nd antibody and the 1st antibody, thereby catch this compound, reached the purpose of object bacteria enrichment if the target Listeria monocytogenes is arranged in the sample to be checked.At this moment, combine 1 anti-compound of object bacteria and do not have excessive 1 of combining target bacterium to resist and all can close, still mix with 2 resistive connections of detecting probe surface.The probe suspension of this enrichment is added on the prepared 1 anti-fixed head of the 2nd step, then combines the probe of O157:H7 in the probe suspension and can be further combine, form the double antibodies sandwich structure with monoclonal antibody generation specificity on the fixed head.This moment is with aseptic washed with de-ionized water, just can be with less than the probe flush away in conjunction with O157:H7.The probe that just only combines O157:H7 that on fixed head, is left.
With eluent (methyl alcohol etc.) with the Escherichia coli O 157 that combines on the fixed head: the probe of H7 elutes.Last magnetic force frame, separate probe and with aseptic washed with de-ionized water 1-2 time, with ion, solvent flush away.The solution that obtains, with nuclear magnetic resonance analyser (NMR20, Niu Mai company or miFe-Ni alloy NMR East China Normal University) measure the T1 and the T2 of solution, with the deionized water is blank, the T1/T2 that solution records is with relatively blank, and there were significant differences, and illustrating has probe to exist in the solution, thereby Escherichia coli O 157: H7 is arranged in the explanation sample, and the amount of probe and the drop-out value of T1/T2 are proportional.The bright probe of speaking more more that descends is many more, thereby side light Escherichia coli O 157: H7 is many more, by the mark-on checking can the detection by quantitative sample in the number of object bacteria.
Claims (6)
1. NMR food-borne pathogens method for quick based on the indirect enrichment of paramagnetic nano Fe-Ni alloy probe, its characterization step is as follows:
1) add specificity the 1st antibody that detects object bacteria in sample, if having object bacteria in the sample, then the combination by antibody antigen forms 1 anti-compound;
2) antibody of the 1st antibody of detection object bacteria, promptly 2 anti-bags are by the preparation of paramagnetic nano Fe-Ni alloy probe;
3) with object bacteria specificity the 1st antibody fixing standby on solid phase carrier;
4) enrichment aimed strain, and separate: the sample of handling in the 1st step, add the paramagnetic nano Fe-Ni alloy probe that the 2nd step made, fully mix concussion reaction a period of time, grasp the after-applied externally-applied magnetic field of object bacteria, because nanometer Fe-Ni alloy has the paramagnetic characteristic, the Fe-Ni alloy probe can gather magnetic field on one side, siphon away supernatant and then can isolate probe, if in the sample to be checked object bacteria is arranged, then at first can be at the 1st when step and 1 anti-formation 1 anti-compound, 1 anti-compound can resist compound with 2 of detecting probe surface again, by externally-applied magnetic field by enrichment, separate, washing, add the paramagnetic nano Fe-Ni alloy probe suspension that a small amount of aseptic deionized water then forms object bacteria after magnetic separates, at this moment, the Fe-Ni alloy probe of catching and not catching 1 anti-compound still mixes;
5) the Fe-Ni alloy probe suspension of enrichment being added to the 2nd step has fixed on the solid phase carrier of monoclonal antibody, if exist object bacteria then to form double antibodies sandwich, with aseptic washed with de-ionized water, the probe that does not then grab object bacteria is just washed off, if there is not object bacteria, then all probes are all washed off;
6) afterwards, washing with the probe of eluant, eluent with the double antibodies sandwich on the fixed head, fall ion and solvent with the method for externally-applied magnetic field separate probe with aseptic washed with de-ionized water, is exactly the probe of catching object bacteria if also there is probe;
7) this part probe, the suspension that adds aseptic deionized water formation probe, carrying out the relaxation time of nuclear magnetic resonance measures, under fixing field intensity, the T1/T2 of aseptic deionized water is a steady state value, with aseptic deionized water is blank, the relaxation time T1/T2 of the suspension that records compares aseptic deionized water remarkable reduction, then explanation contains probe, thereby the target pathogenic bacteria are arranged in the indirect proof sample, the amount of probe and the decline of T1/T2 are proportional, can quantitatively go out the amount of object bacteria by adding the scalar quantity probe indirectly.
2. the NMR food-borne pathogens method for quick based on the indirect enrichment of paramagnetic nano Fe-Ni alloy probe according to claim 1 is characterized in that described NMR nano-probe is the nanometer Fe-Ni alloy material with paramagnetic characteristic.
3. the NMR food-borne pathogens method for quick based on the indirect enrichment of paramagnetic nano Fe-Ni alloy probe according to claim 1, what it is characterized in that described object bacteria finally detects the variation of evaluation method based on the relaxation time characterisitic parameter of nuclear magnetic resonance technique.
4. the NMR food-borne pathogens method for quick based on the indirect enrichment of paramagnetic nano Fe-Ni alloy probe according to claim 1, it is characterized in that described NMR relaxation time characteristic, be meant spin-lattice relaxation time (T1) and spin spin relaxation time (T2).
5. the NMR food-borne pathogens method for quick based on the indirect enrichment of paramagnetic nano Fe-Ni alloy probe according to claim 1, it is characterized in that described relaxation time characteristic, T1 adopts 180 °-τ-90 ° of pulse method to measure, and T2 adopts cpmg sequence row method to measure.
6. the NMR food-borne pathogens method for quick based on the indirect enrichment of paramagnetic nano Fe-Ni alloy probe according to claim 1 is characterized in that described 1 is anti-for detecting the specific antibody of object bacteria, monoclonal antibody preferably, 2 anti-be the antibody of the 1st antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310097635.3A CN103217449B (en) | 2013-03-26 | 2013-03-26 | A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni alloy probe indirect enrichment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310097635.3A CN103217449B (en) | 2013-03-26 | 2013-03-26 | A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni alloy probe indirect enrichment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103217449A true CN103217449A (en) | 2013-07-24 |
| CN103217449B CN103217449B (en) | 2016-02-24 |
Family
ID=48815431
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310097635.3A Active CN103217449B (en) | 2013-03-26 | 2013-03-26 | A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni alloy probe indirect enrichment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103217449B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885822A (en) * | 2017-03-31 | 2017-06-23 | 南昌大学 | A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Co Pd Al O nano-probes |
| CN106885817A (en) * | 2017-03-11 | 2017-06-23 | 南昌大学 | A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe Ni nano-probes |
| CN106885818A (en) * | 2017-03-22 | 2017-06-23 | 南昌大学 | A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on CoZrO nano-probes |
| CN106885820A (en) * | 2017-03-30 | 2017-06-23 | 南昌大学 | A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Co Cr nano-probes |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1094938A (en) * | 1993-04-23 | 1994-11-16 | 布里斯托尔-迈尔斯斯奎布公司 | The in-vivo measurement method of the detectable biological exogenous compounds concentration of nuclear magnetic resonance, NMR |
| CN101509919A (en) * | 2009-03-12 | 2009-08-19 | 湖南工业大学 | Method for producing water- soluble magnetic nanoparticle for detecting SQUID |
| CN102323408A (en) * | 2011-05-31 | 2012-01-18 | 上海师范大学 | Method for rapid detection of enterobacter sakazakii |
| US20130195767A1 (en) * | 2010-05-26 | 2013-08-01 | Ralph Weissleder | Magnetic Nanoparticles |
-
2013
- 2013-03-26 CN CN201310097635.3A patent/CN103217449B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1094938A (en) * | 1993-04-23 | 1994-11-16 | 布里斯托尔-迈尔斯斯奎布公司 | The in-vivo measurement method of the detectable biological exogenous compounds concentration of nuclear magnetic resonance, NMR |
| CN101509919A (en) * | 2009-03-12 | 2009-08-19 | 湖南工业大学 | Method for producing water- soluble magnetic nanoparticle for detecting SQUID |
| US20130195767A1 (en) * | 2010-05-26 | 2013-08-01 | Ralph Weissleder | Magnetic Nanoparticles |
| CN102323408A (en) * | 2011-05-31 | 2012-01-18 | 上海师范大学 | Method for rapid detection of enterobacter sakazakii |
Non-Patent Citations (1)
| Title |
|---|
| 陈艳: "纳米材料在食源性致病菌检测和生物成像中的应用研究", 《中国优秀硕士学位论文全文数据库信息科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885817A (en) * | 2017-03-11 | 2017-06-23 | 南昌大学 | A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Fe Ni nano-probes |
| CN106885818A (en) * | 2017-03-22 | 2017-06-23 | 南昌大学 | A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on CoZrO nano-probes |
| CN106885820A (en) * | 2017-03-30 | 2017-06-23 | 南昌大学 | A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Co Cr nano-probes |
| CN106885822A (en) * | 2017-03-31 | 2017-06-23 | 南昌大学 | A kind of method that anaerobic bacteria Inhibitors form Chinese Traditional Medicinal Herbs are screened based on Co Pd Al O nano-probes |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103217449B (en) | 2016-02-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103217448B (en) | A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano Fe probe | |
| CN103217449B (en) | A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni alloy probe indirect enrichment | |
| CN103207202B (en) | A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano Fe-Ni alloy/C probe | |
| CN103217451B (en) | A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano nano-Fe-Ni-Co alloy | |
| CN103207273A (en) | Paramagnetic nano Fe-Co alloy probe based quick detecting method for NMR (nuclear magnetic resonance) food-borne pathogenic bacteria | |
| CN103217450B (en) | A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment | |
| CN103217530B (en) | NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Co alloy probe indirect enrichment | |
| CN103185796B (en) | Food-borne pathogenic bacteria quick detection method based on Gamma-Fe2O3@Au nano particle indirect enrichment and immunomagnetic separation | |
| CN103175858B (en) | A kind of based on Fe3O4The NMR food-borne pathogen rapid detection of nano particle indirect enrichment | |
| CN103353461A (en) | Rapid NMR food-borne pathogenic bacteria detection method based on paramagnetic nanometer Ni-Co alloy probe indirect enrichment | |
| CN103207198A (en) | NMR detection method for food-borne pathogenic bacteria based on Fe3O4 nano-material | |
| CN103091347B (en) | A kind of based on γ-Fe 2o 3the NMR food-borne pathogen rapid detection of@Au composite Nano probe | |
| CN103226115B (en) | A kind of NMR food-borne pathogen rapid detection based on indirect enrichment of paramagnetic nano-Ni probe | |
| CN103115934B (en) | A kind of based on Fe 3o 4the NMR food-borne pathogen rapid detection of@Au composite nanoparticle | |
| CN103207203B (en) | A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano Co probe | |
| CN103364428B (en) | A kind of based on γ-Fe2O3The method for quick of NMR nano-probe food-borne pathogens | |
| CN103217447B (en) | A kind of NMR Methods for Fast Detection of Foodborne Pathogenic Bacteria based on paramagnetic nano Ni-Co alloy probe | |
| CN103245686B (en) | A kind of NMR food-borne pathogen rapid detection based on indirect enrichment of paramagnetic nano-Ni probe indirect enrichment | |
| CN103226116B (en) | A kind of NMR food-borne pathogen rapid detection based on indirect enrichment of paramagnetic nano-Co probe | |
| CN103207201B (en) | A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano Fe probe indirect enrichment | |
| CN103134932B (en) | Food-borne pathogenic bacteria rapid detection method based on gamma-Fe203@AU nanometer material immune magnet separation | |
| CN105372277A (en) | CoFe2O4 nanoparticles based NMR method for rapid detection of food-borne pathogenic bacteria | |
| CN103185774A (en) | Food-borne pathogenic bacteria quick detection method based on Fe3O4 nano particle indirect enrichment and immunomagnetic separation | |
| CN103149333B (en) | A kind of based on Fe3O4The Methods for Fast Detection of Foodborne Pathogenic Bacteria that the indirect enrichment immunity of Au nano particle magnetic separates | |
| CN103196937A (en) | Quick NMR (nuclear magnetic resonance) food-borne pathogenic bacteria detection method based on Fe3O4 at Au nano-particle indirect enrichment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201029 Address after: Room a1310, No. 109, Shazhou West Road, yangshe Town, Zhangjiagang City, Suzhou City, Jiangsu Province Patentee after: Suzhou auxiliary Survey Technology Service Co., Ltd Address before: 999 No. 330000 Jiangxi province Nanchang Honggutan University Avenue Patentee before: Nanchang University |
|
| TR01 | Transfer of patent right |