CN103217450B - A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment - Google Patents
A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment Download PDFInfo
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Abstract
Based on a NMR food-borne pathogen rapid detection for paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment, belong to technical field of quick detection of pathogenic bacteria for food safety.The present invention depends on the magnetic resonance detection method that may be used for pathogenic bacteria in food liquid sample of foundation, utilize 1 anti-target acquisition bacterium, the 1 antibody paramagnetic nano nano-Fe-Ni-Co alloy that is 2 anti-bags are produced resisted is utilized to carry out enrichment, separation to object bacteria, utilize the paramagnetic properties of nanometer Fe-Ni-Co alloy on the impact in the relaxation time of nuclear magnetic resonance deamplification, whether detect in sample containing object bacteria.Different concrete corresponding relations is: paramagnetic nano nano-Fe-Ni-Co alloy, demonstrate linear relationship under certain condition, namely nanometer Fe-Ni-Co alloy content is large, spin-lattice relaxation time and the spin spin relaxation time value of sample are less, quantitatively can detect object bacteria in certain limit.The method may be used for the quick detection of harmful pathogenic bacteria in food samples, thus can as the rapid screening of large quantities of measuring samples.
Description
Technical field
The present invention relates to the quick detection side of a kind of pathogenic bacteria, particularly relate to a kind of NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment.
Background technology
Ultimate principle: monoclonal antibody or antigen molecule are combined by covalent bond with enzyme molecule, this combination can not change immunological characteristic and the biologically active of monoclonal antibody, antigen and enzyme, and specific monoclonal antibody only can be combined with specific antigen.Fe-Ni-Co alloy material has ferromagnetism, there will be paramagnetic properties to a certain extent when particle diameter is little.Namely there is no there is no magnetic during externally-applied magnetic field, and show certain magnetic when there being externally-applied magnetic field, may be used for Magneto separate.Meanwhile, paramagnet is very remarkable on the impact of NMR signal, and the paramagnet of trace will make NMR signal show change.Therefore can build paramagnetic specificity Fe-Ni-Co alloy nano probe biology sensor, detect from the angle of magnetic resonance.
Its main principle steps: 1. add detection object bacteria in the sample to which 1 resists, if there is object bacteria in sample, then form 1 anti-compound by the combination of antibody antigen, now 1 has resisted signal amplification.2. commercially paramagnetic nano Fe-Ni-Co alloy material, also can prepare nano level Fe-Ni-Co alloy by additive method.Use silane coupling agent, its general formula is: Y (CH
2) nSiX
3.Herein, n is 0-3; X is hydrolyzable group; Y is organo-functional group.X is chloro, methoxyl, ethoxy, acetoxyl group etc. normally, generates silanol (Si (OH) during the hydrolysis of these groups
3), and be combined with dead matter, form siloxane.Y is vinyl, amino, epoxy radicals, methacryloxy, sulfydryl.These reactive groups can react with organic substance and combine.Therefore, by using silane coupling agent, can erect " molecular bridge " between dead matter and the interface of organic substance, link together the material of two kinds of character great disparities the performance improving compound substance and the effect increasing bonding strength.Can realize surface-functionalized by modified antibodies, form specific immunity probe, then close unnecessary avtive spot.Because nanometer nano-Fe-Ni-Co alloy has paramagnetic properties, therefore, can be separated by externally-applied magnetic field the antibody not having to join.What nanometer Fe-Ni-Co alloy material was modified is 1 anti-antibody, and namely 2 resist.3. method certain for anti-for object bacteria specificity 1 employing is fixed on ELISA Plate surface, and unnecessary avtive spot is closed for subsequent use.4. at the sample of the 1st step process, add the paramagnetic nano nano-Fe-Ni-Co alloy that the 2nd step is obtained, abundant mixing concussion reaction a period of time, capture the after-applied externally-applied magnetic field of object bacteria, because nanometer Fe-Ni-Co alloy has paramagnetic properties, nano-Fe-Ni-Co alloy can gather magnetic field on one side, siphons away supernatant and then can isolate probe.If have object bacteria in measuring samples, then first can when the 1st step and the anti-compound of 1 anti-formation 1,1 anti-compound can again with 2 anti-compounds of detecting probe surface, by externally-applied magnetic field by enrichment, separation.The paramagnetic nano nano-Fe-Ni-Co alloy suspension that deionized water aseptic on a small quantity then forms object bacteria is added after washing, Magneto separate.Now, the excess probe caught object bacteria and do not catch object bacteria still mixes.5. by the above-mentioned probe mixed, be added to the ELISA Plate surface of the 3rd step, then captured object bacteria probe will with ELISA Plate surface monoclonal antibodies generation specific binding, formed double antibodies sandwich, with aseptic washed with de-ionized water can will not occur combine probe wash-out.6. adopt eluant, eluent to be washed by the specific nano immunological probe of the combination on immobilization carrier again, wash ion, solvent by the method for Magneto separate.If this part probe exists, capture the probe of object bacteria exactly.Because Fe-Ni-Co alloy has paramagnetic properties, very responsive for resonance instrument, for other molecules, the Fe-Ni-Co alloy of trace significantly can reduce 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, spin-lattice relaxation time and spin spin relaxation time are fixing.Eluent is placed in nuclear magnetic resonance analyser, contrasts with aseptic deionized water control group.The explanation that remarkable generation spin-lattice relaxation time and spin spin relaxation time value reduce has probe to exist, thus has pathogenic bacteria to detect in side light food samples.Probe content and spin-lattice relaxation time and spin spin relaxation time value reduce proportional.By mark-on, quantitatively object bacteria can be detected.In the method, nano-Fe-Ni-Co alloy is the means of separation and concentration, and simultaneously the paramagnetic properties that has of Fe-Ni-Co alloy, can be used as again the probe quantitatively detected.The major advantage of the method is exactly quick, highly sensitive.2-3 days even time of several days is cultivated relative to the microorganism of pathogenic bacteria.The method depends primarily on the pretreatment time of sample, and magnetic resonance detection only needs a few minutes.All food standards, pathogenic bacteria all must not detect.Therefore, the positive-selecting of extensive measuring samples can be done by the method, can quantitatively detect to a certain extent.At present, this method of bibliographical information is not also had both at home and abroad.
Summary of the invention
Based on a NMR food-borne pathogen rapid detection for paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment, for evaluating various different food samples.The method is a kind of objective method effectively detecting harmful pathogenic bacteria in food, thus greatly reduces the screening time of food samples harmful pathogenic bacteria to a certain extent.
A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment, utilize nuclear magnetic resonance analyser to the response sensibility of paramagnetic material, propose the correlation metric of NMR (Nuclear Magnetic Resonance) relaxation Parameters variation and Fe-Ni-Co alloy nano particle paramagnetic immunological probe content.Different pathogenic bacteria detect lower limit difference.
What the method depended on foundation can be used for the enrichment of harmful pathogenic bacteria specificity paramagnetic nano nano-Fe-Ni-Co alloy, separation in food samples, from the angle of NMR (Nuclear Magnetic Resonance) relaxation signal parameter change, detects the harmful pathogenic bacteria in sample.Adopt the coupling paramagnetic nano nano-Fe-Ni-Co alloy of monoclonal antibody specific, the specific pathogenetic bacterium in sample can be carried out enrichment.Due to nuclear magnetic resonance analyser SPIN-LATTICE RELAXATION efficiency and spin-spin relaxation efficiency very responsive to Fe-Ni-Co alloy nano particle, namely in deionized water, there is the paramagnetic Fe-Ni-Co alloy nano particle of trace, then the spin-lattice relaxation time (T1) of water and/spin-spin relaxation (T2) will significantly decline.Under certain condition, the paramagnetic properties of paramagnetic immunological probe makes the relaxation decay signal spin-lattice relaxation time of nuclear magnetic resonance and spin spin relaxation time produce linear reduction.By quantitatively detecting the immunological probe content in sample, thus detrimental bacterial content in food samples can be gone out by indirect quantification.The pathogenic bacteria content detected and probe content linear correlation, degree of fitting is better.Final with the corresponding relation between paramagnetic immunological probe and pathogenic bacteria for tie, determine the pathogenic bacteria clump count in food samples.And all food standards, pathogenic bacteria all must not detect.Therefore, the method can do the positive-selecting of extensive measuring samples, can quantitatively detect to a certain extent.
The present invention is achieved in that step is as follows:
1) add detection object bacteria in the sample to which 1 resists, if there is object bacteria in sample, then forms 1 anti-compound by the combination of antibody antigen.
2) detect 1 antibody resisted of object bacteria, namely 2 anti-bags are by the preparation of paramagnetic nano nano-Fe-Ni-Co alloy; .
3) what object bacteria specificity 1 resisted in ELISA Plate is fixing for subsequent use.
4) enrich target bacterium, and be separated: the 1st) sample of step process, add the paramagnetic nano nano-Fe-Ni-Co alloy that the 2nd step is obtained, abundant mixing concussion reaction a period of time, capture the after-applied externally-applied magnetic field of object bacteria, due to the paramagnetic properties of nanometer Fe-Ni-Co alloy, then nano-Fe-Ni-Co alloy is just pooled to magnetic field, siphons away supernatant and then can isolate probe.If have object bacteria in measuring samples, then first can the 1st) step time and the anti-compound of 1 anti-formation 1,1 anti-compound can again with 2 anti-compounds of detecting probe surface, by externally-applied magnetic field by enrichment, separation.The paramagnetic nano nano-Fe-Ni-Co alloy suspension that deionized water aseptic on a small quantity then forms object bacteria is added after washing, Magneto separate.Now, the excess probe caught object bacteria and do not catch object bacteria still mixes.
5) the probe suspension of enrichment is added to the 3rd) step make secure in the ELISA Plate of monoclonal antibody, if there is object bacteria, form double antibodies sandwich; By aseptic washed with de-ionized water, then the probe not grabbing object bacteria is just washed off, if there is not object bacteria, then all probes are all washed off.
6) after, wash with the probe of eluant, eluent by the double antibodies sandwich in ELISA Plate, drop off son and solvent by the method for externally-applied magnetic field separate probe by aseptic washed with de-ionized water, if also there is probe is exactly the probe catching object bacteria.This part probe, add the suspension that aseptic deionized water forms probe, the relaxation time of carrying out nuclear magnetic resonance measures, with aseptic deionized water for blank, the relaxation time spin-lattice relaxation time of the suspension recorded compares aseptic deionized water with spin spin relaxation time have remarkable reduction, then illustrate containing probe, thus have object bacteria in indirect proof sample, the decline of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is proportional, can by quantitative probe to a certain extent indirect quantification go out the amount of object bacteria.
Described NMR probe is the nanoscale Fe-Ni-Co alloy material with paramagnetic properties, and nanometer particle size is less than 1000 nanometers.
Described object bacteria finally detect the change of evaluation method based on the relaxation time characterisitic parameter of nuclear magnetic resonance technique.
Described relaxation time characteristic, refers to spin-lattice relaxation time and spin spin relaxation time.
Described 1 resists the monoclonal antibody for detecting object bacteria.2 to resist be 1 anti-antibody.
Beneficial effect of the present invention: the invention provides a kind of method objectively detecting the harmful pathogenic bacteria in food fast, is characterized in that the magnetic resonance detection method that can be used for detecting paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment depending on foundation.The method can objectively detect harmful pathogenic bacteria in food effectively, confirms compared to the biological culture of pathogenic bacteria, and the method has the advantage detected fast, may be used for the rapid screening of extensive sample.
Embodiment
Example 1
Whether it is measured containing harmful pathogenic bacteria---Listeria monocytogenes in inspection food samples.
1. nanometer Fe-Ni-Co alloy immunological probe preparation: the rabbit anti-igg monoclonal antibody of 1 anti-employing Listeria monocytogenes, 2 resist the goat anti-rabbit igg for Listeria monocytogenes.Fe-Ni-Co alloy nano particle can be commercially.Such as Beijing Deco Dao Jin Science and Technology Co., Ltd. or Shanghai Chao Wei nano material company, 20nm, ratio 1:1:1, respective purity 99.5%.
Silicon dioxide coated nanometer Fe-Ni-Co alloy: get 47.5g sodium silicate, be dissolved in beaker with deionized water is 12-13 with salt acid for adjusting pH value.Getting 5.0g nanometer Fe-Ni-Co alloy joins in this beaker, mechanical raking (with glass bar) 5min.By ultrasonic for mixed liquor 30min, stir in good time.Be warmed up to 85 C, dropwise add salt acid for adjusting pH value 6-7, generate precipitation.Magneto separate limit, limit spends deionized water precipitation, washs 3-4 time.Then, precipitation is scattered in 250mL methyl alcohol.Above process in triplicate, ensures that silicon is attached on Fe-Ni-Co alloy.
Amino containing silane Fe-Ni-Co alloy nano-material: obtained silicon dioxide coated nanometer Fe-Ni-Co alloy is joined in 25mL methyl alcohol, uses 1mLH
2o and methanol dilution are to 150mL.Then the mixing of 150mL glycerine is added.Ultrasonic 30min, transfers in the 500mL there-necked flask of stirring apparatus.Add 10mL amino silicane coupling agent (AEAPS), under 80-90 C after rapid stirring 3h, migrate out product.Product spends deionized water 3 times, methanol wash 2 times (using Buchner funnel suction filtration).Vacuum drying.It should be noted that suction filtration is owing to there being glycerine, so slow, can suck supernatant liquid by good time suction pipe after taking out a period of time, suction filtration process about needs 6-8h.The amino containing silane Fe-Ni-Co alloy nano-material vacuum drying 12h finally will obtained.
Antibody modification: after getting a certain amount of amination Fe-Ni-Co alloy nano particle, add the goat anti-rabbit igg antibody of excessive Listeria monocytogenes, 26 C are hatched, washing, add excessive 1% bovine serum albumin (BSA), 22 C, the active room of confining surface, washing, resuspension.Because nanometer Fe-Ni-Co alloy has paramagnetic properties, externally-applied magnetic field is separated, and nanometer Fe-Ni-Co alloy will be pooled to magnetic field, siphons away supernatant, and washing, then wash away unnecessary antibody, BSA.Preparation paramagnetic nano immunological probe be kept at 4 DEG C stand-by.
2. monoclonal antibody is fixed: can adopt conventional ELISA Plate fixing means, also can adopt following methods.With clean cover glass 5 × 5mm
2square, coating machine first sprays one deck Cr (2 – 4nm) in order to help fixing gold.Be used in surface sputtering again and spray one deck nm of gold, then adopt 200 microlitre 2mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) nm of gold.Add Listeria monocytogenes first antibody, rabbit anti-igg monoclonal antibody, fix on a glass also 37 C by 100 μ L100 μ g/mL monoclonal antibodies by e and hatch 45min.Add 1% bovine serum albumin (BSA), 22 C, 1 hour, avtive spot remaining on plate is carried out close and dry.
3. food samples is carried out pre-service, adopt FDA enrichment if desired, sample is filtered, increases the pre-service such as bacterium activation, obtain measuring samples.Resist adding 1 in measuring samples, the rabbit anti-igg of Listeria monocytogenes.If there is Listeria monocytogenes in measuring samples, will with the anti-compound of 1 anti-formation 1.Fully shake after the goat anti-rabbit igg probe of the 2nd antibody Listeria monocytogenes the 1st step obtained adds.Upper magnetic frame separate probe, adds the suspension that deionized water aseptic on a small quantity obtains probe.Now, if there is target Listeria monocytogenes in measuring samples, then by the 2nd antibody and 1 anti-interaction, thus catches this compound, reach the object of object bacteria enrichment.Now, the 1 anti-compound combining object bacteria and there is no combining target bacterium excessive 1 anti-all can with 2 anti-bindings of detecting probe surface, still mix.The probe suspension of this enrichment is added on 1 antienzyme target prepared by the 2nd step, then combine in probe suspension Listeria monocytogenes probe can further with the monoclonal antibody generation specific binding in ELISA Plate, form double antibodies sandwich structure.Now by aseptic washed with de-ionized water, just the probe not in conjunction with Listeria monocytogenes can be washed away.The probe just only combining Listeria monocytogenes remaining in ELISA Plate.
4. with eluent (methyl alcohol etc.), the probe combining Listeria monocytogenes in ELISA Plate is eluted.Upper magnetic frame, separate probe is also cleaned 1-2 time, is washed away by ion.The solution obtained, measures spin-lattice relaxation time and the spin spin relaxation time of solution by nuclear magnetic resonance analyser (NMR20, Niu Mai company).With aseptic deionized water for blank, the spin-lattice relaxation time that solution records and spin spin relaxation time are compared with blank, and there were significant differences, illustrate in solution and have probe to exist, thus have Listeria monocytogenes in interpret sample.The drop-out value of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is proportional.The bright probe of more speaking more declined is more, thus side light Listeria monocytogenes is more, quantitatively can be detected the number of object bacteria in sample by mark-on checking.All food standards all must not detect Listeria monocytogenes, and whether the method can detect in sample fast containing Listeria monocytogenes.
Embodiment
example 2
Whether measure food samples containing harmful pathogenic bacteria---Escherichia coli O 157: H7.
1. nanometer Fe-Ni-Co alloy immunological probe preparation: the rabbit anti-igg monoclonal antibody of 1 anti-employing O157:H7,2 resist the goat anti-rabbit igg for O157:H7, can be monoclonal antibody also can be resist more.Fe-Ni-Co alloy nano particle can be commercially.Such as Beijing Deco Dao Jin Science and Technology Co., Ltd. or Shanghai Chao Wei nano material company, 20nm, ratio 1:1:1, respective purity 99.5%.
Silicon dioxide coated nanometer Fe-Ni-Co alloy: get 47.5g sodium silicate, be dissolved in beaker with deionized water is 12-13 with salt acid for adjusting pH value.Getting 5.0g nanometer Fe-Ni-Co alloy joins in this beaker, mechanical raking (with glass bar) 5min.By ultrasonic for mixed liquor 30min, stir in good time.Be warmed up to 85 C, dropwise add salt acid for adjusting pH value 6-7, generate precipitation.Magneto separate limit, limit spends deionized water precipitation, washs 3-4 time.Then, precipitation is scattered in 250mL methyl alcohol.Above process in triplicate, ensures that silicon is attached on Fe-Ni-Co alloy.
Amino containing silane Fe-Ni-Co alloy nano-material: obtained silicon dioxide coated nanometer Fe-Ni-Co alloy is joined in 25mL methyl alcohol, uses 1mLH
2o and methanol dilution are to 150mL.Then the mixing of 150mL glycerine is added.Ultrasonic 30min, transfers in the 500mL there-necked flask of stirring apparatus.Add 10mL amino silicane coupling agent (AEAPS), under 80-90 C after rapid stirring 3h, migrate out product.Product spends deionized water 3 times, methanol wash 2 times (using Buchner funnel suction filtration).Vacuum drying.It should be noted that suction filtration is owing to there being glycerine, so slow, can suck supernatant liquid by good time suction pipe after taking out a period of time, suction filtration process about needs 6-8h.The amino containing silane Fe-Ni-Co alloy nano-material vacuum drying 12h finally will obtained.
Antibody modification: get a certain amount of amination Fe-Ni-Co alloy nano particle, adds excessive 2 and resists, be i.e. the goat anti-rabbit igg antibody of Escherichia coli O 157: H7,26 C are hatched, wash after, add excessive 1% bovine serum albumin (BSA), 22 C, the active room of confining surface, washing, resuspension.Because nanometer Fe-Ni-Co alloy has paramagnetic properties, externally-applied magnetic field is separated, and nanometer Fe-Ni-Co alloy will be pooled to magnetic field, siphons away supernatant, and washing, then wash away unnecessary antibody, BSA.Preparation paramagnetic nano immunological probe be kept at 4 DEG C stand-by.
2. monoclonal antibody is fixed: can adopt conventional ELISA Plate fixing means, also can adopt following methods.With clean cover glass 5 × 5mm
2square, coating machine first sprays one deck Cr (2 – 4nm) in order to help fixing gold.Be used in surface sputtering again and spray one deck nm of gold, then adopt 200 microlitre 2mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) nm of gold.Add 1 to resist, i.e. rabbit anti-igg monoclonal antibody, fix on a glass also 37 C by 100 μ L100 μ g/mL monoclonal antibodies by e and hatch 45min.Add bovine serum albumin avtive spot remaining on plate is carried out close and dry.
3. food samples is carried out pre-service, adopt FDA enrichment if desired, sample is filtered, increases the pre-service such as bacterium activation, obtain measuring samples.Resist adding 1 in measuring samples, the rabbit anti-igg of O157:H7.If there is O157:H7 in measuring samples, will with the anti-compound of 1 anti-formation 1.By the 2nd obtained for the 1st step antibody, fully shake after the goat anti-rabbit igg probe of O157:H7 adds.Upper magnetic frame separate probe, adds the suspension that deionized water aseptic on a small quantity obtains probe.Now, if there is target Listeria monocytogenes in measuring samples, then by the 2nd antibody and 1 anti-interaction, thus catches this compound, reach the object of object bacteria enrichment.Now, the 1 anti-compound combining object bacteria and there is no combining target bacterium excessive 1 anti-all can with 2 anti-bindings of detecting probe surface, still mix.The probe suspension of this enrichment is added on 1 antienzyme target prepared by the 2nd step, then combine in probe suspension O157:H7 probe can further with the monoclonal antibody generation specific binding in ELISA Plate, form double antibodies sandwich structure.Now by aseptic washed with de-ionized water, just the probe not in conjunction with O157:H7 can be washed away.The probe just only combining O157:H7 remaining in ELISA Plate.
4. ELISA Plate will combine Escherichia coli O 157 with eluent (methyl alcohol etc.): the probe of H7 elutes.Upper magnetic frame, ion, solvent also by aseptic washed with de-ionized water 1-2 time, wash away by separate probe.The solution obtained, with nuclear magnetic resonance analyser (NMR20, Niu Mai company or miFe-Ni-Co alloy NMR East China Normal University) measure T1 and T2 of solution, take deionized water as blank, the spin-lattice relaxation time that solution records and spin spin relaxation time compare with blank, and there were significant differences, illustrate in solution and have probe to exist, thus having Escherichia coli O 157 in interpret sample: H7, the drop-out value of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is proportional.The bright probe of more speaking more declined is more, thus side light Escherichia coli O 157: H7 is more, quantitatively can be detected the number of object bacteria in sample by mark-on checking.
Claims (3)
1., based on a NMR food-borne pathogen rapid detection for paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment, its characterization step is as follows:
1) add the specificity 1 detecting object bacteria in the sample to which to resist, if there is object bacteria in sample, then form 1 anti-compound by the combination of antibody antigen;
2) detect 1 antibody resisted of object bacteria, namely 2 anti-bags are by the preparation of paramagnetic nano nano-Fe-Ni-Co alloy;
3) what object bacteria specificity 1 resisted in ELISA Plate is fixing for subsequent use;
4) enrich target bacterium, and be separated: the 1st) sample of step process, add the paramagnetic nano nano-Fe-Ni-Co alloy that the 2nd step is obtained, abundant mixing concussion reaction a period of time, capture the after-applied externally-applied magnetic field of object bacteria, because nanometer Fe-Ni-Co alloy has paramagnetic properties, nano-Fe-Ni-Co alloy can gather magnetic field, siphons away supernatant and then can isolate probe; If have object bacteria in measuring samples, then first can when the 1st step and the anti-compound of 1 anti-formation 1,1 anti-compound can again with 2 anti-compounds of detecting probe surface, by externally-applied magnetic field by enrichment, separation; The paramagnetic nano nano-Fe-Ni-Co alloy suspension that deionized water aseptic on a small quantity then forms object bacteria is added after washing, Magneto separate; Now, to catch and the nano-Fe-Ni-Co alloy of not catching 1 anti-compound still mixes;
5) the nano-Fe-Ni-Co alloy suspension of enrichment being added to the 3rd) step secures in the ELISA Plate of monoclonal antibody, if there is object bacteria, form double antibodies sandwich, by aseptic washed with de-ionized water, the probe then not grabbing object bacteria is just washed off, if there is not object bacteria, then all probes are all washed off;
6) after, wash with the probe of eluant, eluent by the double antibodies sandwich in ELISA Plate, drop off son and solvent by the method for externally-applied magnetic field separate probe by aseptic washed with de-ionized water, if also there is probe is exactly the probe catching object bacteria;
7) the 6th) probe of step gained, adds the suspension that aseptic deionized water forms probe, and the relaxation time of carrying out nuclear magnetic resonance measures; Under fixing field intensity, the spin-lattice relaxation time of aseptic deionized water and spin spin relaxation time are steady state values, with aseptic deionized water for blank, the relaxation time spin-lattice relaxation time of the suspension recorded compares aseptic deionized water with spin spin relaxation time have remarkable reduction, then illustrate containing probe, thus have object bacteria in indirect proof sample, the decline of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is proportional, by adding scalar quantity probe and indirect quantification goes out the amount of object bacteria.
2. the NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment according to claim 1, what it is characterized in that described object bacteria finally detects the change of evaluation method based on the relaxation time of nuclear magnetic resonance technique.
3. the NMR food-borne pathogen rapid detection based on paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment according to claim 1, is characterized in that described 1 resists monoclonal antibody for detecting object bacteria; 2 to resist be 1 anti-antibody.
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