CN103353461B - A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano Ni-Co alloy probe indirect enrichment - Google Patents
A kind of NMR food-borne pathogen rapid detection based on paramagnetic nano 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 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 bacterium in food liquid sample of foundation, utilize 1 to resist and catch object bacteria, object bacteria is carried out enrichment, separation by the paramagnetic nano Ni-Co alloy probe utilizing the 1 anti-anti-bag in antibody that is 2 to be produced, utilize the paramagnetic properties of Ni nanoparticle-Co alloy on the impact in the relaxation time of nucleus magnetic resonance deamplification, whether detect out in sample containing object bacteria. Different concrete corresponding relations is: paramagnetic nano Ni-Co alloy probe, demonstrate linear relationship under certain condition, namely Ni nanoparticle-Co alloy content is big, and spin-lattice relaxation time and the spin spin relaxation time value of sample are more little, can detection by quantitative object bacteria in certain limit. The method may be used for the quick detection of harmful pathogenic bacteria in food samples, such that it is able to as the rapid screening of large quantities of sample to be checked.
Description
Technical field
The present invention relates to the method for quick of a kind of pathogenic bacterium, particularly relate to a kind of NMR food-borne pathogen rapid detection based on paramagnetic nano Ni-Co alloy probe indirect enrichment.
Background technology
Ultimate principle: monoclonal antibody or antigen molecule are combined by covalent linkage with enzyme molecule, this kind of combination can not change immunological characteristic and the biological activity of monoclonal antibody, antigen and enzyme, and the monoclonal antibody of specificity only can be combined with the antigen of specificity. Ni-Co alloy material has ferromegnetism, there will be paramagnetic properties to a certain extent when particle dia 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, it is possible to for Magneto separate. Meanwhile, paramagnetic substance is very remarkable on the impact of NMR signal, and the paramagnetic substance of trace will make NMR signal show change. Therefore can build the specificity Ni-Co alloy nano probe biosensor of paramagnetism, detect from the angle of mr.
Its main principle steps: 1. add detection object bacteria in the sample to which 1 resists, if there is object bacteria in sample, then forms 1 anti-mixture by the combination of antibody antigen, and now 1 has resisted signal amplification. 2. commercially paramagnetic nano Ni-Co alloy material, it is also possible to prepare nano level Ni-Co alloy by additive method. Using silane coupling agent, its general formula is: Y (CH2)nSiX3. Herein, n is 0-3; X is the group of hydrolyzable; Y is organo-functional group. X is chlorine base, methoxyl group, oxyethyl group, acetoxyl group etc. normally, generates silicon alcohol (Si (OH) when these groups are hydrolyzed3), and be combined with inorganic substance, form siloxanes. Y is vinyl, amino, epoxy group(ing), methacryloxypropyl base, sulfydryl. These reactive groups can react with organic substance and combine.Therefore, with the use of silane coupling agent, can erect between inorganic substance and the interface of organic substance " molecule bridge ", the effect of the performance improving matrix material that the material of two kinds of character great disparities is linked together and increase bonding strength. Can realize surface-functionalized by modified antibodies, form specific immunity probe, then close unnecessary avtive spot. Owing to Ni nanoparticle-Co alloy probe has paramagnetic properties, therefore, it is possible to be there is no the antibody joined by externally-applied magnetic field separation. What Ni nanoparticle-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 enzyme plate surface, and unnecessary avtive spot is closed for subsequent use. 4. the 1st) sample that processed of step, add the 2nd) the obtained paramagnetic nano Ni-Co alloy probe of step, fully mixing concussion reaction for some time, capture the after-applied externally-applied magnetic field of object bacteria, owing to Ni nanoparticle-Co alloy has paramagnetic properties, Ni-Co alloy probe can gather magnetic field on one side, siphons away supernatant liquor and then can isolate probe. If sample to be checked has object bacteria, then first can the 1st) step time and the 1 anti-anti-mixture of formation 1,1 anti-mixture can again with the 2 of detecting probe surface anti-compounds, by externally-applied magnetic field by enrichment, separation. Add, after washing, Magneto separate, the paramagnetic nano Ni-Co alloy probe suspension liquid that deionized water aseptic on a small quantity then forms object bacteria. Now, the excess probe caught object bacteria and do not catch object bacteria still mixes. 5. by the above-mentioned probe mixed, it is added to the enzyme plate surface of the 3rd step, then captured object bacteria probe will with enzyme plate surface monoclonal antibody generation specific binding, formed double antibodies sandwich, with aseptic washed with de-ionized water can will not occur combine probe wash-out. 6. adopt eluent to wash by the specific nano immunological probe of the combination on immobilization carrier again, wash ion, solvent by the method for Magneto separate. This part probe is if it is present, be exactly the probe having captured object bacteria. Owing to Ni-Co alloy has paramagnetic properties, very responsive for resonance instrument, for other molecules, the Ni-Co alloy of trace can the spin lattice relaxation time of the aseptic deionized water of much slower and spin spin relaxation time, and aseptic deionized water is under certain even field intensity, spin-lattice relaxation time and spin spin relaxation time are fixing. Elutriant is placed in nuclear magnetic resonance analyser, compares with aseptic deionized water control group. The explanation significantly occurring spin-lattice relaxation time and spin spin relaxation time value to reduce has probe to exist, thus has pathogenic bacterium detection in side light food samples. Probe content and spin-lattice relaxation time and spin spin relaxation time value reduce in direct ratio. By adding mark, it is possible to detection by quantitative object bacteria. In the method, Ni-Co alloy probe is the means of separation and concentration, and the paramagnetic properties that Ni-Co alloy has simultaneously, can be used as again the probe of detection by quantitative. The major advantage of the method is exactly quick, highly sensitive. Relative to microorganism culturing 2-3 days even time of several days of pathogenic bacterium. This method depends primarily on the pretreatment time of sample, and magnetic resonance detection only needs several minutes. All codex alimentariuss, pathogenic bacterium all must not detection. Therefore, the positive-selecting of extensive sample to be checked can be done by the method, to a certain extent can detection by quantitative. At present, document is not also had to report this kind of method both at home and abroad.
Summary of the invention
Based on a NMR food-borne pathogen rapid detection for paramagnetic nano Ni-Co alloy probe indirect enrichment, for various different food samples is evaluated. The method is the method for harmful pathogenic bacteria in a kind of objective effective detection 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 Ni-Co alloy probe indirect enrichment, utilize nuclear magnetic resonance analyser to the response sensibility of paramagnetic substance, it is proposed to the correlation metric of NMR relaxation Parameters variation and Ni-Co alloy nano particle paramagnetic immunological probe content. Different pathogenic bacterium detection lower limits is different.
What the method depended on foundation can be used for the Ni-Co alloy probe enrichment of harmful pathogenic bacteria specificity paramagnetic nano, separation in food samples, from the angle of NMR relaxation signal Parameters variation, detects out the harmful pathogenic bacteria in sample. Adopt the coupling paramagnetic nano Ni-Co alloy probe of monoclonal antibody specific, it is possible to the specific pathogenetic bacterium in sample is carried out enrichment. Owing to nuclear magnetic resonance analyser SPIN-LATTICE RELAXATION efficiency and spin-spin relaxivity are very responsive to Ni-Co alloy nano particle, namely in deionized water, there is the paramagnetic Ni-Co alloy nano particle of trace, then the spin-lattice relaxation time of water and spin-spin relaxation will significantly decline. Under certain condition, the paramagnetic properties of paramagnetic immunological probe makes the relaxation decay signal spin-lattice relaxation time of nucleus magnetic resonance and spin spin relaxation time produce linear reduction. The immunological probe content in sample is gone out, such that it is able to indirectly quantify the detrimental bacterial content in food samples by detection by quantitative. The pathogenic bacterium content detected out is linearly relevant to probe content, and degree of fitting is better. Final taking the corresponding relation between paramagnetic immunological probe and pathogenic bacterium as tie, it is determined that the pathogenic bacterium colony number in food samples. And all codex alimentariuss, pathogenic bacterium all must not detection. Therefore, the method can do the positive-selecting of extensive sample to be checked, to a certain extent can detection by quantitative.
The present invention realizes like this, and 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-mixture by the combination of antibody antigen.
2) detecting 1 anti-antibody of object bacteria, namely 2 anti-bags are by the preparation of paramagnetic nano Ni-Co alloy probe; .
3) what object bacteria specificity 1 resisted on enzyme plate is fixing for subsequent use.
4) enrichment aimed strain, and be separated: the 1st) sample that processed of step, add the paramagnetic nano Ni-Co alloy probe that the 2nd step is obtained, fully mixing concussion reaction for some time, capture the after-applied externally-applied magnetic field of object bacteria, due to the paramagnetic properties of Ni nanoparticle-Co alloy, then Ni-Co alloy probe is just pooled to magnetic field, siphons away supernatant liquor and then can isolate probe. If sample to be checked has object bacteria, then first can the 1st) step time and the 1 anti-anti-mixture of formation 1,1 anti-mixture can again with the 2 of detecting probe surface anti-compounds, by externally-applied magnetic field by enrichment, separation. Add, after washing, Magneto separate, the paramagnetic nano Ni-Co alloy probe suspension liquid that deionized water aseptic on a small quantity then forms object bacteria. Now, the excess probe caught object bacteria and do not catch object bacteria still mixes.
5) the probe suspension liquid of enrichment is added to the 3rd) step make the enzyme plate securing monoclonal antibody on, 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 eluent by the double antibodies sandwich on enzyme plate, drop off son and solvent by aseptic washed with de-ionized water by the method for externally-applied magnetic field separate probe, if also there is probe is exactly the probe catching object bacteria.This part probe, add the suspension liquid that aseptic deionized water forms probe, the relaxation time carrying out nucleus magnetic resonance measures, taking aseptic deionized water as blank, the relaxation time spin-lattice relaxation time of the suspension liquid recorded compares aseptic deionized water with spin spin relaxation time remarkable reduction, then illustrate containing probe, thus indirect proof sample has object bacteria, the decline of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is direct ratio, it is possible to indirectly quantified the amount of object bacteria to a certain extent by quantitative probe.
Described NMR probe is the nano level Ni-Co alloy material with paramagnetic properties, and nanometer particle size is less than 1000 nanometers.
The final detection evaluation method of described object bacteria is based on the change of the relaxation time characteristic parameter of nuclear magnetic resonance technique.
Described relaxation time characteristic, refers to spin-lattice relaxation time (T1) and spin spin relaxation time (T2).
Described 1 resists the specific antibody for detecting object bacteria, it is preferred to monoclonal antibody. 2 to resist be 1 anti-antibody.
The useful effect of the present invention: the present invention provides a kind of method objectively detecting out the harmful pathogenic bacteria in food fast, is characterized in that depending on the magnetic resonance detection method that can be used for detecting paramagnetic nano Ni-Co alloy probe indirect enrichment of foundation. Harmful pathogenic bacteria in food can objective be detected by the method effectively, confirms compared to the biological culture of pathogenic bacterium, and the method has the advantage of detection fast, it is possible to for the rapid screening of extensive sample.
Embodiment
Example 1
Inspection food samples measures it whether containing harmful pathogenic bacteria Listeria monocytogenes.
1. Ni nanoparticle-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. Ni-Co alloy nano particle can be commercially. Such as Beijing De Kedao gold science and technology limited-liability company or Shanghai Chao Wei nano material company, 20nm, ratio 1:1, respective purity 99.5%.
Silicon dioxide coated Ni nanoparticle-Co alloy: get 47.5g water glass, be dissolved in beaker with deionized water is 12-13 with salt acid for adjusting pH value. Getting 5.0g Ni nanoparticle-Co alloy joins in this beaker, mechanical stirring (with glass stick) 5min. By ultrasonic for mixed solution 30min, stir in good time. It is warmed up to 85 C, dropwise adds salt acid for adjusting pH value 6-7, generate precipitation. Magneto separate limit, limit deionized water wash precipitates, and washs 3-4 time. Then, precipitation is scattered in 250mL methyl alcohol. Above process repeats three times, ensures that silicon is attached on Ni-Co alloy.
Amino containing silane Ni-Co alloy nano-material: obtained silicon dioxide coated Ni nanoparticle-Co alloy is joined in 25mL methyl alcohol, uses 1mLH2O and methyl alcohol are diluted to 150mL. Then the mixing of 150mL glycerine is added. Ultrasonic 30min, transfers in the 500mL there-necked flask of whipping appts. Add 10mL amino silicane coupling agent (AEAPS), after stirring 3h fast under 80-90 C, migrate out product. Product deionized water wash 3 times, methanol wash 2 times (taking out filter with Büchner funnel). Vacuum-drying. It is noted that take out filter owing to there being glycerine, thus slow, supernatant liquid can be sucked by good time suction pipe after taking out for some time, take out filtration journey and about need 6-8h. The amino containing silane Ni-Co alloy nano-material vacuum-drying 12h that finally will obtain.
Antibody modification: get a certain amount of amination Ni-Co alloy nano particle, add the goat anti-rabbit igg antibody of excessive Listeria monocytogenes, 26 C hatch, wash after, add excessive 1% bovine serum albumin (BSA), 22 C, close surfactivity room, washing, resuspended floating.Because Ni nanoparticle-Co alloy has paramagnetic properties, externally-applied magnetic field is separated, and Ni nanoparticle-Co alloy will be pooled to magnetic field, siphons away supernatant liquor, and washing, then wash unnecessary antibody, BSA. It is stand-by that the paramagnetic nano immunological probe of preparation is kept at 4 DEG C.
2. monoclonal antibody is fixed: can adopt conventional enzyme plate fixing means, it is also possible to adopt following method. With clean cover glass 5 × 5mm2Square, coating equipment first sprays one layer of Cr (2 4nm) in order to help fixing gold. It is used in surface sputtering spray last layer nanometer gold again, then adopts the 200 micro-2mmol of liter disulfide group-succinimide-propionic esters (DSP) nanometer gold to be modified (DMSO, dimethyl sulfoxide (DMSO) dilution DSP). 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-treatment, adopt FDA to increase bacterium method if desired, sample is filtered, increases the pre-treatment such as bacterium activation, obtain sample to be checked. Resist sample to be checked adds 1, the rabbit anti-igg of Listeria monocytogenes. If there is Listeria monocytogenes in sample to be checked, will with the 1 anti-anti-mixture of formation 1. Fully shake after the goat anti-rabbit igg probe of the 2nd obtained for the 1st step antibody Listeria monocytogenes is added. Upper magnetic force frame separate probe, adds the suspension liquid that deionized water aseptic on a small quantity obtains probe. Now, if sample to be checked has target Listeria monocytogenes, then by the 2nd antibody and 1 anti-interaction, thus catch this mixture, reach the object of object bacteria enrichment. Now, the 1 anti-mixture 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 liquid of this enrichment is added on 1 anti-enzyme plate prepared by the 2nd step, then probe suspension liquid combines Listeria monocytogenes probe can further with the monoclonal antibody generation specific binding on enzyme plate, formed double antibodies sandwich structure. Now by aseptic washed with de-ionized water, so that it may so that not probe in conjunction with Listeria monocytogenes is washed. The probe just only combining Listeria monocytogenes remaining on enzyme plate.
4. with elutriant (methyl alcohol etc.), the probe combining Listeria monocytogenes on enzyme plate is eluted. Upper magnetic force frame, separate probe is also cleaned 1-2 time, is washed by ion. The solution obtained, measures T1 and T2 of solution by nuclear magnetic resonance analyser (NMR20, Niu Mai company). Taking aseptic deionized water as blank, the spin-lattice relaxation time that solution records is with spin spin relaxation time compared with blank, and there were significant differences, illustrates and has probe to exist in solution, thus illustrates and have Listeria monocytogenes in sample. The decline value of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is direct ratio. The bright probe of more speaking more of decline is more many, thus side light Listeria monocytogenes is more many, can the number of object bacteria in detection by quantitative sample by adding mark checking. All codex alimentariuss all must not detection Listeria monocytogenes, whether this method can detect out in sample fast containing Listeria monocytogenes.
Embodiment
Example 2
Whether measure food samples containing harmful pathogenic bacteria Escherichia coli O 157: H7.
1. Ni nanoparticle-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, it is possible to be monoclonal antibody can also be resist more. Ni-Co alloy nano particle can be commercially. Such as Beijing De Kedao gold science and technology limited-liability company or Shanghai Chao Wei nano material company, 20nm, ratio 1:1, respective purity 99.5%.
Silicon dioxide coated Ni nanoparticle-Co alloy: get 47.5g water glass, be dissolved in beaker with deionized water is 12-13 with salt acid for adjusting pH value. Getting 5.0g Ni nanoparticle-Co alloy joins in this beaker, mechanical stirring (with glass stick) 5min. By ultrasonic for mixed solution 30min, stir in good time. It is warmed up to 85 C, dropwise adds salt acid for adjusting pH value 6-7, generate precipitation. Magneto separate limit, limit deionized water wash precipitates, and washs 3-4 time. Then, precipitation is scattered in 250mL methyl alcohol. Above process repeats three times, ensures that silicon is attached on Ni-Co alloy.
Amino containing silane Ni-Co alloy nano-material: obtained silicon dioxide coated Ni nanoparticle-Co alloy is joined in 25mL methyl alcohol, uses 1mLH2O and methyl alcohol are diluted to 150mL. Then the mixing of 150mL glycerine is added. Ultrasonic 30min, transfers in the 500mL there-necked flask of whipping appts. Add 10mL amino silicane coupling agent (AEAPS), after stirring 3h fast under 80-90 C, migrate out product. Product deionized water wash 3 times, methanol wash 2 times (taking out filter with Büchner funnel). Vacuum-drying. It is noted that take out filter owing to there being glycerine, thus slow, supernatant liquid can be sucked by good time suction pipe after taking out for some time, take out filtration journey and about need 6-8h. The amino containing silane Ni-Co alloy nano-material vacuum-drying 12h that finally will obtain.
Antibody modification: get a certain amount of amination 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 hatch, wash after, add excessive 1% bovine serum albumin (BSA), 22 C, close surfactivity room, washing, resuspended floating. Because Ni nanoparticle-Co alloy has paramagnetic properties, externally-applied magnetic field is separated, and Ni nanoparticle-Co alloy will be pooled to magnetic field, siphons away supernatant liquor, and washing, then wash unnecessary antibody, BSA. It is stand-by that the paramagnetic nano immunological probe of preparation is kept at 4 DEG C.
2. monoclonal antibody is fixed: can adopt conventional enzyme plate fixing means, it is also possible to adopt following method. With clean cover glass 5 × 5mm2Square, coating equipment first sprays one layer of Cr (2 4nm) in order to help fixing gold. It is used in surface sputtering spray last layer nanometer gold again, then adopts the 200 micro-2mmol of liter disulfide group-succinimide-propionic esters (DSP) nanometer gold to be modified (DMSO, dimethyl sulfoxide (DMSO) dilution DSP). 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-treatment, adopt FDA to increase bacterium method if desired, sample is filtered, increases the pre-treatment such as bacterium activation, obtain sample to be checked. Resist sample to be checked adds 1, the rabbit anti-igg of O157:H7. If there is O157:H7 in sample to be checked, will with the 1 anti-anti-mixture of formation 1. 2nd antibody obtained by the 1st step, the goat anti-rabbit igg probe of O157:H7 fully shakes after adding.Upper magnetic force frame separate probe, adds the suspension liquid that deionized water aseptic on a small quantity obtains probe. Now, if sample to be checked has target Listeria monocytogenes, then by the 2nd antibody and 1 anti-interaction, thus catch this mixture, reach the object of object bacteria enrichment. Now, the 1 anti-mixture 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 liquid of this enrichment is added on 1 anti-enzyme plate prepared by the 2nd step, then probe suspension liquid combines O157:H7 probe can further with the monoclonal antibody generation specific binding on enzyme plate, formed double antibodies sandwich structure. Now by aseptic washed with de-ionized water, so that it may so that not probe in conjunction with O157:H7 is washed. The probe just only combining O157:H7 remaining on enzyme plate.
4. enzyme plate will combine Escherichia coli O 157 with elutriant (methyl alcohol etc.): the probe of H7 elutes. Upper magnetic force frame, separate probe and by aseptic washed with de-ionized water 1-2 time, ion, solvent are washed. The solution obtained, with nuclear magnetic resonance analyser (NMR20, Niu Mai company or miNi-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 and have probe to exist in solution, thus illustrate and sample has Escherichia coli O 157: H7, the decline value of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is direct ratio. The bright probe of more speaking more of decline is more many, thus side light Escherichia coli O 157: H7 is more many, can the number of object bacteria in detection by quantitative sample by adding mark checking.
Claims (3)
1., based on a NMR food-borne pathogen rapid detection for paramagnetic nano Ni-Co alloy probe indirect enrichment, its characterization step is as follows:
1) specificity 1 adding detection object bacteria in the sample to which resists, if there is object bacteria in sample, then forms 1 anti-mixture by the combination of antibody antigen;
2) detecting 1 anti-antibody of object bacteria, namely 2 anti-bags are by the preparation of paramagnetic nano Ni-Co alloy probe;
3) what object bacteria specificity 1 resisted on enzyme plate is fixing for subsequent use;
4) enrichment aimed strain, and be separated: the 1st) sample that processed of step, add the 2nd) the obtained paramagnetic nano Ni-Co alloy probe of step, fully mixing concussion reaction for some time, capture the after-applied externally-applied magnetic field of object bacteria, owing to Ni nanoparticle-Co alloy has paramagnetic properties, Ni-Co alloy probe can gather magnetic field, siphons away supernatant liquor and then isolates probe; If sample to be checked has object bacteria, then first can when the 1st step and the 1 anti-anti-mixture of formation 1,1 anti-mixture can again with the 2 of detecting probe surface anti-compounds, by externally-applied magnetic field by enrichment, separation; Add, after washing, Magneto separate, the paramagnetic nano Ni-Co alloy probe suspension liquid that deionized water aseptic on a small quantity then forms object bacteria; Now, catch and the Ni-Co alloy probe of not catching 1 anti-mixture still mixes;
5) the Ni-Co alloy probe suspension liquid of enrichment being added to the 3rd) step secures on the enzyme 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 eluent by the double antibodies sandwich on enzyme plate, drop off son and solvent by aseptic washed with de-ionized water by the method for externally-applied magnetic field separate probe, if also there is probe is exactly the probe catching object bacteria;
7) the 6th) probe of step gained, adds the suspension liquid that aseptic deionized water forms probe, and the relaxation time carrying out nucleus 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, taking aseptic deionized water as blank, the relaxation time spin-lattice relaxation time of the suspension liquid recorded compares aseptic deionized water with spin spin relaxation time remarkable reduction, then illustrate containing probe, thus indirect proof sample has object bacteria, the decline of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is direct ratio, indirectly quantifies the amount of object bacteria by adding demarcation amount probe.
2. the NMR food-borne pathogen rapid detection based on paramagnetic nano Ni-Co alloy probe indirect enrichment according to claim 1, is characterized in that the change of the final detection evaluation method of described object bacteria based on the relaxation time of nuclear magnetic resonance technique.
3. the NMR food-borne pathogen rapid detection based on paramagnetic nano Ni-Co alloy probe indirect enrichment according to claim 1, is characterized in that described 1 resists the specific monoclonal antibody for detecting object bacteria; 2 to resist be 1 anti-antibody.
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| CN102323408A (en) * | 2011-05-31 | 2012-01-18 | 上海师范大学 | Method for rapid detection of enterobacter sakazakii |
| WO2011150212A3 (en) * | 2010-05-26 | 2012-03-15 | The General Hospital Corporation | Magnetic nanoparticles |
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| WO2011150212A3 (en) * | 2010-05-26 | 2012-03-15 | The General Hospital Corporation | Magnetic nanoparticles |
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