CN103217447A - Paramagnetic nano-Ni-Co alloy probe-based NMR food borne pathogen rapid detection method - Google Patents
Paramagnetic nano-Ni-Co alloy probe-based NMR food borne pathogen rapid detection method Download PDFInfo
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Abstract
The invention provides a paramagnetic nano-Ni-Co alloy probe-based NMR (nuclear magnetic resonance) food borne pathogenic bacteria rapid detection method, and belongs to technical field of food safety pathogenic bacteria rapid detection. The present invention can detect whether the sample includes the target pathogenic bacteria depending on the establishment of NMR detection method for pathogenic bacteria in food samples by using the influence of paramagnetic characteristics of the paramagnetic nano-Ni-Co alloy probe on the relaxation time of the NMR attenuation signal. Different specific correspondence relationships are that: the paramagnetic nano-Ni-Co alloy probe displays a linear relationship under certain conditions, i.e. when the content of nano-Ni-Co alloy is larger, the T2/T1 of the sample is smaller; and the target bacteria in a certain range can be quantitatively detected. The method can be used for the rapid detection of harmful pathogenic bacteria in food samples, thus can be used for rapid screening of a large number of samples to be tested.
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 nanometer Ni-Co 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 Ni-Co 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 Ni-Co alloy nano probe biology sensor, do detection from the angle of magnetic resonance.
The principle steps that it is main: 1. detect the preparation of the specificity paramagnetic nano Ni-Co alloy probe of object bacteria.Buy nanometer Ni-Co alloy material from the market, also can prepare nano level Ni-Co 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 Ni-Co alloy probe has the paramagnetic characteristic, therefore, can separate the antibody that does not have on the connection by externally-applied magnetic field.2. the monoclonal antibody specific with another part object bacteria adopts certain method to be fixed on surface of solid phase carriers, and with unnecessary avtive spot sealing, standby.3. the specific immunity probe with the 1st step modified adds sample to be checked, and after fully object bacteria was grasped in the mixing concussion, last magnetic force frame separated.Because nanometer Ni-Co alloy has the paramagnetic characteristic, probe can be pooled to magnetic field on one side, siphons away supernatant and then can isolate probe.If in the sample to be checked object bacteria is arranged, then by the enrichment of probe institute.Remove the magnetic force frame and add the probe suspension that a small amount of aseptic deionized water then forms object bacteria; At this moment, the probe of catching object bacteria and not catching object bacteria still mixes.4. with the above-mentioned probe that mixes, be added to the surface of solid phase carriers in the 2nd 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 not grab the probe wash-out of object bacteria with aseptic washed with de-ionized water.5. adopt eluant, eluent that the specific nano immunological probe of the combination on the immobilization carrier is washed again, wash ion, solvent with the magnetism separate method of externally-applied magnetic field.If this part probe exists, grasped the probe of object bacteria exactly.Because the Ni-Co alloy has the paramagnetic characteristic, very responsive for the resonance instrument, for other molecules, the nanometer Ni-Co 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.Nanometer Ni-Co alloy probe is the means of separation and concentration in this method, and the paramagnetic characteristic that has of nanometer Ni-Co 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 paramagnetic nano Ni-Co 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 paramagnetic nano Ni-Co alloy probe, nuclear magnetic resonance analyser propose the correlativity index of variation of NMR (Nuclear Magnetic Resonance) relaxation parameter and Ni-Co alloy nano particle content to the response susceptibility of paramagnetic material.Different pathogenic bacteria detect the lower limit difference.
This method depends on harmful pathogenic bacteria specificity paramagnetic nano Ni-Co 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 Ni-Co 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 Ni-Co alloy nano particle, promptly in deionized water, the paramagnetic Ni-Co 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) preparation of the specificity paramagnetic nano Ni-Co alloy probe of detection object bacteria.
2) it is standby the object bacteria monoclonal antibody specific to be fixed on surface of solid phase carriers.
3) immunological probe enrichment aimed strain, and separate: the immunological probe that the 1st step was made adds sample to be checked, and after fully object bacteria was grasped in the mixing concussion, last magnetic force frame separated.Because nanometer Ni-Co alloy has the paramagnetic characteristic, probe can be pooled to magnetic field on one side, siphons away supernatant and then can isolate probe.If in the sample to be checked object bacteria is arranged, then by the enrichment of probe institute.Remove the magnetic force frame and add the probe suspension that a small amount of aseptic deionized water then forms object bacteria; At this moment, the probe of catching object bacteria and not catching object bacteria still mixes.
4) the probe suspension with enrichment is added on the solid phase carrier of having fixed monoclonal antibody specific of the 2nd 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.
5) 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.
6) 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 nanometer Ni-Co 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.
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 paramagnetic nano Ni-Co 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
The check food samples is measured it and whether is contained harmful pathogenic bacteria---Listeria monocytogenes.
1. nanometer Ni-Co alloy immunological probe preparation: buy nanometer Ni-Co alloy material 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 Ni-Co 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 Ni-Co 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 Ni-Co alloy.
Amino containing silane Ni-Co alloy nano-material: the silicon dioxide coated nanometer Ni-Co 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 Ni-Co alloy nano-material vacuum drying 12h that obtains.
Antibody modification: get a certain amount of amination Fe nanometer particles, add excessive Listeria monocytogenes antibody, 26 ℃ hatch, wash after, add excessive 1% bovine serum albumin (BSA), 22 ℃, the active room of confining surface, washing, resuspension.Because of nanometer Ni-Co alloy has the paramagnetic characteristic, externally-applied magnetic field separates, and nanometer Ni-Co 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 Listeria monocytogenes monoclonal monoclonal antibody, be 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.After adding, the immune monoclonal antibody probe that the 1st step was made fully shook several minutes.With magnetic force frame separate probe, add the suspension that a small amount of aseptic deionized water obtains probe.At this moment, if the target Listeria monocytogenes is arranged in the sample to be checked, then just reached the purpose of object bacteria enrichment by the specific reaction of immunological probe.The probe suspension of this enrichment is added on the prepared monoclonal antibody 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, 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 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.
Embodiment
Example 2
Measure food samples and whether contain harmful pathogenic bacteria---Escherichia coli O 157: H7.
1. nanometer Ni-Co alloy immunological probe preparation: buy nanometer Ni-Co alloy material 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 Ni-Co 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 Ni-Co 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 Ni-Co alloy.
Amino containing silane Ni-Co alloy nano-material: the silicon dioxide coated nanometer Ni-Co 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 Ni-Co alloy nano-material vacuum drying 12h that obtains.
Antibody modification: get a certain amount of amination Fe nanometer particles, add excessive Escherichia coli O 157: H7 antibody, 26 ℃ hatch, wash after, add excessive 1% bovine serum albumin (BSA), 22 ℃, the active room of confining surface, washing, resuspension.Because of nanometer Ni-Co alloy has the paramagnetic characteristic, externally-applied magnetic field separates, and nanometer Ni-Co 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 O157:H7 monoclonal antibody, be 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, sample is filtered, increases pre-service such as bacterium activation, obtain sample to be checked.After adding, the immune monoclonal antibody probe that the first step is made fully shook several minutes.Last magnetic force frame separate probe adds the suspension that low amounts of water obtains probe.At this moment, if target Escherichia coli O 157: H7 is arranged in the sample to be checked, then just reached the purpose of object bacteria enrichment by the specific reaction of immunological probe.The probe suspension of this enrichment is added on the prepared monoclonal antibody fixed head of the 2nd step, then combine the probe of Escherichia coli O 157: 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 not in conjunction with Escherichia coli O 157: the probe flush away of H7.The probe that just only combines Escherichia coli O 157: 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) T1 and the T2 of mensuration 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 (5)
1. NMR food-borne pathogens method for quick based on paramagnetic nano Ni-Co alloy probe, its characterization step is as follows:
1) preparation of the paramagnetic nano Ni-Co alloy probe of detection object bacteria;
2) with object bacteria monoclonal antibody specific fixing standby on solid phase carrier;
3) enrichment aimed strain, and separate: the paramagnetic nano Ni-Co alloy probe that the 1st step was made adds sample to be checked, fully mix concussion, grasp after the object bacteria by applying externally-applied magnetic field, because nanometer Ni-Co alloy has the paramagnetic characteristic, then the Ni-Co alloy probe can gather magnetic field on one side, siphons away supernatant and then can isolate probe; If in the sample to be checked object bacteria is arranged,, add the paramagnetic nano Ni-Co alloy probe suspension that a small amount of aseptic deionized water then forms object bacteria then by the enrichment of probe institute;
4) the Ni-Co 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;
5) 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;
6) 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 paramagnetic nano Ni-Co alloy probe according to claim 1 is characterized in that described NMR nano-probe is the nanoscale Ni-Co alloy material with paramagnetic characteristic.
3. the NMR food-borne pathogens method for quick based on paramagnetic nano Ni-Co 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 paramagnetic nano Ni-Co alloy probe according to claim 1 is characterized in that described NMR relaxation time characteristic, is meant spin-lattice relaxation time (T1) and spin spin relaxation time (T2).
5. the NMR food-borne pathogens method for quick based on paramagnetic nano Ni-Co alloy probe according to claim 1 is characterized in that described relaxation time characteristic, and T1 adopts 180 °-τ-90 ° of pulse method to measure, and T2 adopts cpmg sequence row method to measure.
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