CN103115934B - A kind of based on Fe 3o 4the NMR food-borne pathogen rapid detection of@Au composite nanoparticle - Google Patents
A kind of based on Fe 3o 4the NMR food-borne pathogen rapid detection of@Au composite nanoparticle Download PDFInfo
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- CN103115934B CN103115934B CN201310032117.3A CN201310032117A CN103115934B CN 103115934 B CN103115934 B CN 103115934B CN 201310032117 A CN201310032117 A CN 201310032117A CN 103115934 B CN103115934 B CN 103115934B
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Abstract
A kind of based on Fe
3o
4the NMR food-borne pathogen rapid detection of@Au composite nanoparticle, belongs 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, utilizes Fe
3o
4@Au composite nano materials prepares immunomagnetic beads enrich target bacterium pointedly, utilizes Fe
3o
4whether the paramagnetic of@Au composite nanoparticle and superparamagnetic characteristic, on the impact in nuclear magnetic resonance deamplification relaxation time, detect in sample containing object bacteria.Different concrete corresponding relations is: paramagnetic immunomagnetic beads, demonstrates linear relationship under certain condition, and namely immunomagnetic beads content is large, T2 or the T1 value of sample is 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 method for quick of a kind of pathogenic bacteria, particularly relate to a kind of based on Fe
3o
4the NMR food-borne pathogen rapid detection of@Au composite nanoparticle.
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.Its main principle steps: the 1. magnetic Fe that utilized two-step approach to prepare
3o
4@Au core-shell nano, is realized by modified antibodies surface-functionalized, monoclonal antibody is coupled at magnetic bead surfaces, forms specific immunity magnetic bead, and is closed by unnecessary avtive spot.2. adopt certain method to be fixed on ELISA Plate surface the monoclonal antibody specific of another part object bacteria, and unnecessary avtive spot is closed.3. the specific immunity magnetic bead of preparation is used for capturing enrich target bacterium, by externally-applied magnetic field by Beads enrichment out, now, combines the magnetic bead of object bacteria and do not have the magnetic bead of combining target bacterium still to mix.4. by the above-mentioned magnetic bead mixed, be added to the ELISA Plate surface of the 2nd step, then captured object bacteria magnetic bead 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 magnetic bead wash-out.5. adopt eluant, eluent to be washed by the specific nano immunomagnetic beads of the combination on immobilization carrier again, wash ion, solvent.If this part magnetic bead exists, capture the magnetic bead of object bacteria exactly.Due to Fe
3o
4@Au composite nanoparticle has paramagnetic and superparamagnetic characteristic, very responsive for resonance instrument, for other molecules, and the Fe of trace
3o
4@Au composite nanoparticle 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, T1 or T2 is fixing.Eluent is placed in nuclear magnetic resonance analyser, contrasts with aseptic deionized water control group.The explanation that remarkable generation T1 or T2 value reduces has magnetic bead to exist, thus has pathogenic bacteria to detect in side light food samples.Magnetic bead content and T1 or T2 value reduce proportional.By mark-on, quantitatively object bacteria can be detected.Fe in the method
3o
4@Au composite nanoparticle magnetic bead is the means of separation and concentration, simultaneously Fe
3o
4the paramagnetic that@Au composite nanoparticle has and superparamagnetic characteristic, 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.Therefore, can do the positive-selecting of extensive measuring samples by the method, the positive sample detected also needs to cultivate with microorganism to confirm.At present, also there is no this method of bibliographical information both at home and abroad, but adopt immunomagnetic beads to carry out the report of the enrichment of pathogenic bacteria, the enrichment of object etc. or a lot, but all do not adopt nuclear magnetic resonance to do further detection.
Summary of the invention
The object of this invention is to provide a kind of based on Fe
3o
4the method for quick of the food-borne pathogens of@Au composite nanoparticle, 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 based on Fe
3o
4the nuclear magnetic resonance technique of@Au composite nanoparticle detects the method for the harmful pathogenic bacteria in food fast, utilizes nuclear magnetic resonance analyser to the response sensibility of paramagnetic, superparamagnetic material, proposes NMR (Nuclear Magnetic Resonance) relaxation Parameters variation and Fe
3o
4the correlation metric of@Au composite nanoparticle superparamagnetic immunomagnetic beads content.Different pathogenic bacteria detect lower limit difference.
What the method depended on foundation can be used for the enrichment of harmful pathogenic bacteria characteristic immunomagnetic beads, 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 superparamagnetic immunomagnetic beads of monoclonal antibody specific, the specific pathogenetic bacterium in sample can be carried out enrichment; Because nuclear magnetic resonance analyser SPIN-LATTICE RELAXATION efficiency and spin-spin relaxation efficiency are to Fe
3o
4@Au composite nanoparticle is very responsive, namely in deionized water, there is the superparamagnetic Fe of trace
3o
4@Au composite nanoparticle, then the spin-lattice relaxation time (T1) of water and/spin-spin relaxation (T2) will significantly decline.Under certain condition, the paramagnetic properties of superparamagnetic immunomagnetic beads makes relaxation decay signal T2 or T1 of nuclear magnetic resonance produce linear reduction.By quantitatively detecting the immunomagnetic beads content in sample, thus detrimental bacterial content in food samples can be gone out by indirect quantification.The pathogenic bacteria content detected and magnetic bead content linear correlation, degree of fitting is better.Final with the corresponding relation between superparamagnetic immunomagnetic beads and pathogenic bacteria for tie, determine the pathogenic bacteria clump count in food samples.
The present invention is achieved in that step is as follows:
1) preparation of the specific immunity magnetic bead of object bacteria is detected;
2) object bacteria monoclonal antibody specific is fixed on ELISA Plate surface;
3) immunomagnetic beads enrich target bacterium, and be separated: the immunomagnetic beads that the 1st step is obtained adds measuring samples, abundant mixing concussion, by applying externally-applied magnetic field after capturing object bacteria, then magnetic bead is just pooled to magnetic field on one side, if siphon away supernatant then can isolate in magnetic bead measuring samples and have object bacteria, then by the enrichment of magnetic bead institute, add the magnetic bead suspension that deionized water aseptic on a small quantity then forms object bacteria;
4) the magnetic bead suspension of enrichment is added to securing in the ELISA Plate of monoclonal antibody of the 2nd step making, if there is object bacteria, forms double antibodies sandwich; By aseptic washed with de-ionized water, then the magnetic bead not grabbing object bacteria is just washed off, if there is not object bacteria, then all magnetic beads are all washed off;
5) after, wash with the magnetic bead of eluant, eluent by the double antibodies sandwich in ELISA Plate, the method being separated magnetic bead with externally-applied magnetic field drops off son and solvent by aseptic washed with de-ionized water, if also there is magnetic bead is exactly the magnetic bead catching object bacteria;
6) this part magnetic bead, add the suspension that aseptic deionized water forms magnetic bead, the relaxation time of carrying out nuclear magnetic resonance measures, with aseptic deionized water for blank, the relaxation time T1 of the suspension recorded or T2 compare aseptic deionized water remarkable reduction, then illustrate containing magnetic bead, thus have object bacteria in indirect proof sample, the amount of magnetic bead and the decline of T1 or T2 proportional, the amount of object bacteria can be gone out by indirect quantification by quantitative magnetic bead.
Described Fe
3o
4@Au material, nanometer particle size is less than 1000 nanometers.
Described object bacteria finally detect the change of evaluation method based on the relaxation time parameter of nuclear magnetic resonance technique.
In the described relaxation time, refer to spin-lattice relaxation time (T1) and spin spin relaxation time (T2).
Beneficial effect of the present invention: the invention provides a kind of method objectively detecting the harmful pathogenic bacteria in food fast, it is characterized in that the magnetic resonance detection method that can be used for detection of superparamagnetic immunomagnetic beads 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 in inspection food samples.
1. immunomagnetic beads preparation:
Adopt the Fe of " two-step approach " synthetic kernel shell structure
3o
4/ Au nano particle.First by molysite [n (Fe
3+)/n (Fe
2+)=2/1.2] mix, be placed in 50 DEG C of calibration cells, add 2 mol/L NaOH under stirring, control pH value of solution and be about 11, remain unchanged to pH, then be warming up to 80 DEG C of slaking 1h, whole reaction is carried out under nitrogen protection.Precipitation is through magnetic separation, and redistilled water cleaning, for several times to neutral, obtains Fe
3o
4suspending liquid, constant volume also measures its solid content.Measure appropriate Fe
3o
4seed suspension liquid and HAuCl
4solution mixing 1h, then adds excessive oxammonium hydrochloride (80mmol/L), reacts 1h, obtain the Fe of nucleocapsid structure under stirring
3o
4/ Au magnetic composite particle.Again add HAuCl
4, it is coated to carry out secondary.Magnetic separation, carries out purification process with 1mol/L hydrochloric acid solution, to remove the Fe be not wrapped by
3o
4, redistilled water cleaning, for several times to neutral, is kept in Dark Place.
Immunity Fe
3o
4prepared by@Au: Fe
3o
4after adding excessive Listeria antibody after@Au concentrates, hatch, washing, add the active room of excessive BSA confining surface, washing, resuspension, be kept at 4 DEG C stand-by.Also following methods can be adopted: adopt 200 microlitre 2 mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) nm of gold.Add Listeria monoclonal monoclonal antibody, to be fixed on Au by e by 100 μ L 100 μ g/mL monoclonal antibodies and 37 C hatch 45 min.Add 1% bovine serum albumin (BSA), 22 C, 1 hour, remaining avtive spot is carried out close and dry.
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 – 4 nm) in order to help fixing gold.Be used in surface sputtering again and spray one deck nm of gold, then adopt 200 microlitre 2 mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) nm of gold.Add Listeria monoclonal monoclonal antibody, fix on a glass also 37 C by 100 μ L 100 μ g/mL monoclonal antibodies by e and hatch 45 min.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.Fully concussion several minutes is carried out after being added by the immunizing monoclonal antibody magnetic bead that the first step is obtained.Upper magnetic frame is separated magnetic bead, adds the suspension that deionized water aseptic on a small quantity obtains magnetic bead.Now, if there is target Listeria in measuring samples, then just reached the object of object bacteria enrichment by the specific reaction of immunomagnetic beads.The magnetic bead suspension of this enrichment is added in the monoclonal antibody ELISA Plate prepared by the 2nd step, then combine in magnetic bead emulsion listerial magnetic bead 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 can will not wash away in conjunction with listerial magnetic bead.What ELISA Plate was left just only combines listerial magnetic bead.
4. with eluent (methyl alcohol etc.), the listerial magnetic bead that combines in ELISA Plate is eluted.Upper magnetic frame, is separated magnetic bead and cleans 1-2 time, being washed away by ion.The solution obtained, measures T1 or T2 of solution by nuclear magnetic resonance analyser (NMR20, Niu Mai company or miniNMR East China Normal University).With aseptic deionized water for blank, T1 or T2 that solution records is compared with blank, and there were significant differences, illustrates in solution and has magnetic bead to exist, thus have Listeria in interpret sample, the amount of magnetic bead and the drop-out value of T1 or T2 proportional.The bright magnetic bead of more speaking more declined is more, thus side light Listeria is more, quantitatively can be detected the number of object bacteria in sample by mark-on checking.
Example 2
Whether measure food samples containing harmful pathogenic bacteria Escherichia coli O 157: H7.
1. adopt the Fe of " two-step approach " synthetic kernel shell structure
3o
4@Au composite nanoparticle.First by molysite [n (Fe
3+)/n (Fe
2+)=2/1.2] mix, be placed in 50 DEG C of calibration cells, add 2 mol/L NaOH under stirring, control pH value of solution and be about 11, remain unchanged to pH, then be warming up to 80 DEG C of slaking 1h, whole reaction is carried out under nitrogen protection.Precipitation is through magnetic separation, and redistilled water cleaning, for several times to neutral, obtains Fe
3o
4suspending liquid, constant volume also measures its solid content.Measure appropriate Fe
3o
4seed suspension liquid and HAuCl4 solution mixing 1h, then add excessive oxammonium hydrochloride (80mmol/L), react 1h, obtain the Fe of nucleocapsid structure under stirring
3o
4/ Au magnetic composite particle.Again add HAuCl
4, it is coated to carry out secondary.Magnetic separation, carries out purification process with 1mol/L hydrochloric acid solution, to remove the Fe be not wrapped by
3o
4, redistilled water cleaning, for several times to neutral, is kept in Dark Place.
Immunity Fe
3o
4prepared by@Au: Fe
3o
4after adding excessive O157:H7 antibody after@Au concentrates, hatch, washing, add the active room of excessive BSA confining surface, washing, resuspension, be kept at 4 DEG C stand-by.Also following methods can be adopted: adopt 200 microlitre 2 mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) nm of gold.Add O157:H7 monoclonal monoclonal antibody, to be fixed on Au by e by 100 μ L 100 μ g/mL monoclonal antibodies and 37 C hatch 45 min.Add 1% bovine serum albumin (BSA), 22 C, 1 hour, avtive spot remaining on plate is carried out close and dry.
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 – 4 nm) in order to help fixing gold.Be used in surface sputtering again and spray one deck nm of gold, then adopt 200 microlitre 2 mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) nm of gold.Add O157:H7 monoclonal antibody, fix on a glass also 37 C by 100 μ L 100 μ g/mL monoclonal antibodies by e and hatch 45 min.Add bovine serum albumin avtive spot remaining on plate is carried out close and dry.
3. food samples carried out pre-service, sample is filtered, increase the pre-service such as bacterium activation, obtain measuring samples.Fully concussion several minutes is carried out after being added by the immunizing monoclonal antibody magnetic bead that the first step is obtained.Upper magnetic frame is separated magnetic bead, adds the emulsion that a small amount of water obtains magnetic bead.Now, if there is target Escherichia coli O 157 in measuring samples: H7, then just reached the object of object bacteria enrichment by the specific reaction of immunomagnetic beads.The magnetic bead suspension of this enrichment is added in the monoclonal antibody ELISA Plate prepared by the 2nd step, then combine Escherichia coli O 157 in magnetic bead suspension: the magnetic bead of H7 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 can by conjunction with Escherichia coli O 157: the magnetic bead of H7 washes away.What ELISA Plate was left just only combines Escherichia coli O 157: the magnetic bead of H7.
4. ELISA Plate will combine Escherichia coli O 157 with eluent (methyl alcohol etc.): the magnetic bead of H7 elutes.Upper magnetic frame, is separated magnetic bead and by aseptic washed with de-ionized water 1-2 time, ion, solvent is washed away.The solution obtained, with nuclear magnetic resonance analyser (NMR20, Niu Mai company or miniNMR, East China Normal University) measure T1 or T2 of solution, take deionized water as blank, T1 or T2 that solution records compares with blank, there were significant differences, illustrate in solution and have magnetic bead to exist, thus have Escherichia coli O 157 in interpret sample: H7, the amount of magnetic bead and the drop-out value of T1 or T2 proportional.The bright magnetic bead 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 (4)
1. one kind based on Fe
3o
4the NMR food-borne pathogen rapid detection of@Au composite nanoparticle, its characterization step is as follows:
1) preparation of the specific immunity magnetic bead of object bacteria is detected;
2) by object bacteria monoclonal antibody specific fixing in ELISA Plate;
3) immunomagnetic beads enrich target bacterium, and be separated: by the 1st) the obtained immunomagnetic beads of step adds measuring samples, abundant mixing concussion, by applying externally-applied magnetic field after capturing object bacteria, then magnetic bead is just pooled to magnetic field on one side, siphons away supernatant and then can isolate magnetic bead, if having object bacteria in measuring samples, then by the enrichment of magnetic bead institute, add the magnetic bead suspension that deionized water aseptic on a small quantity then forms object bacteria;
4) the magnetic bead suspension of enrichment being added to the 2nd) 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, then the magnetic bead not grabbing object bacteria is just washed off, if there is not object bacteria, then all magnetic beads are all washed off;
5) after, wash with the magnetic bead of eluant, eluent by the double antibodies sandwich in ELISA Plate, be separated the method for magnetic bead with externally-applied magnetic field, drop off son and solvent by aseptic washed with de-ionized water, if also there is magnetic bead is exactly the magnetic bead catching object bacteria;
6) by the 5th) magnetic bead of step gained adds the suspension that aseptic deionized water forms magnetic bead, carries out NMR relaxation time mensuration; Under fixing field intensity, T1 or T2 of aseptic deionized water is a steady state value, with aseptic deionized water for blank, the relaxation time T1 of the suspension recorded or T2 compare aseptic deionized water remarkable reduction, then illustrate containing magnetic bead, thus have object bacteria in indirect proof sample, the amount of magnetic bead and the decline of T1 or T2 proportional, can by adding scalar quantity magnetic bead and indirect quantification goes out the amount of object bacteria.
2. according to claim 1 based on Fe
3o
4the NMR food-borne pathogen rapid detection of@Au composite nanoparticle, is characterized in that described Fe
3o
4@Au composite nanoparticle, be namely the magnetic core-shell composite nanoparticle material of Shell Materials with gold, nanometer particle size is less than 1000 nanometers.
3. according to claim 1 based on Fe
3o
4the NMR food-borne pathogen rapid detection of@Au composite nanoparticle, 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.
4. according to claim 1 based on Fe
3o
4the method for quick of the NMR food-borne pathogens of@Au composite nanoparticle, is characterized in that the described NMR relaxation time, refers to spin-lattice relaxation time (T1) and spin spin relaxation time (T2).
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| CN108371942A (en) * | 2018-02-12 | 2018-08-07 | 湖北工业大学 | A kind of composite magnetic nano-particle Fe3O4@Au/MPA/NTA-Ni2+And its it prepares and applies |
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