CN103185796A - Food-borne pathogenic bacteria quick detection method based on Gamma-Fe2O3@Au nano particle indirect enrichment and immunomagnetic separation - Google Patents
Food-borne pathogenic bacteria quick detection method based on Gamma-Fe2O3@Au nano particle indirect enrichment and immunomagnetic separation Download PDFInfo
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Abstract
A food-borne pathogenic bacteria quick detection method based on Gamma-Fe2O3@Au nano particle indirect enrichment and immunomagnetic separation, and belongs to the technical field of quick pathogen detection for food security. On dependence of a method for detecting pathogen in a food liquid sample, the specificity of an antibody I is combined with the target bacteria, the Gamma-Fe2O3@Au composite nano particle is utilized to prepare immuomagnetic beads of an antibody II to enrich target strains marked by the antibody I, the nano magnetic beads of the target bacteria are captured through separation, and then subjected to nitration to be converted into ferrous ions, and the ferrous ions are detected to indirectly detect whether the sample contains target pathogen. Under a certain condition, the quantity of the ferrous ions and the target bacteria show linear relation, so that the target bacteria can be quantificationally detected within a certain range. The food-borne pathogenic bacteria quick detection method can be used for quick detection of harmful pathogen in the food sample, and can be used for quick screening of large quantities of samples to be detected.
Description
Technical field
The present invention relates to the fast detecting side of a kind of pathogenic bacteria, relate in particular to a kind of based on γ-Fe
2O
3The food-borne pathogens method for quick that the indirect enrichment immunity of @Au nano particle magnetic separates.
Background technology
Ultimate principle: monoclonal antibody or antigen molecule and enzyme molecule are by covalent bonds, and 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.The principle steps that it is main: 1. in sample to be checked, add the 1st antibody of object bacteria, if there is object bacteria, then closes with 1 resistive connection and form 1 anti-compound; 2. utilize seed mediated growth method to prepare magnetic
γ-Fe
2O
3The @Au core-shell nano, surface-functionalized by the modified antibodies realization, be the 2 anti-magnetic bead surfaces that are coupled at 1 anti-antibody, form the specific immunity magnetic bead, and unnecessary avtive spot is sealed.2. the specificity 1 anti-monoclonal antibody with another part object bacteria adopts certain method to be fixed on surface of solid phase carriers, and with unnecessary avtive spot sealing.3. 2 anti-specific immunity magnetic beads with the preparation of the 2nd step join in the sample to be checked in the 1st step, be used for grasping the enrichment object bacteria, by externally-applied magnetic field magnetic bead is separated, at this moment, combine 1 anti-compound of object bacteria and do not have excessive 1 of combining target bacterium to resist and all can close with 2 resistive connections of magnetic bead surfaces, still mix.4. with the above-mentioned magnetic bead that mixes, be added to the surface of solid phase carriers in the 2nd step, the magnetic bead that has then grasped object bacteria will be combined with surface of solid phase carriers 1 anti-generation specificity, form double antibodies sandwich, can will the magnetic bead wash-out of combination not take place with aseptic washed with de-ionized water.5. adopt eluant, eluent that the specific nano immunomagnetic beads of the combination on the immobilization carrier is washed again, wash ion, solvent.If this part magnetic bead exists, grasped the magnetic bead of object bacteria exactly.6. add nitric acid and sulfuric acid (chloroazotic acid) and carry out nitration reaction, if this part magnetic bead exists, then
γ-Fe
2O
3Be converted into ferric ion and ferrous ion.Pathogenic bacteria all must not detect in all food standards.Whether exist ferric ion just can detect by detection and whether have object bacteria in the sample.By mark-on, can quantitatively detect object bacteria within the specific limits.In this method
γ-Fe
2O
3The @Au magnetic bead is the means of separation and concentration, also is simultaneously the carrier that quantitatively detects, and has played a signal and has amplified.The major advantage of this method be exactly fast, sensitivity is higher relatively.Cultivate the time of 2-3 days even several days with respect to the microorganisms of pathogenic bacteria.The method depends primarily on the The pretreatment time.Therefore, can do the positive-selecting of extensive sample to be checked with this method, the positive sample that detects also needs to cultivate with microorganism and prove conclusively.At present, also do not have this method of bibliographical information both at home and abroad, but the report that adopts immunomagnetic beads to carry out the enrichment of pathogenic bacteria, the enrichment of object etc. still is a lot, but does not adopt the method for check ferric ion to do further detection.
Summary of the invention
The purpose of this invention is to provide a kind of based on γ-Fe
2O
3The food-borne pathogens method for quick that the indirect enrichment immunity of @Au nano particle magnetic separates, be 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 based on
γ-Fe
2O
3The immune magnetic of the indirect enrichment of @Au composite nanoparticle separates the food-borne pathogens method for quick, the immunomagnetic beads of catching by separation, make it be converted into ferric ion, the correlativity index of mover iron ion and object bacteria is by detecting ferric ion quantitative objective bacterium indirectly.Different pathogenic bacteria detect the lower limit difference.
This method depends on harmful pathogenic bacteria characteristic immunomagnetic beads enrichment, the separation in the food samples of can be used for of foundation.Adopt 1 anti-mark of object bacteria to form 1 anti-compound; Adopt the i.e. 2 super paramagnetic immunomagnetic beadses that resist of coupling 1 antiantibody, the specificity pathogenic bacteria in the sample can be carried out enrichment and separation; Separate the magnetic bead that captures object bacteria by the design double antibodies sandwich, again the magnetic bead nitration reaction is converted into ferric ion and ferrous ion.Adopt Phen absorption photometry, potassium rhodanide colourimetry etc. can detect the amount of ferric ion, thereby can calculate the amount of magnetic bead, by adding the scalar quantity magnetic bead in the amount that to a certain degree quantitatively goes out object bacteria indirectly.Catch the magnetic bead content of object bacteria by quantitative test, thereby can quantitatively go out the harmful pathogenic bacteria content in the food samples indirectly.Detected pathogenic bacteria content and magnetic bead content linear dependence, degree of fitting is better.Final is tie with the corresponding relation between immunomagnetic beads and pathogenic bacteria, determines the pathogenic bacteria clump count in the food samples.
The present invention is achieved in that step is as follows:
1) in sample to be checked, adds the 1st antibody of object bacteria, if there is object bacteria, then closes with 1 resistive connection and form 1 anti-compound;
2) the 2 anti-i.e. preparations of the antibody immune magnetic beads of the 1st antibody;
3) object bacteria specificity 1 anti-monoclonal antibody is fixed on surface of solid phase carriers;
4) immunomagnetic beads enrichment aimed strain, and separate: the 2 anti-immunomagnetic beadses that the 2nd step was made add the 1st sample to be checked that goes on foot, fully mix concussion, grasp after the object bacteria by applying externally-applied magnetic field, then magnetic bead just is pooled to magnetic field on one side, siphon away supernatant and then can isolate magnetic bead if in the sample to be checked object bacteria is arranged, then by the enrichment of magnetic bead institute, add the magnetic bead suspension that a small amount of aseptic deionized water then forms object bacteria; At this moment, combine 1 anti-compound of object bacteria and do not have excessive 1 of combining target bacterium to resist and all can close with 2 resistive connections of magnetic bead surfaces, still mix.
4) the magnetic bead suspension with enrichment is added on the solid phase carrier of having fixed 1 anti-monoclonal antibody of the 3rd step making, if exist object bacteria then to form double antibodies sandwich; With aseptic washed with de-ionized water, the magnetic bead that does not then grab object bacteria is just washed off, if there is not object bacteria, then all magnetic beads are all washed off;
5) afterwards, washing with the magnetic bead of eluant, eluent with the double antibodies sandwich on the fixed head, separate the method for magnetic bead with externally-applied magnetic field and fall ion and solvent with aseptic washed with de-ionized water, is exactly the magnetic bead of catching object bacteria if also there is magnetic bead;
6) add nitric acid and sulfuric acid (chloroazotic acid) and carry out nitration reaction, if this part magnetic bead exists, then be converted into ferric ion and ferrous ion by reaction.All food standards, pathogenic bacteria all must not detect, and just indirectly detected target pathogenic bacteria if detected ferric ion this moment.After excessive acid neutralization, adopt Phen absorption photometry, potassium rhodanide colourimetry etc. can detect the amount of ferric ion, thereby can calculate the amount of magnetic bead, by adding the scalar quantity magnetic bead in the amount that to a certain degree quantitatively goes out object bacteria indirectly.
Described nano-probe is
γ-Fe
2O
3The @Au particle namely is the magnetic core shell composite nanometer particle of shell particle with the gold, and nanometer particle size is less than 1000 nanometers.
The evaluation method that finally detects of described object bacteria is based on the concentration that whether detects ferric ion and ferric ion.
The monoclonal antibody of described 1 anti-finger object bacteria, the antibody of 2 anti-finger the 1st antibody can be that monoclonal antibody also can be to resist more.
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 detecting the target pathogenic bacteria indirectly by the check ferric ion.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.Can quantitatively detect to a certain extent.
Embodiment
Example 1
Measure it in the check food samples and whether contain harmful pathogenic bacteria---Listeria.
1. 2 anti-immunomagnetic beads preparations: the anti-IgG monoclonal antibody of the listerial rabbit of 1 anti-employing, 2 is anti-for listerial goat anti-rabbit igg, can be that monoclonal antibody also can be to resist more.
γ-Fe
2O
3Nano particle can adopt the chemical coprecipitation preparation, also can adopt the additive method preparation.After 1mL is diluted
γ-Fe
2O
3With mix with volume 0.1 mol/L sodium citrate, be diluted to 20mL, stir and to add excessive 80 mmol/L NH down
2Drip 0.1% (weight ratio) HAuCl behind the OHHCl solution again
4Solution 2mL reacts 1 h and namely obtains by the washing separation
γ-Fe
2O
3@Au.
Immunity
γ-Fe
2O
3The @Au preparation:
γ-Fe
2O
3@Au concentrates the back and adds excessive goat anti-rabbit igg antibody, after hatching, washing, adds the active room of excessive BSA confining surface, washing, resuspension, be kept at 4 ℃ stand-by.Also can adopt following method: adopt 200 microlitres, 2 mmol disulfide group-succinimide-propionic esters (DSP) that nm of gold is modified (DMSO, dimethyl sulfoxide (DMSO) dilution DSP).Add the Listeria goat anti-rabbit igg antibody, be about to 100
L100
The g/mL goat anti-rabbit igg antibody be fixed on by e that Au goes up and 37 ℃ hatch 45 min.Add 1% bovine serum albumin (BSA), 22 ℃, 1 hour, remaining avtive spot is sealed and drying.
2. 1 anti-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 anti-IgG monoclonal of listerial rabbit 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.The 1st antibody, the anti-IgG of listerial rabbit will be added in the sample to be checked.If have the Listeria in the sample to be checked, will with 1 anti-formation 1 anti-compound.Fully shake after the listerial goat anti-rabbit igg magnetic bead of the 1st the 2nd antibody that makes of step added.Last magnetic force frame separates magnetic bead, adds the suspension that a small amount of aseptic deionized water obtains magnetic bead.At this moment, if the target Listeria is arranged in the sample to be checked, then pass through the interaction of the 2nd antibody and the 1st antibody, thereby catch this compound, reached the purpose of object bacteria enrichment.At this moment, combine 1 anti-compound of object bacteria and do not have excessive 1 of combining target bacterium to resist and all can close with 2 resistive connections of magnetic bead surfaces, still mix.The magnetic bead suspension of this enrichment is added on the prepared 1 anti-fixed head of the 2nd step, then combine in the magnetic bead emulsion listerial magnetic bead further the monoclonal antibody generation specificity on fixed head be combined, form the double antibodies sandwich structure.Just can will do not have in conjunction with listerial magnetic bead flush away with aseptic washed with de-ionized water this moment.Just only combine listerial magnetic bead what fixed head was left.
4. with eluent (methyl alcohol etc.) the listerial magnetic bead that combines on the fixed head is eluted.Last magnetic force frame, the separation magnetic bead also cleans 1-2 time, with ion, solvent flush away.Add nitric acid and sulfuric acid and carry out nitration reaction, if there is magnetic bead, then reaction makes it to be converted into ferric ion and ferrous ion.All food standards, pathogenic bacteria all must not detect, and have detected ferric ion and have just detected the target pathogenic bacteria indirectly.Neutralization reaction is neutral to pH value, adding the reductive agent oxammonium hydrochloride makes ferric ion all be converted into ferrous ion, adopt the Phen absorption photometry can detect the amount of ferric ion, thereby can calculate the amount of magnetic bead, by adding the scalar quantity magnetic bead in the amount that to a certain degree indirectly quantitatively goes out object bacteria.
Embodiment
Example 2
Measure food samples and whether contain harmful pathogenic bacteria Escherichia coli O 157: H7.
1.2 anti-immunomagnetic beads preparation: the anti-IgG monoclonal antibody of rabbit of 1 anti-employing O157:H7,2 anti-ly are the goat anti-rabbit igg of O157:H7, can be that monoclonal antibody also can be to resist more.
γ-Fe
2O
3Nano particle can adopt the chemical coprecipitation preparation, also can adopt the additive method preparation.After 1mL is diluted
γ-Fe
2O
3With mix with volume 0.1 mol/L sodium citrate, be diluted to 20mL, stir and to add excessive 80 mmol/L NH down
2Drip 0.1% (weight ratio) HAuCl behind the OHHCl solution again
4Solution 2mL reacts 1 h and namely obtains by the washing separation
γ-Fe
2O
3@Au.
Immunity
γ-Fe
2O
3The @Au preparation:
γ-Fe
2O
3@Au concentrates the back and adds excessive O157:H7 goat anti-rabbit igg antibody, after hatching, washing, adds the active room of excessive BSA confining surface, washing, resuspension, be kept at 4 ℃ stand-by.Also can adopt following method: adopt 200 microlitres, 2 mmol disulfide group-succinimide-propionic esters (DSP) that nm of gold is modified (DMSO, dimethyl sulfoxide (DMSO) dilution DSP).Add the O157:H7 goat anti-rabbit igg antibody, be about to 100
L100
The g/mL goat anti-rabbit igg antibody be fixed on by e that Au goes up and 37 ℃ hatch 45 min.Add 1% bovine serum albumin (BSA), 22 ℃, 1 hour, remaining avtive spot on the plate is sealed and drying.
2. 1 anti-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 anti-IgG monoclonal antibody of O157:H7 rabbit, 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.The 1st antibody, the anti-IgG of the rabbit of O157:H7 will be added in the sample to be checked.If have O157:H7 in the sample to be checked, will with 1 anti-formation 1 anti-compound.Fully shake after the goat anti-rabbit igg antibody magnetic bead of the 1st the 2nd antibody O157:H7 that makes of step added.Last magnetic force frame separates magnetic bead, adds the emulsion that low amounts of water obtains magnetic bead.At this moment, if target Escherichia coli O 157: H7 is arranged in the sample to be checked, then pass through the interaction of the 2nd antibody and the 1st antibody, thereby catch this compound, reached the purpose of object bacteria enrichment.At this moment, combine 1 anti-compound of object bacteria and do not have excessive 1 of combining target bacterium to resist and all can close with 2 resistive connections of magnetic bead surfaces, still mix.The magnetic bead suspension of this enrichment is added on the prepared 1 anti-fixed head of the 2nd step, and the magnetic bead that then combines Escherichia coli O 157: H7 in the magnetic bead suspension further monoclonal antibody generation specificity on fixed head is combined, and forms the double antibodies sandwich structure.This moment is with aseptic washed with de-ionized water, just can be with not in conjunction with Escherichia coli O 157: the magnetic bead flush away of H7.At the remaining magnetic bead that just only combines Escherichia coli O 157: H7 of fixed head.
With eluent (methyl alcohol etc.) with the Escherichia coli O 157 that combines on the fixed head: the magnetic bead of H7 elutes.Last magnetic force frame, the separation magnetic bead also cleans 1-2 time, with ion, solvent flush away.Add nitric acid and sulfuric acid (chloroazotic acid) and carry out nitration reaction, if there is magnetic bead, then reaction makes it to be converted into ferric ion and ferrous ion.All food standards, pathogenic bacteria all must not detect, and have detected ferric ion and have just detected the target pathogenic bacteria indirectly.Neutralization reaction is neutral to pH value, adding the reductive agent oxammonium hydrochloride makes ferric ion all be converted into ferrous ion, adopt the Phen absorption photometry can detect the amount of ferric ion, thereby can calculate the amount of magnetic bead, by adding the scalar quantity magnetic bead in the amount that to a certain degree indirectly quantitatively goes out object bacteria.
Claims (6)
1. one kind based on γ-Fe
2O
3The food-borne pathogens method for quick that the indirect enrichment immunity of @Au nano particle magnetic separates, its characterization step is as follows:
1) 1 resistive connection that will detect object bacteria and object bacteria closes formation 1 anti-compound;
2) 1 antiantibody of detection object bacteria, i.e. the preparation of 2 anti-specific immunity magnetic beads;
3) with object bacteria 1 anti-monoclonal antibody specific fixing on solid phase carrier;
4) immunomagnetic beads enrichment aimed strain, and separate: the 2 anti-immunomagnetic beadses that the 2nd step was made joined for the 1st step and combine 1 anti-sample to be checked, fully mix concussion, grasp after the object bacteria by applying externally-applied magnetic field, then magnetic bead just is pooled to magnetic field on one side, siphon away supernatant and then can isolate magnetic bead, if in the sample to be checked object bacteria is arranged, object bacteria is at first closed the formation compound with 1 resistive connection, after adding 2 diamagnetic pearls, by 2 anti-and 1 anti-interactions, thereby catch 1 anti-compound, by the enrichment of magnetic bead institute, add the magnetic bead suspension that a small amount of aseptic deionized water then forms object bacteria;
4) the magnetic bead suspension of enrichment being added to the 3rd step has fixed on the solid phase carrier of 1 anti-monoclonal antibody, if exist object bacteria then to form double antibodies sandwich, with aseptic washed with de-ionized water, the magnetic bead that does not then grab object bacteria is just washed off, if there is not object bacteria, then all magnetic beads are all washed off;
5) afterwards, washing with the magnetic bead of eluant, eluent with the double antibodies sandwich on the fixed head, separate the method for magnetic bead with externally-applied magnetic field and fall ion and solvent with aseptic washed with de-ionized water, is exactly the magnetic bead of catching object bacteria if also there is magnetic bead;
6) this part magnetic bead adds analytically pure nitric acid and sulfuric acid (chloroazotic acid) and carries out nitration reaction, makes magnetic bead
γ-Fe
2O
3Change ferric ion and ferrous ion into, in and adopt Phen absorption photometry or potassium rhodanide colourimetry etc. can detect the amount of ferric ion after the excess acid, thereby can calculate the amount of magnetic bead, by adding the scalar quantity magnetic bead in the amount that to a certain degree indirectly quantitatively goes out object bacteria.
2. according to claim 1 based on γ-Fe
2O
3The food-borne pathogens method for quick that the indirect enrichment immunity of @Au nano particle magnetic separates is characterized in that described nano-probe is
γ-Fe
2O
3The @Au particle namely is the magnetic core shell composite nanometer particle of shell particle with the gold, and nanometer particle size is less than 1000 nanometers.
3. according to claim 1 based on γ-Fe
2O
3The food-borne pathogens method for quick that the indirect enrichment immunity of @Au nano particle magnetic separates is characterized in that the evaluation method that finally detects of described object bacteria is the magnetic bead of catching object bacteria by analysis
γ-Fe
2O
3The amount of @Au, the amount of indirect calculation object bacteria.
4. according to claim 1 based on γ-Fe
2O
3The food-borne pathogens method for quick that the indirect enrichment immunity of @Au nano particle magnetic separates is characterized in that by nitration reaction, and the magnetic bead of catching object bacteria is changed into ferric ion, comes quantitative magnetic bead amount and indirect quantitative objective bacterium by the amount that detects ferric ion.
5. according to claim 1 based on γ-Fe
2O
3The food-borne pathogens method for quick that the indirect enrichment immunity of @Au nano particle magnetic separates is characterized in that object bacteria earlier and 1 resistive connection closes the formation compound, and with 2 anti-specific immunity enrichment with magnetic bead, separation, 1 has resisted the signal amplification.
6. according to claim 1 based on γ-Fe
2O
3The food-borne pathogens method for quick that the indirect enrichment immunity of @Au nano particle magnetic separates is characterized in that 1 anti-finger object bacteria monoclonal antibody, and the antibody of 2 anti-finger the 1st antibody can be that monoclonal also can be polyclonal antibody.
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| US11307129B2 (en) | 2020-03-23 | 2022-04-19 | Savannah River Nuclear Solutions, Llc | Automatic gas sorption apparatus and method |
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| US10016745B2 (en) | 2016-06-15 | 2018-07-10 | Savannah River Nuclear Solutions, Llc | Multifunctional nanomaterials and methods of photothermal heating and catalysis using the same |
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