CN105372279A - CoFe2O4 nanoprobe based NMR method for rapid detection of virus - Google Patents
CoFe2O4 nanoprobe based NMR method for rapid detection of virus Download PDFInfo
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- CN105372279A CN105372279A CN201510839528.2A CN201510839528A CN105372279A CN 105372279 A CN105372279 A CN 105372279A CN 201510839528 A CN201510839528 A CN 201510839528A CN 105372279 A CN105372279 A CN 105372279A
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Abstract
A CoFe2O4 nanoprobe based NMR method for rapid detection of a virus belongs to the technical field of food safety rapid detection. The invention is dependent on a nuclear magnetic resonance detection method applicable to virus in the samples, and uses the effect of paramagnetic property of the CoFe2O4 nanoprobe on relaxation time of nuclear magnetic resonance to detect virus in the sample. Different specific corresponding relationship is as below: the CoFe2O4 nanoprobe under certain conditions shows linear relationship, namely when the content of CoFe2O4 nanoprobe is higher, the spin-lattice relaxation time value of the sample is smaller, so as to quantitatively detect the virus in a certain range. The method can be used for rapid detection of virus in samples, therefore the method can be used for rapid screening of a large number of samples to be tested.
Description
Technical field
The present invention relates to a kind of method for quick of virus, is a kind of based on CoFe
2o
4the NMR virus method for quick of nano-probe.
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.CoFe
2o
4nano-probe is the CoFe of antibody modification modification
2o
4nano particle, CoFe
2o
4owing to having Elements C o, there is high saturation and magnetic intensity, particle diameter little to a certain extent time, there will be superparamagnetic characteristic.Therefore, the impact of its particle on the nuclear magnetic resonance spin-Lattice Relaxation Time of surrounding water molecules is very large.This namely this kind of material as the mechanism of contrast preparation.Very micro-nano Co Fe
2o
4nuclear magnetic resonance spin-the Lattice Relaxation Time of water will be caused to decline to a great extent, just can be clear and definite by blank, cause owing to there is this material.Therefore CoFe can be built
2o
4nano-probe biology sensor, detects from the angle of magnetic resonance.
Its main step principle: the specific C oFe 1) detecting target viral
2o
4the preparation of nano-probe.Commercially CoFe
2o
4nano material, also can prepare nano level CoFe by other chemical synthesis process
2o
4.Use silane coupling agent or PEG(polyglycol), modify and improve biocompatibility, its general formula is: Y (CH
2) nSiX
3.Herein, n is 0-3; X is hydrolyzable group; Y is organo-functional group.X is chloro, methoxyl, ethoxy, acetoxyl group etc. normally, generates silanol (Si (OH) during the hydrolysis of these groups
3), and be combined with dead matter, form siloxane.Y is vinyl, amino, epoxy radicals, methacryloxy, sulfydryl.These reactive groups can react with organic substance and combine.Therefore, by using silane coupling agent, can erect " molecular bridge " between dead matter and the interface of organic substance, link together the material of two kinds of character great disparities the performance improving compound substance and the effect increasing bonding strength.Can realize surface-functionalized by modified specificity antibody again, form specific probe, then close unnecessary avtive spot.2) adopt conventional method that the specific monoclonal antibody of target viral to be checked is fixed on ELISA Plate surface, and unnecessary avtive spot is closed, for subsequent use.Monoclonal antibody is fixing in ELISA Plate, and this has been the mature technology of industry.3) measuring samples is added to the 2nd) ELISA Plate securing target viral monoclonal antibody that obtains of step hatches 20mins, if there is target viral in sample, then target viral can be combined by the monoclonal antibody in ELISA Plate, and by washing, then target viral is fixed in ELISA Plate.4) by the 1st) CoFe of the made antibody modification of step
2o
4nano-probe adds the 3rd) in the ELISA Plate that obtains of step, now, if ELISA Plate has target viral, by the specific reaction of probe, then probe can with the target viral generation specific binding in ELISA Plate, forming double antibodies sandwich structure, now by aseptic washed with de-ionized water, just can wash away not having the probe combined.The probe just only having combining target virus remaining in ELISA Plate.If ELISA Plate does not have target viral, then so probe is all washed away.5) add quantitative eluent (30% methyl alcohol) probe combining target viral in ELISA Plate is eluted.The solution obtained, measures the spin-lattice relaxation time of solution by nuclear magnetic resonance analyser (NMR20, Niu Mai company).Above-mentioned steps is repeated as blank using aseptic deionized water, the spin-lattice relaxation time that solution records is compared with blank, and there were significant differences, illustrates in solution and has probe to exist, thus have target viral in interpret sample, the amount of probe and the drop-out value of spin-lattice relaxation time proportional.The bright probe of more speaking more declined is more, thus side light target viral is more, is quantitatively detected the number of target viral in sample by mark-on checking.
This process employs ELISA Plate technique for fixing, double antibodies sandwich technology, nuclear magnetic resonance technique, the design of its step, mainly for effective separation of probe when operating excessive, makes testing result accurate.For adopting, the virus of double antibodies sandwich technology separation is applicable, for the virus that some are special, can not to adopt double antibodies sandwich, then inapplicable.
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 virus.The method depends primarily on the pretreatment time of sample, and magnetic resonance detection only needs a few minutes.Therefore, the positive-selecting of extensive measuring samples can be done by the method, can quantitatively detect to a certain extent.
Summary of the invention
The object of this invention is to provide a kind of based on CoFe
2o
4the NMR virus method for quick of nano-probe, for evaluating various different sample.The method is a kind of objective method effectively detecting virus in sample, thus greatly reduces the screening time of sample virus to a certain extent.
A kind of based on CoFe
2o
4the NMR virus method for quick of nano-probe, nuclear magnetic resonance analyser, to the response sensibility of paramagnetic material, proposes NMR (Nuclear Magnetic Resonance) relaxation Parameters variation and CoFe
2o
4the correlation metric of nano-probe content.
What the method depended on foundation can be used for harmful virus specific C oFe in sample
2o
4the separation of nano-probe, from the angle of NMR (Nuclear Magnetic Resonance) relaxation time variations, detects the harmful virus in sample.Because nuclear magnetic resonance analyser SPIN-LATTICE RELAXATION efficiency and spin-spin relaxation efficiency are to CoFe
2o
4nano-probe is very responsive, namely in deionized water, there is the CoFe of trace
2o
4nano-probe, then the spin-lattice relaxation time of water and spin-spin relaxation will significantly decline.By quantitatively detecting the immunological probe content in sample, thus harmful virus content in sample can be gone out by indirect quantification.The viral level detected and probe content linear correlation, degree of fitting is better.Final with the corresponding relation between immunological probe and virus for tie, determine the viral number in sample.Therefore, the method can do the positive-selecting of extensive measuring samples, can quantitatively detect to a certain extent.
The present invention is achieved by the following technical solutions.
One of the present invention is based on CoFe
2o
4the NMR virus method for quick of nano-probe, step is as follows.
(1) the specific C oFe of target viral is detected
2o
4the preparation of nano-probe.
(2) target viral specific monoclonal antibody is fixed on ELISA Plate surface, and unnecessary avtive spot bovine serum albumin is closed.
(3) measuring samples is added in the obtained ELISA Plate securing target viral monoclonal antibody of step (2) and hatches 20mins, if there is target viral in sample, then target viral can be combined by the monoclonal antibody in ELISA Plate, and by washing, then target viral is fixed in ELISA Plate.
(4) by the CoFe of antibody modification made for step (1)
2o
4nano-probe adds in the ELISA Plate that step (3) obtains, now, if ELISA Plate has target viral, by the specific reaction of probe, then probe can with the target viral generation specific binding in ELISA Plate, forming double antibodies sandwich structure, now by aseptic washed with de-ionized water, just can wash away not having the probe combined.The probe just only having combining target virus remaining in ELISA Plate.If ELISA Plate does not have target viral, then so probe is all washed away.
(5) add quantitative eluent (30% methyl alcohol) probe combining target viral in ELISA Plate is eluted.The solution obtained, measures the spin-lattice relaxation time of solution by nuclear magnetic resonance analyser (NMR20, Niu Mai company).Above-mentioned steps is repeated as blank using aseptic deionized water, the spin-lattice relaxation time that solution records is compared with blank, there were significant differences, illustrate in solution and have probe to exist, thus having target viral in interpret sample, the drop-out value of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is proportional.The bright probe of more speaking more declined is more, thus side light target viral is more, is quantitatively detected the number of target viral in sample by mark-on checking.
Described probe is the CoFe with paramagnetic properties
2o
4nano material, nanometer particle size is less than 200 nanometers.
Described target viral finally detect the change of evaluation method based on the NMR (Nuclear Magnetic Resonance) relaxation time.
Described relaxation time characteristic, refers to spin-lattice relaxation time and spin spin relaxation time.
Beneficial effect of the present invention: the invention provides a kind of method objectively detecting the harmful virus in sample fast, it is characterized in that depending on foundation can be used for detect CoFe
2o
4the magnetic resonance detection method of nano-probe.The method can objectively detect virus in sample effectively, confirms compared to the biological culture of virus, and the method has the advantage detected fast, may be used for the rapid screening of extensive sample.
Embodiment
The present invention will be described further by following examples.
Embodiment 1.
Whether check sample measures it containing virus---bovine viral diarrhea virus.
1, CoFe
2o
4prepared by nano particle: CoFe
2o
4nano particle can be bought from market, CoFe
2o
4nano particle can be bought from market, also can chemically synthesize by list of references.By the CoFe obtained
2o
4with PEG-1500 in mass ratio 1:10 under the dissolving of absolute ethyl alcohol, add in reactor, after sealing, put into baking oven, temperature is adjusted to 90 DEG C, reacts 12 hours; When room temperature is down to by reactor, be generally the 3-4h after closing baking oven.Open reactor, supernatant is gone, stir with absolute ethanol washing; Washed once just carry out once centrifugal, until supernatant clarification.Rotating speed 8000r/min, time 3min; The final products washed are put and naturally dries in an oven, obtain the CoFe that PEG modifies
2o
4nano particle.
Antibody modification: the CoFe taking a certain amount of carboxyl modified
2o
4nano particle, adds EDC and NHS of 2 times of quality, adds each 1mL of PBS of 0.01M.Be placed on blending instrument, rotating speed 15 turns, reaction 45min.) by soak time to sample to carry out 10000r/min centrifugal, time 5min, and then the PBS adding 0.01M to carry out washing centrifugal, repeat 3 times.Centrifugal complete sample is respectively added appropriate anti-bovine viral diarrhea virus antibody, coupling 1h on blending instrument.The 10%BSA confining liquid 45min added after coupling completes.Close after terminating, rotating speed 8000r/min centrifuging 5min, centrifugal with the PBS cleaning of 0.01M, repeat 3 times, then unnecessary antibody, BSA are washed away, finally add PBS and carry out resuspended.The CoFe of the resistance modification of preparation
2o
4nano particle is immunological probe, be kept at 4 DEG C stand-by.
2, monoclonal antibody is fixed: can adopt conventional ELISA Plate fixing means, also can adopt following methods.With clean cover glass 5 × 5mm
2square, coating machine first sprays one deck Cr (2 – 4nm) in order to help fixing gold.Be used in surface sputtering again and spray one deck nm of gold, then adopt 200 microlitre 2mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) nm of gold.Add anti-bovine viral diarrhea virus 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, fecal specimens etc. is carried out pre-service, adopt FDA enrichment if desired, sample is filtered, increases the pre-service such as bacterium activation, obtain measuring samples.Measuring samples is added in the ELISA Plate securing specific monoclonal antibody that the 2nd step obtains and hatches 20mins, if there is target viral in sample, then can be combined by the monoclonal antibody in ELISA Plate, by washing, then target viral is fixed in ELISA Plate.
4, by the CoFe of antibody modification made for the 1st step
2o
4nano-probe adds in the ELISA Plate that the 3rd step obtains, and now, if ELISA Plate has target viral, by the specific reaction of immunological probe, then probe with the target viral generation specific binding in ELISA Plate, can form double antibodies sandwich structure.Now by aseptic washed with de-ionized water, just can wash away not having the probe combined.The probe just only combining virus remaining in ELISA Plate.
5, add quantitative eluent (30% methyl alcohol) probe combining virus in the ELISA Plate of the 4th step is eluted.The solution obtained, measures the spin-lattice relaxation time of solution by nuclear magnetic resonance analyser (NMR20, Niu Mai company).Above-mentioned steps is repeated as blank using aseptic deionized water, the spin-lattice relaxation time that solution records is compared with blank, there were significant differences, illustrate in solution and have probe to exist, thus having target viral in interpret sample, the drop-out value of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is proportional.The bright probe of more speaking more declined is more, thus side light virus is more, is quantitatively detected the number of target viral in sample by mark-on checking.
Embodiment 2.
Whether working sample is containing virus---pig parvoviral.
1, CoFe
2o
4prepared by nano particle: CoFe
2o
4nano particle can be bought from market, also can chemically synthesize by list of references.By the CoFe obtained
2o
4with PEG-1500 in mass ratio 1:10 under the dissolving of absolute ethyl alcohol, add in reactor, after sealing, put into baking oven, temperature is adjusted to 90 DEG C, reacts 12 hours; When room temperature is down to by reactor, be generally the 3-4h after closing baking oven.Open reactor, supernatant is gone, stir with absolute ethanol washing; Washed once just carry out once centrifugal, until supernatant clarification.Rotating speed 8000r/min, time 3min; The final products washed are put and naturally dries in an oven, obtain the CoFe that PEG modifies
2o
4nano particle.
Antibody modification: the CoFe taking a certain amount of carboxyl modified
2o
4nano particle, adds EDC and NHS of 2 times of quality, adds each 1mL of PBS of 0.01M.Be placed on blending instrument, rotating speed 15 turns, reaction 45min.) by soak time to sample to carry out 10000r/min centrifugal, time 5min, and then the PBS adding 0.01M to carry out washing centrifugal, repeat 3 times.Centrifugal complete sample is respectively added pig parvoviral monoclonal antibody, coupling 1h on blending instrument.The 10%BSA confining liquid 45min added after coupling completes.Close after terminating, rotating speed 8000r/min centrifuging 5min, centrifugal with the PBS cleaning of 0.01M, repeat 3 times, then unnecessary antibody, BSA are washed away, finally add PBS and carry out resuspended.The CoFe of the resistance modification of preparation
2o
4nano particle is immunological probe, be kept at 4 DEG C stand-by.
2, monoclonal antibody is fixed: can adopt conventional ELISA Plate fixing means, also can adopt following methods.With clean cover glass 5 × 5mm
2square, coating machine first sprays one deck Cr (2 – 4nm) in order to help fixing gold.Be used in surface sputtering again and spray one deck nm of gold, then adopt 200 microlitre 2mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) nm of gold.Add pig parvoviral monoclonal antibody, fix on a glass also 37 C by 100 μ L100 μ g/mL pig parvoviral antibody 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, sample is carried out pre-service, adopt FDA enrichment if desired, sample is filtered, increases the pre-service such as bacterium activation, obtain measuring samples.Measuring samples is added in the ELISA Plate securing large monoclonal antibody that the 2nd step obtains and hatches 20mins, if there is target viral in sample, then can be combined by the monoclonal antibody in ELISA Plate, by washing, then target viral is fixed in ELISA Plate.
4, by the CoFe of antibody modification made for the 1st step
2o
4nano-probe adds in the ELISA Plate that the 3rd step obtains, and now, if ELISA Plate has pig parvoviral, by the specific reaction of immunological probe, then probe with the target viral generation specific binding in ELISA Plate, can form double antibodies sandwich structure.Now by aseptic washed with de-ionized water, just the probe not in conjunction with pig parvoviral can be washed away.The probe just only combining pig parvoviral remaining in ELISA Plate.
5, add quantitative eluent (30% methyl alcohol) probe combining virus in ELISA Plate is eluted.The solution obtained, measures the spin-lattice relaxation time of solution by nuclear magnetic resonance analyser (NMR20, Niu Mai company).Above-mentioned steps is repeated as blank using aseptic deionized water, the spin-lattice relaxation time that solution records is compared with blank, there were significant differences, illustrate in solution and have probe to exist, thus having pig parvoviral in interpret sample, the drop-out value of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is proportional.The bright probe of more speaking more declined is more, thus side light pig parvoviral is more, quantitatively can be detected the number of target viral in sample by mark-on checking.
Claims (3)
1. one kind based on CoFe
2o
4the NMR virus method for quick of nano-probe, its step is as follows:
(1) CoFe of target viral is detected
2o
4the preparation of nano-probe;
(2) by fixing for subsequent use in ELISA Plate of target viral specific monoclonal antibody, and unnecessary avtive spot bovine serum albumin is closed;
(3) be added to by measuring samples in the ELISA Plate obtained by step (2), hatch a period of time, if containing target viral in measuring samples, then can be combined by the monoclonal antibody in ELISA Plate, after aseptic washed with de-ionized water, target viral is fixed in ELISA Plate;
(4) by CoFe obtained for step (1)
2o
4nano-probe suspension is added to step (3) and secures in the ELISA Plate of target viral, if there is target viral, forms double antibodies sandwich, and by aseptic washed with de-ionized water, then the probe do not combined just is washed off; If there is not target viral, then all probes are all washed off;
(5) wash with the probe of eluant, eluent by the double antibodies sandwich in ELISA Plate, if also there is probe is exactly the probe catching target viral;
(6) suspension of the probe of step (5) gained, the relaxation time of carrying out nuclear magnetic resonance measures; Above-mentioned steps is repeated as blank using aseptic deionized water, the relaxation time spin-lattice relaxation time of the suspension recorded compares aseptic deionized water with spin spin relaxation time have remarkable reduction, then illustrate containing probe, thus have target viral in indirect proof sample; The decline of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is proportional, by adding scalar quantity probe and indirect quantification goes out the amount of target viral.
2. according to claim 1 based on CoFe
2o
4the NMR virus method for quick of nano-probe, is characterized in that described probe is nanoscale CoFe
2o
4material, its nanometer particle size is less than 500 nanometers.
3. according to claim 1 based on CoFe
2o
4the NMR virus method for quick of nano-probe, is characterized in that the described NMR (Nuclear Magnetic Resonance) relaxation time, refers to spin-lattice relaxation time and spin spin relaxation time.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0186616A1 (en) * | 1984-11-23 | 1986-07-02 | Schering Aktiengesellschaft | Magnetic particles for diagnostic purposes |
| CN103207273A (en) * | 2013-03-26 | 2013-07-17 | 南昌大学 | Paramagnetic nano Fe-Co alloy probe based quick detecting method for NMR (nuclear magnetic resonance) food-borne pathogenic bacteria |
| CN103217451A (en) * | 2013-03-26 | 2013-07-24 | 南昌大学 | NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Ni-Co alloy probes |
| CN103217448A (en) * | 2013-03-26 | 2013-07-24 | 南昌大学 | NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe probes |
| CN103217450A (en) * | 2013-03-26 | 2013-07-24 | 南昌大学 | NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment |
| CN103217530A (en) * | 2013-03-26 | 2013-07-24 | 南昌大学 | NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Co alloy probe indirect enrichment |
-
2015
- 2015-11-27 CN CN201510839528.2A patent/CN105372279A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0186616A1 (en) * | 1984-11-23 | 1986-07-02 | Schering Aktiengesellschaft | Magnetic particles for diagnostic purposes |
| CN103207273A (en) * | 2013-03-26 | 2013-07-17 | 南昌大学 | Paramagnetic nano Fe-Co alloy probe based quick detecting method for NMR (nuclear magnetic resonance) food-borne pathogenic bacteria |
| CN103217451A (en) * | 2013-03-26 | 2013-07-24 | 南昌大学 | NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Ni-Co alloy probes |
| CN103217448A (en) * | 2013-03-26 | 2013-07-24 | 南昌大学 | NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe probes |
| CN103217450A (en) * | 2013-03-26 | 2013-07-24 | 南昌大学 | NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Ni-Co alloy probe indirect enrichment |
| CN103217530A (en) * | 2013-03-26 | 2013-07-24 | 南昌大学 | NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Co alloy probe indirect enrichment |
Non-Patent Citations (1)
| Title |
|---|
| 陈瑾: "高温多元醇法制备超顺磁CoFe2O4纳米颗粒磁共振造影剂", 《无机材料学报》 * |
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Application publication date: 20160302 |