CN104749366A - Method for rapidly detecting pathogenic bacteria - Google Patents
Method for rapidly detecting pathogenic bacteria Download PDFInfo
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- CN104749366A CN104749366A CN201310749891.6A CN201310749891A CN104749366A CN 104749366 A CN104749366 A CN 104749366A CN 201310749891 A CN201310749891 A CN 201310749891A CN 104749366 A CN104749366 A CN 104749366A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/3103—Atomic absorption analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N2021/3129—Determining multicomponents by multiwavelength light
Abstract
The invention provides a method for rapidly detecting pathogenic bacteria. The method is characterized in that firstly, a difunctional composite nanosphere is prepared; quantum dots with different sizes and the composite functional nanosphere are connected with a monoclonal antibody capable of specifically recognizing target bacteria, so as to obtain immune quantum dot probes capable of carrying out antigen-antibody reaction with antigens on the surface of the target bacteria, an immune composite nanosphere probe, and other probes The composite nanosphere with the composite structure can be used as a carrier for immunologically recognizing and separating the pathogenic bacteria and also can be used as a signal enhancer element of the immune quantum dot probe, secondary amplification of a detection signal is realized, and the optical strength can be detected under the corresponding wavelength and multiple target microorganisms can be detected. According to the method, the detection time less than or equal to 2h can be greatly shortened, the sensitivity is improved and multiple pathogenic bacteria can be detected simultaneously through immunoseparation of the magnetic nanoparticles and the signal amplification effect of the quantum dots having dual labels, and the method is suitable for on-site rapid detection of foods and environment samples and can be popularized and applied in grass roots.
Description
Technical field
The present invention relates to a kind of method of quick detection various pathogens, relate to a kind of application of modulating the preparation of dual-functional nanometer ball and detecting fast at multiple food-borne pathogens or rather, also namely described difunctional composite Nano ball, may be used for the aspects such as medical diagnosis, food security, environmental monitoring.Belong to microorganism detection field.
Background technology
The health of the bacterial pathogen serious harm mankind.Food-borne pathogenic microorganism is of a great variety, lacks sensitive, convenient, special Fast Detection Technique, is the one of the main reasons that food security cannot obtain effective guarantee.Therefore develop for quick, sensitive, the reliable detection method of pathogenic bacteria and scene, portable detecting instrument, be food security and national security ensure in the urgent need to.
Select training method to detect the existence of pathogenic bacteria with traditional microorganism, although result is reliable, be regarded as the goldstandard of microorganism detection.But traditional culture of microorganism length consuming time, complex steps, the multiple nutrient culture media of needs and reagent, cannot meet the active demand that some accidents of society detect fast to microorganism field.Therefore arise at the historic moment based on molecular biological various detection method.These methods sum up and mainly contain three classes: the detection that (1) reacts based on chain polymerization.The method is responsive, accurate, quick, alternative pathogeny detection; (2) based on immunoreactive detection.These class methods are economical and practical, favorable reproducibility, and fast, the reaction principle of the commercially produced product that current most of food microorganisms detect fast is based on sandwich immunoassay, and namely immune complex comprises sessile antibody, target bacteria and two anti-labels.(3) biology sensor.It is to biological, biological derivative is relevant analysis device, associating or integrated physicochemical transducer or changes micro-system.Compared with traditional detection method, biology sensor has that volume is little, cost is low, highly sensitive, selectivity and antijamming capability strong and respond the advantages such as fast, being with a wide range of applications in food safety detection, is an important development trend of food safety detection.
In recent years, Many researchers has done a lot of useful exploration based on immunology principle in exploitation pathogenic microbes detect new method and new unit.A kind of quick, overdelicate original position immune analysis method of the based optical Development of Nanometer Material that Tan seminar (Proc.Natl.Acad.Sci.2004) uses antibody to connect, this method does not need amplification and enrichment just can reach detection single bacterium.Pividori seminar (Anal.Chem.2009) utilization in 2009 constructs a kind of Magnetic Sensor by graphite-epoxy compound, by the method for immunity while pathogenic bacteria are caught in enrichment, salmonella can be detected by Electrochemical Detection or double-tagging PCR.This new method has sensitive, fast advantage.But these methods are all confined to detect a kind of pathogenic bacteria in once analyzing, and in testing process, need expensive instrument and equipment and comparatively high detection expense.
The method simultaneously detecting various bacteria in an analytic process more can meet the demand of detection pathogenic bacteria quick, sensitive, special at present, effectively can reduce time and cost-saving.The method that Recent study person have developed multiple multivariate analysis detects 2-3 kind pathogenic bacteria simultaneously, detects various pathogens as utilized multiplex PCR in once analyzing; DNA microarray technology based on nucleic acid hybridization principle can detect various pathogens; Utilize immunochemistry mode to comprise Enzyme Linked Immunoadsorbent Assay, immune Magneto separate, use the specific antibody of organic dyestuff mark bacterium to carry out fluorescence analysis etc.Especially to have particle diameter little for magnetic-particle, and specific surface area is large, and there are many dangling bonds on surface, can give the advantages such as its multiple reactive function base in surface by copolymerization, surface modification; Biocompatibility is good simultaneously, is conducive to the fixing of biomolecule (as: enzyme, nucleic acid, antibody etc.); Be easy to assemble rapidly under magnetic fields, so be widely used in field of bioanalysis.
It is reported, once someone have developed a kind of DNA microarray technology, and it can detect several bacterium simultaneously, but DNA array is confined to the structure of some arrays more.The people such as Magliulo (J.Agric.FoodChem.2007) have developed a kind of simple and quick compound sandwich enzyme immunoassay method, can be used to detect four kinds of bacteriums simultaneously.They use 96 hole polystyrene titer plate, comprise four sub-holes in each hole, four kinds of corresponding monoclonal antibodies of bacterium are fixed on sub-hole.After adding testing sample, bacterium can specificly be combined on corresponding sub-hole, add the potpourri of horseradish peroxidase (HRP) labeled monoclonal antibody corresponding to four kinds of bacteriums subsequently, just can detect HRP activity by the low photoelectronic imaging device of a kind of chemiluminescence.This method is simple and quick, for the detectability of often kind of bacterium all 10
4-10
5cFU/mL.Tan seminar (Bioconjugate Chem.2007) has developed a kind of new method can monitoring complicated pathogenic bacteria sample based on silica fluorescent nano particle, uses colorful Fluorescence Resonance Energy to shift and is no more than 30min detection time.By changing the ratio being embedded in the inner three kinds of dyestuffs of silica nano material, obtain the nano particle sending unique color under a kind of single excitation wavelength.Then combine to the monoclonal antibody of three kinds of pathogenic bacteria and identify corresponding pathogenic bacteria.This method is fast sensitive, and specificity is good, but the preparation method of this nano particle has limited to and can only detect three kinds of pathogenic bacteria, and the consumption of nano particle very high (particle/bacterium=10000/1).Within 2006, Durst seminar (Talanta2006) the array immunization analytical approach delivered based on Protein G-lipid nanometer vesica detects three kinds of pathogenic bacteria (enterohemorrhagic Escherichia coli, salmonella, Listeria Monocytogenes) simultaneously.In pure culture liquid, the detectability of three kinds of bacteriums is respectively 100,500 and 1.2 × 10
5cFU/mL, and in mixed-culture medium, be 3.1 × 10 respectively
3, 7.8 × 10
4, 7.9 × 10
5cFU/mL.
The detection utilizing quantum dot to carry out pathogenic bacteria is also a class detection method emerging in recent years.Quantum dot is also known as semiconductor nanocrystals body, and be a kind of semiconductor nanoparticle that can accept exciting light generation fluorescence be made up of II-VI race or III-group Ⅴ element, its particle diameter is generally about 1-100nm.Because it has unique quantum size effect and surface effect, show excellent spectral signature and photochemical stability.Quantum dot is utilized to carry out fluorescence labeling, compare traditional organic dye molecule and there is many advantages, it is characterized by: the excitation spectrum of (1) quantum dot is wider and in continuous distribution, and emission spectrum width is narrow and be symmetric, can reduce spectra overlapping, make to distinguish multi-fluorescence group becomes possibility simultaneously.Due to its Color tunable, namely the quantum dot of different size can be sent the light of different colours by the optical excitation of single wavelength, its emission wavelength is not from 400nm-2 μm etc., may be used for structure, to detect multi-component fluorescence probe analytical system quantum dot fluorescence probe fluorescence efficiency high simultaneously, photochemical stability is strong, the most frequently used organic dyestuff rhodamine 6G of fluorescence intensity ratio is high more than 20 times, and stability is its more than 100 times.(2) good biocompatibility.Be combined with various biomolecules by the surface of quantum dot, several functions group can be obtained, make biochemical analysis more flexible.(3) emitting semiconductor quanta point material has good nonlinear optical property, can carry out deep non-invasive mark by probe.Based on antigen-antibody combination principle, the quantum dot fluorescence antibody probe that streptavidin is modified such as Hahn in 2005 detects O157: H7, its detection sensitivity will exceed two orders of magnitude than using common organic fluorescent dye probe, and the fluorescence radiation time reaches a few hours, the several seconds being much better than common dye is luminous.Simultaneously due to the amplification of streptavidin-biotin system, further increase detection sensitivity, achieve single celled detection.Hahn in 2008 etc. again further adopt Flow Cytometry in suspension pathogenic O157: H7 and non-pathogenic Escherichia coli detect.The people such as Li in 2006 utilize the quantum dot-labeled of two kinds of different sizes, and binding immunoassay magnetic bead detects the carrying out of two kinds of pathogenic bacteria simultaneously.Although there is a lot of new method in various pathogens context of detection, in how improving detection sensitivity and shortening detection time, remain problem demanding prompt solution.
Usually, composite nanostructure refers to the function nano material comprising two or more, it represent the multifunctional nano system that a class is important.Quantum dot and magnetic nanoparticle being combined into composite nanostructure not only can stop superparamagnetic nanoparticle to be assembled, the bio-toxicity of quantum dot can also be alleviated, have the premium properties of quantum dot and magnetic nanoparticle simultaneously concurrently, thus be expected to for the sensitive quick detection of various pathogens provides new thinking and approach.The present invention attempts to use for reference above-mentioned thinking, provides a kind of method of quick detection various pathogens.
Summary of the invention
The object of the present invention is to provide a kind of method of quick detection various pathogens, it is the preparation of difunctional composite Nano ball that described method comprises, described difunctional composite Nano ball is the quantum dot and the magnetic nanoparticle that first adopt silicon dioxide to embed different size simultaneously, builds the multiple composite Nano ball with different optical characteristic and superparamagnetism.Then, respectively the quantum dot of different size is connected with the monoclonal antibody with specific properties identification object bacteria with Composite Double function nano-ball after difunctional composite Nano ball surface modification, obtains severally to carry out the immune quantum dot probe of antigen-antibody reaction and immune composite Nano talent scout pin with object bacteria surface antigen.The nanosphere probe of this immune composite structure both can be separated the carrier of various pathogens as Immune discrimination, again can as the signal enhancer of immune quantum dot probe, the secondary realizing detection signal amplifies, and just can realize the detection of various pathogens (objective microbe) by detecting its optical strength under respective wavelength.The method passes through the amplification of signal effect of magnetic nanoparticle immunity separation and quantum dot " dual label ", greatly can shorten detection time, improves sensitivity, and detects while realizing various pathogens.Be suitable for food, the field quick detection of environmental sample and basic unit's popularization and application.
The present invention relates to a kind of preparation of modulating dual-functional nanometer ball and the application detected fast at multiple food-borne pathogens thereof.The syntheti c route of immunity composite Nano talent scout pin as shown in Figure 1.Concrete be exactly the quantum dot of certain size and Superparamagnetic Iron Oxide nano particle are embedded to obtain difunctional composite Nano ball simultaneously, then surface modification is carried out, it is the functional group utilizing difunctional composite Nano ball surface, with coupling agent, difunctional composite Nano ball is connected with the corresponding antibody of target pathogenic bacteria, obtains a kind of immune composite Nano talent scout pin with optical property; In like manner, change the size of quantum dot, use same method to obtain the immune composite Nano talent scout pin of other optical property.
Then antibody corresponding to described several frequently seen pathogenic bacteria for the quantum dot of different size is connected and obtain immune quantum dot probe, when connection antibody, note the size one_to_one corresponding with embedded quantum dots in difunctional composite Nano ball.
By described immune composite Nano probe mixing, add bacterium liquid to be measured, immunity Magneto separate object bacteria not of the same race, not isoimmunization quantum dot probe is added after buffer solution, carry out identification marking, by the characteristic absorption peak of different size immunity quantum dot probe, and the secondary singal amplification of corresponding size quantum dot in immune composite Nano probe, just can realize the detection of four kinds of bacterium to be measured.Testing process as shown in Figure 2.The method modulates by this immune Magneto separate that dual-functional nanometer ball can realize the also namely described object bacteria of bacterium to be measured or pathogenic bacteria on the one hand, specifically tested multiple food-borne bacterium uses four kinds of immune composite Nano probes, and their transmitted wave is penetrated and is respectively 481nm, 518nm, 565nm and 610nm; Also namely respectively enterohemorrhagic sun bacillus antibody, antibodies toward salmonella, Listeria Monocytogenes antibody and Campylobacter spp antibody four kinds of pathogenic bacteria and corresponding quantum dot are connected to form four kinds of quantum dot probe.The secondary realizing optical signalling on the other hand amplifies, and significantly improves the sensitivity (10 of pathogenic microbes detect
2cfu/mL), greatly shorten detection time (≤2 hours), and detect while realizing plurality of target pathogenic bacteria.
Wherein, 1) build several dual-functional nanometer ball simultaneously with magnetic and different optical performance, i.e. the quantum dot of embedded magnetic nano particle and different size in silica, obtain the difunctional composite Nano ball that size uniformity, monodispersity are good.The preparation method of described difunctional composite Nano ball, it comprises the following steps:
(1) water, ethanol, ammoniacal liquor mix according to a certain percentage, and upper limit volume ratio is 33:16:1, and lower limit volumetric ratio is 20:16:15;
(2) quantum dot of magnetic nanoparticle and certain size is added;
(3) (upper limit volume ratio is 4:21 to add the mixed liquor of tetraethoxysilance and ethanol; Lower limit volumetric ratio is 1:24), stirring reaction, generates nanosphere;
(4) Magneto separate and ethanol purge, vacuum drying, the range of size of gained composite Nano ball is at 50nm-600nm.
2) utilize difunctional composite Nano ball to detect the method for food-borne pathogens fast, comprise the following steps:
(1) several dual-functional nanometer ball simultaneously with magnetic and different optical performance is built; Build several immune dual-functional nanometer talent scout pin afterwards, namely respectively the monoclonal antibody of specific recognition target food-borne pathogens is connected with dual-functional nanometer ball;
(2) build immune quantum dot probe, namely respectively the monoclonal antibody of specific recognition target food-borne pathogens and corresponding quantum dot are connected to form immune quantum dot probe; The antibody marked in the antibody that this probe marks, quantum dot size and immune composite Nano ball, the size one_to_one corresponding of embedded quantum dots, to form similar sandwich structure with immune composite Nano talent scout pin, target food-borne pathogens;
3) sandwich immunoassay is adopted to detect target food-borne pathogens.Concrete steps comprise:
Step a): the pre-service of sample
Carry out corresponding pre-service according to the character of different sample, be prepared into suitable sample solution 1mL-5mL.
Step b): the enrichment of target pathogenic bacteria
Several immune dual-functional nanometer talent scout pin is joined in sample solution, hatches altogether, fully after reaction, Magneto separate removing supernatant, buffer solution twice, to reach the object removing miscellaneous bacteria, thus obtains the buffer solution comprising target pathogenic bacteria and nanosphere probe.
Step c): immune quantum dot probe mark
A certain amount of corresponding immune quantum dot probe is added in sample described in step b), hatch altogether, after abundant reaction, Magneto separate removing supernatant, buffer solution twice, unnecessary quantum dot probe can be removed, obtain the buffer solution forming similar sandwich structure (quantum dot probe-target pathogenic bacteria-nanosphere probe).
Step d): fluorometric assay
Solution described in step c) is put in fluorescence spectrophotometer, by the intensity of optical detection characteristic absorption peak under respective wavelength, thus realizes the detection of multiple bacterium to be measured.
Accompanying drawing explanation
The syntheti c route schematic diagram of the immune composite Nano probe of Fig. 1 tetra-kinds;
Fig. 2 detects the detection schematic diagram of four kinds of pathogenic bacteria based on the identification of different size immunity quantum dot probe, immune composite Nano probe Magneto separate and secondary singal amplification simultaneously.
Embodiment
Below by the description of specific embodiment, set forth substantive distinguishing features of the present invention and progress significantly further, but the present invention is only confined to described embodiment by no means.
The preparation of the difunctional silicon-based nano ball of embodiment 1: four kind of different optical character
In the present invention, dual-functional nanometer ball passes through improvement
method prepares.Specifically under certain density ammonia-catalyzed, ethyl orthosilicate to be hydrolyzed and to obtain in ethanolic solution.The dual-functional nanometer ball meeting testing requirement is obtained by regulating the relative concentration of ammoniacal liquor, ethyl orthosilicate, ethanol, water, magnetic nanoparticle and quantum dot.Add the quantum dot (as emission wavelength 481nm, 518nm, 565nm and 610nm) with different optical characteristic, thus change the optical property of difunctional composite Nano ball, the dual-functional nanometer ball obtaining four kinds of different optical character (is labeled as nanosphere
481, nanosphere
518, nanosphere
565, nanosphere
610).A typical building-up process is as follows:
24.75mL water, 16.25mL ethanol and 9.0mL ammoniacal liquor fully mix, and then add 20 μ L superparamag-netic iron oxides and 20 μ L cadmium telluride quantum dots, then add rapidly the mixed liquor of 4.5mL ethyl orthosilicate and 45.5mL ethanol, stirring reaction 2 hours, namely generates nanosphere.By Magneto separate and ethanol purge three times, dried in vacuo overnight, obtains dual-functional nanometer ball.
Embodiment 2: the structure of four kinds of immune dual-functional nanometer talent scout pins of catching enterohemorrhagic Escherichia coli (E.coli O157:H), salmonella (Salmonellae), Listeria Monocytogenes (Listeria), Campylobacter spp (Campylobacter) respectively
First above-mentioned four kinds of difunctional composite Nano balls carry out surface modification, and be specifically dissolved in 50mL pyridine by 0.5g nanosphere, add 0.2g succinic anhydride and 0.02g4-dimethylamino naphthyridine, stirring at normal temperature is spent the night, Magneto separate, cleans twice respectively with methyl alcohol and water.
Then respectively by enterohemorrhagic Escherichia coli antibody, antibodies toward salmonella, Listeria Monocytogenes antibody and Campylobacter spp antibody and above four kinds of dual-functional nanometer balls covalently bound, obtain four kinds of immune dual-functional nanometer talent scout pins and (be labeled as nanosphere
481-E, nanosphere
518-S, nanosphere
565-L, nanosphere
610-C).Its operation steps is as follows:
Get 500 μ g nanospheres in 1.5mL centrifuge tube, add 1mL cleaning buffer solution, mix abundant washing, after Magneto separate washing twice, be resuspended in 250 μ L2-(N-morpholinyl) ethyl sulfonic acid, add 500 μ g1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and 750 μ g N-hydroxy-succinamides more respectively, 37 DEG C activate 15 minutes, then with MES cleaning twice, the antibody of 50 μ g is added after resuspended, room temperature reaction 2h, by antibody coupling in nanosphere surface, obtains immunonanospheres.To wash after coupling magnetic bead 2 times with PBS, add 50 μ l and contain in the phosphate buffer (pH=7.4) of 1% bovine serum albumin(BSA), be stored in 4 DEG C of refrigerators stand-by.
Embodiment 3: the structure of four kinds of immune quantum dot probe marking enterohemorrhagic Escherichia coli (E.coli O157:H), salmonella (Salmonellae), Listeria Monocytogenes (Listeria), Campylobacter spp (Campylobacter) respectively
Respectively the monoclonal antibody of specific recognition target food-borne pathogens enterohemorrhagic Escherichia coli antibody, antibodies toward salmonella, Listeria Monocytogenes antibody and Campylobacter spp antibody and corresponding quantum dot are connected to form four kinds of immune quantum dot probe and (are labeled as quantum dot
481-E, quantum dot
518-S, quantum dot
565-L, quantum dot
610-C).The antibody marked in the antibody that this probe marks, quantum dot size and immune composite Nano ball, the size one_to_one corresponding of embedded quantum dots, can form similar, target food-borne pathogens with immune composite Nano talent scout pin and form similar sandwich structure.Its operation steps is as follows:
Get 500 μ L quantum dots in 1.5mL centrifuge tube, add 1mL cleaning buffer solution, mix abundant washing, after centrifuging washing twice, be resuspended in 250 μ L2-(N-morpholinyl) ethyl sulfonic acid, add 500 μ g1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and 750 μ g N-hydroxy-succinamides more respectively, 37 DEG C activate 15 minutes, then with MES cleaning twice, the antibody of 50 μ g is added after resuspended, room temperature reaction 2h, by antibody coupling in nanosphere surface, obtains immunonanospheres.To wash after coupling magnetic bead 2 times with PBS, add 50 μ l and contain in the phosphate buffer (pH=7.4) of 1% bovine serum albumin(BSA), be stored in 4 DEG C of refrigerators stand-by.
Embodiment 4: detect four kinds of target pathogenic bacteria in food based on difunctional immunonanospheres fast: enterohemorrhagic Escherichia coli (E.coli O157:H), salmonella (Salmonellae), Listeria Monocytogenes (Listeria), Campylobacter spp (Campylobacter)
A) pre-service of milk sample
By centrifugal for milk (3000 turns, 5 minutes), remove supernatant, add the phosphate buffer (pH=7.4) of 1-10mL.
B) enrichment of target pathogenic bacteria
By four kinds of immune dual-functional nanometer talent scout pin nanospheres
481-E, nanosphere
518-S, nanosphere
565-L, nanosphere
610-C mixed in equal amounts, joins in sample solution, hatches altogether, and at turbine mixer mixing 1-5 minute, Magneto separate, careful removing supernatant, washes twice with phosphate buffer (pH=7.4), then adds the phosphate buffer (pH=7.4) of 1-10mL.
C) immune quantum dot probe mark
Four kinds of immune quantum dot probe quantum dots are added above-mentioned stating in sample
481-E, quantum dot
518-S, quantum dot
565-L, quantum dot
610-C, at turbine mixer mixing 1-5 minute, Magneto separate, careful removing supernatant, obtains nanosphere
481-E/ enterohemorrhagic Escherichia coli/quantum dot
481-E, nanosphere
518-S/ salmonella/quantum dot
518-S, nanosphere
565-L/ Listeria Monocytogenes/quantum dot
565-L, nanosphere
610-C/ Campylobacter spp/quantum dot
610-C four kinds of potpourris, wash twice with phosphate buffer (pH=7.4).
D) fluorometric assay
Above-mentioned solution is put in fluorescence spectrophotometer, by the intensity of (481nm, 518nm, 565nm and 610nm) optical detection characteristic absorption peak under respective wavelength, thus realizes the detection of multiple bacterium to be measured.
Claims (9)
1. one kind is detected the method for various pathogens fast, it is characterized in that it being first the preparation of difunctional composite Nano ball, described difunctional composite Nano ball is the quantum dot and the magnetic nanoparticle that adopt silicon dioxide to embed different size simultaneously, builds the multiple difunctional composite Nano ball with different optical characteristic and superparamagnetism; Then, after difunctional composite Nano ball surface modification, respectively the dual-functional nanometer ball that the quantum dot of different size and magnetic nano-balls are formed is connected with the monoclonal antibody with specific recognition object bacteria, obtains severally to carry out the immune quantum dot probe of antigen-antibody reaction and immune composite Nano talent scout pin with object bacteria surface antigen; Finally by the detection detecting its optical strength and just can realize various pathogens under respective wavelength.
2., by method according to claim 1, it is characterized in that:
A) magnetic nanoparticle embedded in the difunctional composite Nano ball described in is through the Fe of finishing
3o
4nano particle or γ-Fe
2o
3nano particle; The quantum dot of embedding is through cadmium telluride quantum dot or the CdSe quantum dots of finishing, and described quantum dot has several different optical characteristics and size, thus forms several difunctional composite Nano ball with different optical character;
The preparation of the difunctional composite Nano ball b) is by through improving
method, concrete steps comprise:
(1) water, ethanol, ammoniacal liquor mix according to a certain percentage; Upper limit volume ratio is 33:16:1, and lower limit volumetric ratio is 20:16:15;
(2) magnetic nanoparticle and quantum dot is added;
(3) add the mixed liquor of tetraethoxysilance and ethanol, stirring reaction, generate nanosphere; The upper limit volume ratio of mixed liquor is 4:21, and lower limit volumetric ratio is 1:24;
(4) Magneto separate and ethanol purge, vacuum drying.
3., by the method described in claim 1 or 2, it is characterized in that described difunctional composite Nano ball range of size is 50-600nm.
4. by method according to claim 1, it is characterized in that difunctional composite Nano ball surface modification is the functional group utilizing difunctional composite Nano ball surface, with coupling agent, difunctional composite Nano ball is connected with the corresponding monoclonal antibody of target pathogenic bacteria, obtains the immune composite nano-line probe with certain optical property.
5., by the method described in claim 1 or 4, it is characterized in that adding the quantum dot with different optical characteristic, to change the optical property of difunctional composite Nano ball.
6. by the method described in claim 1 or 4, it is characterized in that described different immune composite Nano probe mixing, add bacterium liquid to be measured, immunity Magneto separate different target bacterium adds different immune quantum dot probe after buffer solution, carry out identification marking, by the characteristic absorption peak of different size immunity quantum dot probe, realize the detection of bacterium to be measured, specifically tested multiple food-borne bacterium uses four kinds of immune composite Nano probes, and their transmitted wave is penetrated and is respectively 481nm, 518nm, 565nm and 610nm; Also namely respectively enterohemorrhagic sun bacillus antibody, antibodies toward salmonella, Listeria Monocytogenes antibody and Campylobacter spp antibody four kinds of pathogenic bacteria and corresponding quantum dot are connected to form four kinds of quantum dot probe.
7. by method according to claim 6, it is characterized in that described immune composite Nano talent scout pin is separated the carrier of various pathogens as Immune discrimination or strengthens as the signal of immune quantum dot, the immune Magneto separate realizing bacterium to be measured and the secondary realizing optical signalling amplify.
8., by the method described in claim 1 or 6, it is characterized in that described method comprises:
(1) build several there is the dual-functional nanometer ball of magnetic and different optical performance simultaneously after, build four kinds of immune dual-functional nanometer talent scout pins, namely respectively the monoclonal antibody of specific recognition target food-borne pathogens be connected with dual-functional nanometer ball;
(2) build immune quantum dot probe, namely respectively the monoclonal antibody of specific recognition target food-borne pathogens and corresponding quantum dot are connected to form immune quantum dot probe; The antibody marked in the antibody that probe marks, quantum dot size and immune composite Nano ball, the size one_to_one corresponding of embedded quantum dots, to form similar sandwich structure with immune composite Nano talent scout pin, target food-borne pathogens;
(3) sandwich immunoassay is adopted to detect target food-borne pathogens; Detecting step comprises:
Step a): the pre-service of sample
Carry out corresponding pre-service according to the character of different sample, be prepared into suitable sample solution 1mL-5mL;
Step b): the enrichment of target pathogenic bacteria
Several immune dual-functional nanometer talent scout pin is joined in sample solution, hatches altogether, fully after reaction, Magneto separate removing supernatant, buffer solution twice, to reach the object removing miscellaneous bacteria, thus obtains the buffer solution comprising target pathogenic bacteria and nanosphere probe;
Step c): immune quantum dot probe mark
A certain amount of corresponding immune quantum dot probe is added in sample described in step b), hatch altogether, after abundant reaction, Magneto separate removing supernatant, buffer solution twice, unnecessary quantum dot probe can be removed, obtain the buffer solution of the similar sandwich structure forming quantum dot probe-target pathogenic bacteria-nanosphere probe;
Step d): fluorometric assay
Solution described in step c) is put in fluorescence spectrophotometer, by the intensity of optical detection characteristic absorption peak under respective wavelength, thus realizes the detection of multiple bacterium to be measured.
9., by the method described in claim 1,2,4,6 or 7, it is characterized in that the sensitivity of described method detection pathogenic bacteria is 10
2cfu/mL, detection time≤2 hours, and detect while realizing plurality of target pathogenic bacteria.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105527427A (en) * | 2016-01-19 | 2016-04-27 | 南昌大学 | Method for fast detecting Listeria monocytogenes |
| WO2017036359A1 (en) * | 2015-08-28 | 2017-03-09 | 北京纳米能源与***研究所 | Device and method for measuring mechanical property of cell |
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| CN111989571A (en) * | 2018-04-23 | 2020-11-24 | 杰宜斯科技有限公司 | Biosensor comprising linker material and quantum dot beads and target antigen detection method using the same |
| CN111989571B (en) * | 2018-04-23 | 2024-04-12 | 杰宜斯科技有限公司 | Biosensor comprising linker material and quantum dot beads and target antigen detection method using the same |
| CN109856389A (en) * | 2019-01-08 | 2019-06-07 | 阜阳师范学院 | The preparation of magnetic nanoparticle based on quantum dot and its application in the detection of polynary food-borne pathogens |
| CN113372439A (en) * | 2021-06-09 | 2021-09-10 | 中国农业大学 | Manganese metal organic framework biological composite material and application thereof in detection of food-borne pathogenic bacteria |
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