CN105548551B - A kind of method of quick detection vibrio parahemolyticus - Google Patents

A kind of method of quick detection vibrio parahemolyticus Download PDF

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CN105548551B
CN105548551B CN201610032264.4A CN201610032264A CN105548551B CN 105548551 B CN105548551 B CN 105548551B CN 201610032264 A CN201610032264 A CN 201610032264A CN 105548551 B CN105548551 B CN 105548551B
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nanoparticle
bqy
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vibrio parahemolyticus
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CN105548551A (en
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赖卫华
王景云
刘道峰
邓省亮
山珊
熊勇华
魏华
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Nanchang University
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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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria

Abstract

The invention provides a kind of detection method of quick detection vibrio parahemolyticus.Scheme is by Fe3O4/Ru(bqy)3 2+Nanoparticle enrichment of bacterial, prepare test strips, sample detection.The step of eluting vibrio parahemolyticus from immunomagnetic beads is eliminated, improves capture rate；Eliminate Ru (bqy)3 2+Nanoparticle is sprayed on the step on pad, and immunological response is more homogeneous, and the coefficient of variation is small when quantitatively detecting；Reduce workload and living contaminants probability.Detection scheme sensitivity is very high, stability is fine.

Description

A kind of method of quick detection vibrio parahemolyticus
Technical field
The present invention relates to microorganism detection field, specifically using Fe3O4/Ru(bqy)3 2+Nanoparticle integrated immune magnetic bead is caught Obtain technology and immunochromatography quick detection vibrio parahemolyticus.
Technical background
Vibrio parahemolyticus is one of common food-borne pathogens, and the bacterium is mainly derived from the marine products such as shrimp, fish and shellfish Product.Reported according to national food origin disease monitoring net, having been poisoned by food to China since 1998 as caused by vibrio parahemolyticus is in Ascendant trend, and exceed Salmonella food poisoning and leap to the first, turn into the foodborne bacterial pathogenses of most serious.The human consumption bacterium The marine product of infection can trigger the gastrointestinal diseases such as enterogastritis and septicemia, be China part coastal area food poisoning case in Primary pathogen.
Find vibrio parahemolyticus so far from nineteen fifty, detect both at home and abroad the method for the bacterium still using normal isolation culture method as It is main.The method complex operation, detection cycle length (4-7d), have not adapted to the needs of hygienic emergency detection；ELISA method, PCR method And hexavalent chrome bio-removal is high to vibrio parahemolyticus detection sensitivity, but their longer detection times of needs, costliness Instrument and technical professional.Therefore it is necessary to establish quick, sensitive detection method.
Colloidal gold immuno-chromatography test paper strip with its simple to operate, quick (10-15min), it is accurate the features such as turn into basic unit sieve The important tool of choosing, limited yet with the optical signalling of collaurum, the sensitivity of colloidal gold immuno-chromatography test paper strip is not high, right The test limit of vibrio parahemolyticus is normally no higher than 105CFU/mL, this defect limit it in food and detection of agricultural products Application.Therefore, the sensitivity for improving test strips provides the detection for vibrio parahemolyticus to simple, efficient approach.
The content of the invention
Present invention aims at provide a kind of quick, sensitive, easy vibrio parahemolyticus qualitative and quantitative analysis technology.
Concrete scheme of the present invention is as follows：
A kind of method of quick detection vibrio parahemolyticus, comprises the following steps：
1) preparation of nanoparticle：
A. 0.4-0.8mmolFeCl is added3·6H2O and 0.2-1.6mmol FeCl2·4H2O to 100mL deionized water In, nitrogen is passed through into solution and is heated to 80-120 DEG C, then by 3-7mL 25% NH3·H2O is added in mixed liquor, React 2h；The solid matter for isolating black from reaction solution with permanent magnet is cleaned 3~5 times with high purity water, obtains Fe3O4Nanometer Particle；
B. 12mg Fe are taken3O4Nano-particle is resuspended with the mixed liquor of 1-10mL deionized waters and 20mL ethanol, first slow 0.3-0.9mL NH is added under conditions of stirring4OH solution, then 10-300uL tetraethyl orthosilicates are dissolved in 50uL ethanol solutions In be added dropwise, 12h is reacted at room temperature, in Fe3O4Nanoparticle surface forms layer of silicon dioxide, clear with deionized water solution Wash several times, redissolved in ethanol solution and obtain the Fe of coated with silica3O4Nano-particle；
C. 10-300uL tetraethyl orthosilicate, 20mL absolute ethyl alcohols, 1-10mL deionized waters and 1mL 0.5-3mg/L's Phenanthroline connection ruthenium (Ru (bqy)3 2+) mixing, by the Fe of mixed solution addition 0.5mL coated with silica3O4Nano-particle, finally Add 600-900 μ L NH4OH, 3h is stirred vigorously, obtains Fe3O4/Ru(bqy)3 2+Nanoparticle, cleaned with deionized water standby With；
D. 1mL mercaptopropyl trimethoxysilane is added in 10mL ethanol solution, redissolves Fe with the mixture3O4/ Ru(bqy)3 2+Nanoparticle, after 300rpm stirs 12h at normal temperatures, then 80 DEG C of 300rpm stirring 1h, centrifugation obtain silanization Fe3O4/Ru(bqy)3 2+Nanoparticle；
E. by the Fe of silanization3O4/Ru(bqy)3 2+Nanoparticle is added to comprising 0.06g NaHCO3, 0.08g dodecanes The 50mL of base benzene sulfonic acid sodium salt, 0.05mL styrene, the 0.15mL acrylic acid and 0.5g potassium persulfate solutions aqueous solution, water-bath 70 DEG C, stirred under 200r/min, the Fe of carboxylated is obtained after reaction 5h3O4/Ru(bqy)3 2+Nanoparticle；
2) Fe of 0.5-2mg carboxylated is taken3O4/Ru(bqy)3 2+Nanoparticle add 1mL coupling buffers in, regulation pH to 5-10,0.05-0.18mg 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) activated carboxyl is added, and The anti-vibrio parahemolyticus monoclonal antibodies of 50-300 μ g, in 37 DEG C of temperature, it is placed on 10-15rpm gyroscope and is coupled 60-120min, Magneto separate 3-5min, abandons supernatant；3-5 is rinsed after with cleaning buffer solution, takes 1mL sealers to mix closing with nanoparticle 0.5-1h, obtain Fe3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody；
The coupling buffer compound method is as follows：By 3mL 19.07g/mL borax and 7mL 12.37g/mL boric acid After mixing, 10 times of volumes are diluted；
The cleaning buffer solution compound method is as follows：Weigh 0.43g 2- (N- morpholines) ethyl sulfonic acids (MES) and be dissolved in 200mL Sterile distilled water in, tune pH is 5.5-6.0；
The closing agent compounding method is as follows：100mg bovine serum albumin(BSA)s (BSA) are taken to add 1mL phosphate (PBS) buffering Liquid is made into sealer；
3) vibrio parahemolyticus is cultivated, is 10 by bacterium solution adjustment concentration6CFU/mL、105CFU/mL、104CFU/mL、 103CFU/mL, respectively take 1mL standby；Take testing sample solution 1mL, each concentration bacterium solution 1mL, respectively with 100-150 μ g Fe3O4/Ru (bqy)3 2+Nanoparticle-monoclonal antibody, in 37 DEG C of temperature, gyroscope rotating speed 10-15rpm mixing is incubated 30-60min, magnetic point after incubation After 3-5min, supernatant is abandoned, after being cleaned with PBS, redissolves and Fe is obtained in PBS3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody- Bacterium；
4) preparation of immuno-chromatographic test paper strip：By sample pad with pH 8.50.1M Tris-HCl buffer solutions (1%BSA, 0.5%Tween-20) immersion treatment, 60 DEG C of air dry ovens are placed in, are taken out after 2h standby；By vibrio parahemolyticus rabbit it is more anti-and The anti-mouse secondary antibody of donkey is sprayed onto on nitrocellulose membrane respectively as detection line (T lines) and nature controlling line (C lines), and concentration is 1-2mg/mL, Discharge rate is 0.75uL/cm, 37 DEG C be dried in vacuum overnight taking-up be placed in it is standby in dry cylinder；Filter pad, sample pad, nitric acid is fine Dimension film, blotting paper are pasted onto on PVC bottom plates successively, and the wide test strips of 4mm are cut into after posting, are loaded；The examination that will be prepared Paper slip is fitted into aluminium foil bag, adds drier to seal, and is placed in dry cylinder and saves backup；
5) detection of the test strips to sample：The Fe that will be collected into3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody-bacterium is diluted to 50-150 μ g/mL, take 100 μ L to be added drop-wise in test strips well, after 10-15min, it is glimmering to read instrument record T lines, C lines with fluorescence The value of luminous intensity and T/C；
6) qualitative analysis：The result that detects by an unaided eye carries out qualitative analysis, and the colour developing of T lines then illustrates there is parahemolyticas arc in sample Bacterium, T lines do not develop the color, and illustrate do not have vibrio parahemolyticus or the amount containing vibrio parahemolyticus to be less than 10 in sample3CFU/ mL；
7) quantitative analysis：The fluorescence intensity of instrument measurement T lines, C lines, and T/C values are read using fluorescence, with the dense of different bacterium It is that ordinate draws standard curve to spend for abscissa, T/C values, determines the vibrio parahemolyticus quantity in common sample.
Step 1) the Fe3O4/Ru(bqy)3 2+Nanoparticle particle diameter is 80-210nm；
Step 3) the immuno-chromatographic test paper strip be pasted successively in adhesive base filter paper, sample pad, nitrocellulose membrane, Blotting paper forms.Use vibrio parahemolyticus Fe3O4/Ru(bqy)3 2+Nanoparticle immuno-chromatographic test paper strip, while use fluorescence Read the method that instrument quantitatively detects, it is characterised in that：The vibrio parahemolyticus solution of series concentration known to preparation, is read with fluorescence Instrument measures its corresponding fluorescence intensity, establishes standard curve according to this series of values and corresponding concentration, then will detect sample Test strips be put into fluorescence read instrument in, according to fluorescence read instrument export numerical value, looking into canonical plotting can draw in sample The content of vibrio parahemolyticus.
The invention has the advantages that：
1) present invention has the advantages that simple to operate, detection time is short (10-15min), is adapted for Site Detection；
2) technical solution of the present invention detection stability is good, and detection sensitivity is high, and test limit can reach 103CFU/mL.Using Fe3O4/Ru(bqy)3 2+Nanoparticle, not only the superparamagnetism energy with magnetic nano-particle, enrichment method is carried out to sample, And there is Ru (bqy)3 2+The strong optical signalling of nanoparticle, Ru (bqy)3 2+The property of the efficient coupled antibody in nanoparticle surface Can, so as to improve the detection sensitivity of test strips.
3) present invention improves capture rate without the step of eluting vibrio parahemolyticus from immunomagnetic beads； Eliminate step immune marker being sprayed on pad, immunological response is more homogeneous, and the coefficient of variation is small when quantitatively detecting； Reduce workload and living contaminants probability.
4) qualitative and quantitative detection can be carried out to object vibrio parahemolyticus simultaneously.
5) label of the invention is the Fe of carboxylated3O4/Ru(bqy)3 2+Nanoparticle, the label mainly pass through chemistry The mode labelled antibody of coupling, compared to traditional collaurum (physisorption), more multispecific antibody can be captured, so as to improve detection Sensitivity, in addition, the rock-steady structure of the label carboxyl modified can improve the stability of material, it is 1 year to improve storage life, tradition Collaurum storage life is 6 months.
6) Fe of the invention3O4/Ru(bqy)3 2+Nanoparticle is due to Fe in core3O4The effect of nano-particle, can be preferably Prevent fluorescent dye Ru (bqy) in shell3 2+Leakage, so as to improve fluorescence intensity.
Brief description of the drawings
Fig. 1 Fe of the present invention3O4/Ru(bqy)3 2+Detection of the nanoparticle immuno-chromatographic test paper strip to vibrio parahemolyticus
Embodiment
Fe prepared by technical solution of the present invention3O4/Ru(bqy)3 2+Nanoparticle is coupled with vibrio parahemolyticus monoclonal antibody, is prepared Immune Fe3O4/Ru(bqy)3 2+Nanoparticle, and vibrio parahemolyticus is detected applied to immuno-chromatographic test paper strip.The examination Pattern of the paper slip based on double antibodies sandwich, spray the anti-mouse secondary antibody work of anti-and donkey more than vibrio parahemolyticus rabbit respectively on nitrocellulose membrane For detection line and nature controlling line, if contain certain density vibrio parahemolyticus in sample, vibrio parahemolyticus will be first With immune Fe3O4/Ru(bqy)3 2+Nanoparticle combines to form Fe3O4/Ru(bqy)3 2+Nanoparticle-Antibody-antigen complex, The complex logistics after testing line by the how anti-capture of vibrio parahemolyticus rabbit in detection line region clustering, gather finite concentration and formed Macroscopic band or test strips read the signal that instrument can detect, unnecessary immune Fe3O4/Ru(bqy)3 2+Nanoparticle It is moved to nature controlling line and macroscopic band is formed by the anti-mouse secondary antibody capture aggregation of donkey, judges it for the positive, if in sample not Containing thing to be checked, Fe is immunized3O4/Ru(bqy)3 2+Nanoparticle only reacted with the anti-mouse secondary antibody of donkey on control line to be formed it is macroscopic Band, detection line do not develop the color, and judge it for feminine gender.If there is no color or no clear signal at nature controlling line, illustrate test paper Bar is defective in quality, test invalidation.
Embodiment is provided below in conjunction with technical scheme.Following examples are to the solution of the present invention with specific experiment The form example of operation, experiment condition and setup parameter therein are not construed as the limitation to basic technical scheme of the present invention.And And protection scope of the present invention is not limited to following embodiments.
Fluorescence reads instrument and is purchased from Shanghai Hu Guo tech equipments Co., Ltd.
Coupling buffer compound method is as follows：It is 12.37g/mL by borax and 7mL concentration that 3mL concentration is 19.07g/mL Boric acid mixing after, dilute 10 times of volumes；
Cleaning buffer solution compound method is as follows：Weigh the nothing that 0.43g 2- (N- morpholines) ethyl sulfonic acids (MES) are dissolved in 200mL In bacterium distilled water, tune pH is 5.5-6.0；
It is as follows to close agent compounding method：Take 100mg bovine serum albumin(BSA)s (BSA) to add 1mL phosphate (PBS) buffer solution to match somebody with somebody Into sealer；
Embodiment one：Use Fe3O4/Ru(bqy)3 2+Nanoparticle immuno-chromatographic test paper strip is to vibrio parahemolyticus in milk Detection
1. prepare the Fe of coupling monoclonal antibody3O4/Ru(bqy)3 2+Nanoparticle
1.1 Fe3O4/Ru(bqy)3 2+The preparation of nanoparticle：Add 0.6mmolFeCl3·6H2O and 0.3mmol FeCl2·4H2In O to 100mL deionized water, nitrogen is passed through into solution and is heated to 90 DEG C, then by 4.7mL's 25% NH3·H2O is added in mixed liquor, reacts 2h.The solid matter high purity water of black is isolated from reaction solution with permanent magnet Cleaning 3~5 times, obtains Fe3O4Nano-particle；Take 12mg Fe3O4Nano-particle 3mL deionized waters and the mixed liquor of 20mL ethanol It is resuspended, 0.5mL NH is first added under conditions of being slowly stirred4OH solution, then 50uL tetraethyl orthosilicates are dissolved in 50uL second It is added dropwise in alcoholic solution, reacts 12h at room temperature, in Fe3O4Nanoparticle surface forms layer of silicon dioxide, uses deionized water Solution cleans several times, is redissolved in ethanol solution and obtains the Fe of coated with silica3O4Nano-particle；100uL positive silicic acid second Ester, 20mL absolute ethyl alcohols, 3mL deionized waters and 1mL 0.5-3mg/L phenanthroline connection ruthenium (Ru (bqy)3 2+) mixing, it will mix Solution adds the Fe of 0.5mL coated with silica3O4Nano-particle, it is eventually adding 750 μ L NH4OH, 3h is stirred vigorously, obtained Fe3O4/Ru(bqy)3 2+Nanoparticle, cleaned with deionized water standby；1mL mercaptopropyl trimethoxysilane is added to 10mL Ethanol solution in, with the mixture redissolve Fe3O4/Ru(bqy)3 2+Nanoparticle, after 300rpm stirs 12h at normal temperatures, then 80 DEG C of 300rpm stir 1h, and centrifugation obtains the Fe of silanization3O4/Ru(bqy)3 2+Nanoparticle；By the Fe of silanization3O4/Ru (bqy)3 2+Nanoparticle is added to comprising 0.06g NaHCO3, 0.08g neopelexes, 0.05mL styrene, The 50mL of the 0.15mL acrylic acid and 0.5g potassium persulfate solutions aqueous solution, 70 DEG C of water-bath, stir under 200r/min, after reacting 5h Obtain the Fe of carboxylated3O4/Ru(bqy)3 2+Nanoparticle；
1.2. coupling reaction：Take the Fe of 1.0mg carboxylated3O4/Ru(bqy)3 2+Nanoparticle adds 1mL coupling buffers In, pH to 8 is adjusted, adds 0.05-0.18mg 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) activation Carboxyl, and the anti-vibrio parahemolyticus monoclonal antibodies of 150 μ g, in 37 DEG C of temperature, it is placed on 10-15rpm gyroscope and is coupled 60- 120min, Magneto separate 3-5min, abandons supernatant；3-5 is rinsed after with cleaning buffer solution, takes 1mL sealers to mix envelope with magnetic bead 0.5-1h is closed, obtains Fe3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody.
2. use immune Fe3O4/Ru(bqy)3 2+Vibrio parahemolyticus in nanoparticle capture milk
Milk is added in 225mL culture mediums after taking 25mL sterilizing, certain density vibrio parahemolyticus is inoculated with, 36 Under conditions of DEG C, concussion and cultivate 8-18h.Bacterial concentration is adjusted to 106CFU/mL、105CFU/mL、104CFU/mL、103CFU/ mL。
Each concentration bacterium solutions of 1mL, 1mL testing sample solutions are taken, is separately added into 120 μ g Fe3O4/Ru(bqy)3 2+Nanoparticle- Monoclonal antibody, 37 DEG C of temperature, rotating speed 10-15rpm mixing are incubated 30-60min；After incubation after Magneto separate 3-5min, supernatant is abandoned, uses PBS After buffer solution for cleaning, redissolve and Fe is obtained in PBS3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody-bacterium.
3. make vibrio parahemolyticus immuno-chromatographic test paper strip
Sample pad is handled with pH 8.50.1M Tris-HCl buffer solutions (1%BSA, 0.5%Tween-20), is placed in 60 DEG C air dry oven, take out after 2h be placed in it is standby in dry cylinder；Vibrio parahemolyticus rabbit is resisted more and the anti-mouse secondary antibody of donkey is sprayed onto nitre Respectively as detection line and nature controlling line on sour tunica fibrosa, concentration is 1.0mg/mL, and discharge rate is 0.75uL/cm, 37 DEG C of vacuum Be dried overnight taking-up be placed in it is standby in dry cylinder；Filter pad, sample pad, nitrocellulose membrane, blotting paper are pasted onto PVC bottoms successively On plate, the wide test strips of 4mm are cut into after posting, are loaded.The test strips prepared are fitted into aluminium foil bag, add drier close Envelope, is placed in dry cylinder and saves backup.
4. being estimated using double-antibody method to sample and using Instrumental results
The Fe being collected into will be captured3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody-bacterium is diluted to 100 μ g/mL, takes 100 μ L drops It is added in test strips well, after 10-15min, instrument record T lines, C lines fluorescence intensity and T/C value is read with fluorescence；With the naked eye Observe result and carry out qualitative analysis, the colour developing of T lines then illustrates there is vibrio parahemolyticus in sample, and T lines do not develop the color, illustrated in sample There is no vibrio parahemolyticus or the amount containing vibrio parahemolyticus to be less than 103CFU/mL。
With reference to the canonical plotting done, the quantity of vibrio parahemolyticus in sample is determined.The model of quantitative testing bacteria concentration It is trapped among 103-106CFU/mL.The inventive method detection is stable, and detection line can be with as little as 103CFU/mL, speed is fast, and effect is good.
Embodiment two：Use Fe3O4/Ru(bqy)3 2+Nanoparticle immuno-chromatographic test paper strip is to vibrio parahemolyticus in beef Detection
1. prepare the Fe of coupling monoclonal antibody3O4/Ru(bqy)3 2+Nanoparticle
1.1 Fe3O4/Ru(bqy)3 2+The preparation of nanoparticle：Add 0.6mmol FeCl3·6H2O and 0.3mmol FeCl2·4H2In O to 100mL deionized water, nitrogen is passed through into solution and is heated to 90 DEG C, then by 4.7mL's 25% NH3·H2O is added in mixed liquor, reacts 2h.The solid matter high purity water of black is isolated from reaction solution with permanent magnet Cleaning 3~5 times, obtains Fe3O4Nano-particle；Take 12mg Fe3O4Nano-particle 3mL deionized waters and the mixed liquor of 20mL ethanol It is resuspended, 0.5mL NH is first added under conditions of being slowly stirred4OH solution, then 50uL tetraethyl orthosilicates are dissolved in 50uL second It is added dropwise in alcoholic solution, reacts 12h at room temperature, in Fe3O4Nanoparticle surface forms layer of silicon dioxide, uses deionized water Solution cleans several times, is redissolved in ethanol solution and obtains the Fe of coated with silica3O4Nano-particle；100uL positive silicic acid second Ester, 20mL absolute ethyl alcohols, 3mL deionized waters and 1mL 0.5-3mg/L phenanthroline connection ruthenium (Ru (bqy)3 2+) mixing, it will mix Solution adds the Fe of 0.5mL coated with silica3O4Nano-particle, it is eventually adding 750 μ L NH4OH, 3h is stirred vigorously, obtained Fe3O4/Ru(bqy)3 2+Nanoparticle, cleaned with deionized water standby；1mL mercaptopropyl trimethoxysilane is added to 10mL Ethanol solution in, with the mixture redissolve Fe3O4/Ru(bqy)3 2+Nanoparticle, after 300rpm stirs 12h at normal temperatures, then 80 DEG C of 300rpm stir 1h, and centrifugation obtains the Fe of silanization3O4/Ru(bqy)3 2+Nanoparticle；By the Fe of silanization3O4/Ru (bqy)3 2+Nanoparticle is added to comprising 0.06g NaHCO3, 0.08g neopelexes, 0.05mL styrene, The 50mL of the 0.15mL acrylic acid and 0.5g potassium persulfate solutions aqueous solution, 70 DEG C of water-bath, stir under 200r/min, after reacting 5h Obtain the Fe of carboxylated3O4/Ru(bqy)3 2+Nanoparticle；
1.2. coupling reaction：Take the Fe of 1.0mg carboxylated3O4/Ru(bqy)3 2+Nanoparticle adds 1mL coupling buffers In, pH to 8 is adjusted, adds 0.05-0.18mg 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) activation Carboxyl, and the anti-vibrio parahemolyticus monoclonal antibodies of 150 μ g, in 37 DEG C of temperature, it is placed on 10-15rpm gyroscope and is coupled 60- 120min, Magneto separate 3-5min, abandons supernatant；3-5 is rinsed after with cleaning buffer solution, takes 1mL sealers to mix envelope with magnetic bead 0.5-1h is closed, obtains Fe3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody.
2. use the Fe of coupling monoclonal antibody3O4/Ru(bqy)3 2+Vibrio parahemolyticus in nanoparticle capture beef
Take the beef meat gruel of 25mg sterilizing to be added in 225mL culture mediums, be inoculated with certain density vibrio parahemolyticus, Under conditions of 36 DEG C, concussion and cultivate 8-18h.Bacterial concentration is adjusted to 106CFU/mL、105CFU/mL、104CFU/mL、 103CFU/mL。
Each concentration bacterium solutions of 1mL, 1mL testing sample solutions are taken, is separately added into 120 μ g Fe3O4/Ru(bqy)3 2+Nanoparticle- Monoclonal antibody, 37 DEG C of temperature, rotating speed 10-15rpm mixing are incubated 30-60min；After incubation after Magneto separate 3-5min, supernatant is abandoned, uses PBS After buffer solution for cleaning, redissolve and Fe is obtained in PBS3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody-bacterium.
3. make vibrio parahemolyticus immuno-chromatographic test paper strip
Sample pad is handled with pH 8.50.1M Tris-HCl buffer solutions (1%BSA, 0.5%Tween-20), is placed in 60 DEG C air dry oven, take out after 2h be placed in it is standby in dry cylinder；Vibrio parahemolyticus rabbit is resisted more and the anti-mouse secondary antibody of donkey is sprayed onto nitre Respectively as detection line and nature controlling line on sour tunica fibrosa, concentration is 1.0mg/mL, and discharge rate is 0.75uL/cm, 37 DEG C of vacuum Be dried overnight taking-up be placed in it is standby in dry cylinder；Filter pad, sample pad, nitrocellulose membrane, blotting paper are pasted onto PVC bottoms successively On plate, the wide test strips of 4mm are cut into after posting, are loaded.The test strips prepared are fitted into aluminium foil bag, add drier close Envelope, is placed in dry cylinder and saves backup.
4. being estimated using double-antibody method to sample and using Instrumental results
The Fe being collected into will be captured3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody-bacterium is diluted to 100 μ g/mL, takes 100 μ L drops It is added in test strips well, after 10-15min, instrument record T lines, C lines fluorescence intensity and T/C value is read with fluorescence；With the naked eye Observe result and carry out qualitative analysis, the colour developing of T lines then illustrates there is vibrio parahemolyticus in sample, and T lines do not develop the color, illustrated in sample There is no vibrio parahemolyticus or the amount containing vibrio parahemolyticus to be less than 103CFU/mL。
With reference to the canonical plotting done, the quantity of vibrio parahemolyticus in sample is determined.The model of quantitative testing bacteria concentration It is trapped among 103-106CFU/mL.The inventive method detection is stable, and test limit can be with as little as 103CFU/mL, speed is fast, and effect is good.

Claims (3)

  1. A kind of 1. method of quick detection vibrio parahemolyticus, it is characterised in that comprise the following steps：
    1) preparation of nanoparticle：
    A. 0.4-0.8mmolFeCl is added3·6H2O and 0.2-1.6mmol FeCl2·4H2In O to 100mL deionized water, to Nitrogen is passed through in solution and is heated to 80-120 DEG C, then by 3-7mL 25% NH3·H2O is added in mixed liquor, reaction 2h；The solid matter for isolating black from reaction solution with permanent magnet is cleaned 3~5 times with high purity water, obtains Fe3O4Nano-particle；
    B. 12mg Fe are taken3O4Nano-particle is resuspended with the mixed liquor of 1-10mL deionized waters and 20mL ethanol, is first being slowly stirred Under conditions of add 0.3-0.9mL NH4OH solution, then by 10-300 μ L tetraethyl orthosilicates be dissolved in 50 μ L ethanol solutions by It is added dropwise to, reacts 12h at room temperature, in Fe3O4Nanoparticle surface forms layer of silicon dioxide, is cleaned with deionized water solution several It is secondary, redissolved in ethanol solution and obtain the Fe of coated with silica3O4Nano-particle；
    C. 10-300 μ L tetraethyl orthosilicate, 20mL absolute ethyl alcohols, 1-10mL deionized waters and 1mL 0.5-3mg/L coffee hello Quinoline connection ruthenium (Ru (bqy)3 2+) mixing, by the Fe of mixed solution addition 0.5mL coated with silica3O4Nano-particle, it is eventually adding 600-900 μ L NH4OH, 3h is stirred vigorously, obtains Fe3O4/Ru(bqy)3 2+Nanoparticle, cleaned with deionized water standby；
    D. 1mL mercaptopropyl trimethoxysilane is added in 10mL ethanol solution, redissolves Fe with the mixture3O4/Ru (bqy)3 2+Nanoparticle, after 300rpm stirs 12h at normal temperatures, then 80 DEG C of 300rpm stirring 1h, centrifugation obtain silanization Fe3O4/Ru(bqy)3 2+Nanoparticle；
    E. by the Fe of silanization3O4/Ru(bqy)3 2+Nanoparticle is added to comprising 0.06g NaHCO3, 0.08g detergent alkylates The 50mL of sodium sulfonate, 0.05mL styrene, the 0.15mL acrylic acid and 0.5g potassium persulfate solutions aqueous solution, 70 DEG C of water-bath, Stirred under 200r/min, the Fe of carboxylated is obtained after reaction 5h3O4/Ru(bqy)3 2+Nanoparticle；
    2) Fe of 0.5-2mg carboxylated is taken3O4/Ru(bqy)3 2+Nanoparticle is added in 1mL coupling buffers, adjusts pH to 5- 10, add 0.05-0.18mg 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) activated carboxyl, and 50- The anti-vibrio parahemolyticus monoclonal antibodies of 300 μ g, in 37 DEG C of temperature, it is placed on 10-15rpm gyroscope and is coupled 60-120min, magnetic point From 3-5min, supernatant is abandoned；3-5 is rinsed after with cleaning buffer solution, takes 1mL sealers to mix closing 0.5- with nanoparticle 1h, obtain Fe3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody；
    The coupling buffer compound method is as follows：3mL 19.07g/mL borax is mixed with 7mL 12.37g/mL boric acid Afterwards, 10 times of volumes are diluted；
    The cleaning buffer solution compound method is as follows：Weigh the nothing that 0.43g 2- (N- morpholines) ethyl sulfonic acids (MES) are dissolved in 200mL In bacterium distilled water, tune pH is 5.5-6.0；
    The closing agent compounding method is as follows：Take 100mg bovine serum albumin(BSA)s (BSA) to add 1mL phosphate (PBS) buffer solution to match somebody with somebody Into sealer；
    3) vibrio parahemolyticus is cultivated, is 10 by bacterium solution adjustment concentration6CFU/mL、105CFU/mL、104CFU/mL、103CFU/mL, Respectively take 1mL standby；Take testing sample solution 1mL, each concentration bacterium solution 1mL, respectively with 100-150 μ g Fe3O4/Ru(bqy)3 2+Receive Meter Wei Qiu-monoclonal antibody, in 37 DEG C of temperature, gyroscope rotating speed 10-15rpm mixing is incubated 30-60min, Magneto separate 3-5min after incubation Afterwards, supernatant is abandoned, after being cleaned with PBS, redissolves and Fe is obtained in PBS3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody-bacterium；
    4) preparation of immuno-chromatographic test paper strip：Sample pad is used and contains 1%BSA, 0.5%Tween-20 pH 8.50.1M Tris- HCl buffer solution immersion treatments, 60 DEG C of air dry ovens are placed in, are taken out after 2h standby；Vibrio parahemolyticus rabbit is resisted more and donkey resists Mouse secondary antibody is sprayed onto on nitrocellulose membrane respectively as detection line (T lines) and nature controlling line (C lines), and concentration is 1-2mg/mL, discharge rate Be 0.75 μ L/cm, 37 DEG C be dried in vacuum overnight taking-up be placed in it is standby in dry cylinder；By filter pad, sample pad, cellulose nitrate Film, blotting paper are pasted onto on PVC bottom plates successively, and the wide test strips of 4mm are cut into after posting, are loaded；The test paper that will be prepared Bar is fitted into aluminium foil bag, adds drier to seal, and is placed in dry cylinder and saves backup；
    5) detection of the test strips to sample：The Fe that will be collected into3O4/Ru(bqy)3 2+Nanoparticle-monoclonal antibody-bacterium is diluted to 50-150 μ g/mL, take 100 μ L to be added drop-wise in test strips well, after 10-15min, instrument record T lines, C line fluorescence intensities are read with fluorescence With T/C value；
    6) qualitative analysis：The result that detects by an unaided eye carries out qualitative analysis, and the colour developing of T lines then illustrates there is vibrio parahemolyticus, T in sample Line does not develop the color, and illustrates do not have vibrio parahemolyticus or the amount containing vibrio parahemolyticus to be less than 10 in sample3CFU/mL；
    7) quantitative analysis：The fluorescence intensity of instrument measurement T lines, C lines, and T/C values are read using fluorescence, using the concentration of different bacterium as Abscissa, T/C values are that ordinate draws standard curve, determine the vibrio parahemolyticus quantity in common sample.
  2. 2. according to the method for claim 1, it is characterised in that the step 1) Fe3O4/Ru(bqy)3 2+Nanoparticle particle diameter For 80-210nm.
  3. 3. according to the method for claim 1, it is characterised in that：Step 3) the immuno-chromatographic test paper strip is in adhesive base On paste successively filter paper, sample pad, nitrocellulose membrane, blotting paper composition.
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