CN104987460A - Amino-contained antibiotic nanometer molecularly imprinted polymer and preparation method and application thereof - Google Patents
Amino-contained antibiotic nanometer molecularly imprinted polymer and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an amino-contained antibiotic nanometer molecularly imprinted polymer and a preparation method and application thereof. An antibiotic is led onto a solid phase carrier glass bead to obtain the glass bead deriving from the amino-contained antibiotic; then, the glass bead is made to react with a functional monomer, a cross-linking agent and a thermal initiator ammonium peroxydisulfate at room temperature, a reaction mixture is poured into a solid phase extraction pipe, the glass bead is washed through cold water, the functional monomer and the cross-linking agent which do not participate in the reaction are removed, the glass bead is washed through hot water to obtain the molecularly imprinted polymer; cooling is performed at room temperature, a microhole ultra centrifugal is used for filtering and separation to obtain an amino-contained antibiotic nanometer molecularly imprinted polymer solution. The amino-contained antibiotic nanometer molecularly imprinted polymer can be used for being applied in the aspect of antibiotics containing a micro amount of amino, and particularly can be used for detection of amino-contained antibiotics in food and surface water. The amino-contained antibiotic nanometer molecularly imprinted polymer is simple, rapid, high in detection specificity and low in detection limit.
Description
Technical field
The invention belongs to minimal feeding technical field, particularly relate to a kind of containing amino microbiotic nanometer molecular imprinting polymer and preparation method thereof and application.
Background technology
Microbiotic is a kind of by microorganism self generation or synthetic, microorganisms producing can be suppressed under lower concentration or kill the organic chemicals of other microorganisms, except the treatment for human and animal's bacterial infection disease, microbiotic is also widely applied in intensive animal husbandry and aquaculture by as short long agent and fodder additives, along with a large amount of antibiotic use, more and more microbiotic that can not be fully absorbed utilization is directly discharged by human and animal excreta, these are containing antibiotic ight soil using to enter between farmland and plant and move by organic fertilizer, in addition these microbiotic are likely by infiltration, runoff, the modes such as leaching move in surface water and groundwater, again environment is flowed into.
Current monitoring method for antibiotic residue detection mainly contains microbial method, immunization, vapor-phase chromatography, high performance liquid chromatography and Liquid Chromatography-Tandem Mass Spectrometry.Microbial method is simple and quick, but detects limit for height, poor specificity; Chromatographic detection length consuming time, in residual separation detection, sample pretreatment process is complicated, and the accuracy of impact analysis; Immunodetection is simple, fast, detection limit is low, can carry out Large-scale Screening, but the preparation of its corresponding antibodies needs great many of experiments animal, and antibody is easy to inactivation in the presence of a harsh environment, affects detected result.
In addition also have and adopt molecular imprinting extraction microbiotic, then coupled HPLC mass-spectrometric technique detects, such as use the Streptomycin sulphate in molecular engram solid phase extraction honey, aqueous favoring mutual effect liquid chromatography-tandem mass spectrometry analyzing and testing Streptomycin sulphate, this molecular imprinting method adopts precipitation polymerization method Synthesis of Molecular Imprinting Polymers, need synthesize in organic phase, reaction times is long, and the polymkeric substance of synthesis becomes block, need through polishing for a long time, screening could be used for Solid-Phase Extraction, molecularly imprinted polymer utilization ratio is not high, need coupled HPLC-mass spectrum serial connection technology in addition, required instrument is valuable.
Summary of the invention
The object of the present invention is to provide and a kind of apply containing amino microbiotic nanometer molecular imprinting polymer and preparation method thereof and detecting, be intended to solve and existingly contain amino microbiotic detection technique and be easy to the problems such as inactivation, detected result be not good.
The present invention is achieved in that a kind of preparation method containing amino microbiotic nanometer molecular imprinting polymer, and the method comprises the following steps:
(1) solid phase carrier granulated glass sphere introduces microbiotic
Joined by 250 ~ 300g granulated glass sphere in the NaOH aqueous solution of 120 ~ 160mL, 1 ~ 4mol/L, ebuillition of heated 10 ~ 15min, taking-up granulated glass sphere washes pH=8 ~ 9 to washing lotion with water, dries and obtains hydroxylation granulated glass sphere;
Hydroxylation granulated glass sphere is joined in the dry toluene of 150mL, then add 3.3mL N-[3-(trimethoxy is silica-based) propyl group] quadrol, after violent shake, kept at room temperature overnight, filtration, acetone rinsing, obtain silanized glass pearl;
100g silanized glass pearl is joined in the PBS of 50mL, pH7.4, then adds 3.5mL glutaraldehyde, react 2 ~ 3h under room temperature, suction filtration, washing, collect Semi-finished glass pearl;
Mixed with Semi-finished glass pearl containing amino microbiotic by PBS and 25mg of 50mL, pH 7.4, after violent shake, kept at room temperature overnight, filters, washes, and obtains containing the derivative granulated glass sphere of amino microbiotic.
(2) nano print polymer of synthetic antibiotic trace
By 1mmol N, N'-dimethylene bisacrylamide, 25 ~ 30mmol NIPA, 18 ~ 20mmol N tert butyl acrylamide and 0.0025 ~ 0.003mmol vinylformic acid are by mixing and being dissolved in 100mL ultrapure water, logical nitrogen 20 ~ 30 minutes, mixture is poured in the described granulated glass sphere derivative containing amino microbiotic, then the aqueous solution of 40 ~ 60mg ammonium peroxydisulfate and 13 ~ 26 μ L Tetramethyl Ethylene Diamines is added, after violent shake, 1 ~ 1.5h is reacted under room temperature, reaction mixture is poured in solid phase extraction tube, with the room temperature water cleaning glass pearl of 6 ~ 8, use 80 ~ 100mL again, 60 ~ 70 heat DEG C water wash, naturally cool to room temperature, obtain containing amino microbiotic nanometer molecular imprinting polymer solution with the centrifugation of micropore ultracentrifugation filtration unit.
Invention further provides and above-mentionedly to detect containing the application in amino microbiotic containing amino microbiotic nanometer molecular imprinting polymer.
Preferably, describedly to comprise the following steps containing amino antibiotic detection:
(1) synthesis of enzyme mark thing horseradish peroxidase-template molecule HRP-T
5 ~ 10mg horseradish peroxidase is dissolved in 0.7 ~ 1.0mL, in the MES damping fluid of pH6.0, add 0.2 ~ 0.4mgEDC and 0.3 ~ 0.6mg NHS again, 15min is reacted under room temperature, with being separated removing damping fluid under micropore ultracentrifugation filtration unit 3500rpm rotating speed, with 3 ~ 5mL, the PBS washing of pH 7.40, collect the horseradish peroxidase after activation, 2 ~ 3h is hatched containing the amino antibiotic PBS liquid of 8 ~ 10mg immediately with 10mL, then wash away with 4.0 ~ 5.0mL PBS damping fluid the microbiotic do not connected, be separated under rotating speed 3500rpm with micropore ultracentrifugation filtration unit and obtain enzyme mark thing HRP-T solution,
(2) canonical plotting makes
Measure different concns containing amino antibiotic absorbancy by enzyme linked immunological competitive adsorption method, set up the canonical plotting of antibiotic concentration-absorbancy;
(3) containing amino antibiotic detection
Pretreated thing to be detected is measured containing amino antibiotic absorbancy by enzyme linked immunological competitive adsorption method, according to this absorbancy and canonical plotting comparison, obtains in thing to be detected containing amino antibiotic type and concentration;
In step (2) and (3), described enzyme linked immunological competitive adsorption method is specially: be coated in 96 hole enzyme plates containing amino microbiotic nanometer molecular imprinting polymer solution described in the claims 1 being 0.04 ~ 0.06mg/ml by 40 μ L, concentration, at room temperature evaporate into dry, wash by PBS solution, add containing 200 ~ 300 μ L containing mass concentration be 1% tensio-active agent Tween 20 and mass concentration be the PBS solution of the bovine serum albumin BSA of 0.1%, hatching 1h, wash by PBS solution, then add the enzyme mark thing HRP-T solution of 60 ~ 100 μ L in each hole and contain amino microbiotic standardized solution incubated at room temperature 1h, with containing mass concentration be 1% Tween 20 and mass concentration be the BSA of 0.1% PBS solution washing, add 60 ~ 100 μ LTMB reagent react 2 ~ 10min, add 0.5mol/L, 60 ~ 100 μ L H
2sO
4solution termination reaction, microplate reader reader reads the absorbancy of each micropore solution in 450nm place.
4, apply as claimed in claim 3, it is characterized in that, before step (2), also comprise the condition optimizing step of enzyme linked immunological competitive adsorption method, the condition optimizing step detailed process of described enzyme linked immunological competitive adsorption method is:
Bag quilt: get 30 ~ 50 μ L, in nanometer polymer solution to 96 hole enzyme plate that concentration is amino microbiotic trace described in 0.04 ~ 0.06mg/ml claims book 1; At room temperature evaporate into dry, wash by PBS solution;
Close: add containing 300 μ L containing tensio-active agent Tween 20 mass concentration be 0.5 ~ 3% and bovine serum albumin BSA mass concentration be the PBS solution of 0.05 ~ 0.3%, hatching 1 ~ 2h; And then wash 3 times by 300 μ L PBS solution;
Application of sample: add 100 μ L enzyme mark thing HRP-T solution, hatching 2h;
Colour developing with measure: with containing mass concentration be 0.5 ~ 3% Tween 20 and mass concentration be that the PBS solution of the BSA of 0.05 ~ 0.3% wash, drying, adds 100 μ LHRP substrates 3,3', 5,5'-tetramethyl benzidine (TMB) reagent, after hatching 10min, add 0.5mol/L H
2sO
4100 μ L solution color development stopping reactions, then measure its absorbancy at 450nm place by microplate reader, determine optimal detection parameter.
5, apply as claimed in claim 4, it is characterized in that, in step (3), described thing to be detected is meat, and described meat comprises fish, chicken; The pre-treatment of this meat comprises: fish sample is scaled, removed the peel, and gets muscle along ridge; Chicken class sample is boned, and gets muscle parts.Sample homogenization treatment becomes the sample to be tested of meat gruel shape.Accurately take sample, be placed in polystyrene centrifuge tube, add acidifying acetonitrile, homogenization treatment, whirlpool mixes, ultrasonic extraction, and with the centrifugal 5 ~ 10min of 4000r/min, supernatant liquor is transferred in evaporative flask; Solvent evaporated under reduced pressure, redissolves with PBS damping fluid, obtains containing amino antibiotic PBS damping fluid.
Preferably, in step (3), described thing to be detected is milk, and the pre-treatment of described milk is: get milk and carry out centrifugation, and collect supernatant liquid, supernatant liquor is transferred in evaporative flask; Solvent evaporated under reduced pressure, redissolves with PBS damping fluid, obtains containing amino antibiotic PBS damping fluid.
Preferably, in step (3), described thing to be detected is water, and the pre-treatment of described water is: 0.45um filter membrane removing suspended substance, and collect supernatant liquid, supernatant liquor is transferred in evaporative flask; Solvent evaporated under reduced pressure, redissolves with PBS damping fluid, obtains containing amino antibiotic PBS damping fluid.
Preferably, describedly Streptomycin sulphate, gentamicin and sulfamethoxazole is comprised containing amino microbiotic.
Compared to the shortcoming and defect of prior art, the present invention has following beneficial effect: the nanometer molecular imprinting polymer of Solid phase synthesis microbiotic trace of the present invention directly can replace antibody, with in bionical enzyme-linked immunosorbent assay water and food (milk, chicken, pork, the flesh of fish, egg etc.) in sulfamido, aminoglycoside, cynnematins etc. are containing amino microbiotic, not only can avoid the defect of above method, and the pre-treatment of this method sample is simple, do not need to be separated, simple and quick, detection specificity is high, detection limit is low, in addition, containing the nanometer polymer of amino microbiotic trace as bionic antibody, in one month, its Detection results is constant.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1 is containing the preparation of amino microbiotic nanometer molecular imprinting polymer solution
(1) solid phase carrier granulated glass sphere introduces microbiotic
Joined by 250g granulated glass sphere in the NaOH aqueous solution of 120mL, 1mol/L, ebuillition of heated 15min, taking-up granulated glass sphere washes the pH=8 to washing lotion with water, dries and obtains hydroxylation granulated glass sphere;
Hydroxylation granulated glass sphere is joined in the dry toluene of 150mL, then add 3.3mL N-[3-(trimethoxy is silica-based) propyl group] quadrol, after violent shake, kept at room temperature overnight, filtration, acetone rinsing, obtain silanized glass pearl;
100g silanized glass pearl is joined in the PBS of 50mL, pH7.4, then adds 3.5mL glutaraldehyde, react 2 ~ 3h under room temperature, suction filtration, washing, collect Semi-finished glass pearl;
Mixed with Semi-finished glass pearl containing amino microbiotic by PBS and 25mg of 50mL, pH 7.4, after violent shake, kept at room temperature overnight, filters, washes, and obtains containing the derivative granulated glass sphere of amino microbiotic;
(2) nano print polymer of synthetic antibiotic trace
By 1mmol N, N'-dimethylene bisacrylamide, 25mmol NIPA, 18mmolN-N-tert-butyl acrylamide and 0.0025mmol vinylformic acid are by mixing and being dissolved in 100mL ultrapure water, logical nitrogen 30 minutes, mixture is poured in the described granulated glass sphere derivative containing amino microbiotic, then the aqueous solution of 40mg ammonium peroxydisulfate and 26 μ L Tetramethyl Ethylene Diamines is added, after violent shake, 1.5h is reacted under room temperature, reaction mixture is poured in solid phase extraction tube, with the room temperature water cleaning glass pearl of 8, use 80mL again, 70 DEG C of hot water drip washing, naturally cool to room temperature, obtain containing amino microbiotic nanometer molecular imprinting polymer solution 1 with the centrifugation of micropore ultracentrifugation filtration unit.
Embodiment 2 is containing the preparation of amino microbiotic nanometer molecular imprinting polymer solution
(1) solid phase carrier granulated glass sphere introduces microbiotic
Joined by 300g granulated glass sphere in the NaOH aqueous solution of 160mL, 2mol/L, ebuillition of heated 10min, taking-up granulated glass sphere washes the pH=9 to washing lotion with water, dries and obtains hydroxylation granulated glass sphere;
Hydroxylation granulated glass sphere is joined in the dry toluene of 150mL, then add 3.3mL N-[3-(trimethoxy is silica-based) propyl group] quadrol, after violent shake, kept at room temperature overnight, filtration, acetone rinsing, obtain silanized glass pearl;
100g silanized glass pearl is joined in the PBS of 50mL, pH7.4, then adds 3.5mL glutaraldehyde, react 2h under room temperature, suction filtration, washing, collect Semi-finished glass pearl;
Mixed with Semi-finished glass pearl containing amino microbiotic by PBS and 25mg of 50mL, pH 7.4, after violent shake, kept at room temperature overnight, filters, washes, and obtains containing the derivative granulated glass sphere of amino microbiotic;
(2) nano print polymer of synthetic antibiotic trace
By 1mmol N, N'-dimethylene bisacrylamide, 30mmol NIPA, 20mmolN-N-tert-butyl acrylamide and 0.003mmol vinylformic acid are by mixing and being dissolved in 100mL ultrapure water, logical nitrogen 20 minutes, mixture is poured in the described granulated glass sphere derivative containing amino microbiotic, then the aqueous solution of 60mg ammonium peroxydisulfate and 13 μ L Tetramethyl Ethylene Diamines is added, after violent shake, 1h is reacted under room temperature, reaction mixture is poured in solid phase extraction tube, with the room temperature water cleaning glass pearl of 6, use 100mL again, 60 DEG C of hot water drip washing, naturally cool to room temperature, with the centrifugation of micropore ultracentrifugation filtration unit obtain concentration be 0.05mg/mL containing amino microbiotic nanometer molecular imprinting polymer solution 2.
Containing amino antibiotic detection in the foods such as embodiment 3 chicken, the flesh of fish
(1) synthesis of enzyme mark thing horseradish peroxidase-template molecule HRP-T
10mg horseradish peroxidase is dissolved in the MES damping fluid of 1.0mL, pH6.0, add 0.4mg EDC and 0.6mg NHS again, 15min is reacted under room temperature, with being separated removing damping fluid under micropore ultracentrifugation filtration unit 3500rpm rotating speed, wash with the PBS of 5mL, pH 7.40, collect the horseradish peroxidase after activation, 2 hours are hatched containing the amino antibiotic PBS damping fluid of 10mg immediately with 10mL, then wash away the microbiotic do not connected with 5.0mLPBS damping fluid, be separated under rotating speed 3500rpm with micropore ultracentrifugation filtration unit and obtain enzyme mark thing HRP-T solution;
(2) condition optimizing of enzyme linked immunological competitive adsorption method is explored
Bag quilt: getting concentration prepared by 30 μ L embodiments 1 is in nanometer polymer solution 1 to the 96 hole enzyme plate of the amino microbiotic trace of 0.04mg/ml; At room temperature evaporate into dry, with 300 μ L PBS solution washings;
Close: add containing 300 μ L containing tensio-active agent Tween 20 mass concentration be 0.5% and bovine serum albumin BSA mass concentration be the PBS solution of 0.3%, hatching 2h; And then wash 3 times by 300 μ L PBS solution;
Application of sample: add 100 μ L enzyme mark thing HRP-T solution, hatching 2h;
Colour developing with measure: containing Tween 20 mass concentration be 0.5% and BSA mass concentration be 0.3% PBS solution 300 μ L wash, drying, add 100 μ LHRP substrates 3,3', 5,5'-tetramethyl benzidine (TMB) reagent, after hatching 10min, add 0.5mol/L H
2sO
4100 μ L solution color development stopping reactions, then measure its absorbancy at 450nm place by microplate reader, determine enzyme linked immunological competitive adsorption method optimal detection parameter.
(3) enzyme linked immunological competitive adsorption method measures the making of microbiotic typical curve
Be that 0.04mg/ml contains amino microbiotic nanometer molecular imprinting polymer solution 1 and is coated in 96 hole enzyme plates by the concentration in 40 μ L embodiments 1, at room temperature evaporate into dry, with 300 μ L PBS solution washings, add containing 300 μ L containing tensio-active agent Tween 20 mass concentration be 3% and bovine serum albumin BSA mass concentration be the PBS solution of 0.05%, hatching 1h, 3 times are washed by 300 μ L PBS solution, then add in each hole the enzyme mark thing solution of 100 μ L and different concns containing amino microbiotic standardized solution incubated at room temperature 1h, with 300 μ L contain Tween 20 mass concentration be 3% and BSA mass concentration be 0.05% PBS solution washing, add 100 μ LTMB reagent react 10min, add 100 μ L, 0.5mol/L H
2sO
4solution termination reaction, microplate reader reader reads the absorbancy of each micropore solution in 450nm place, sets up the canonical plotting of antibiotic concentration-absorbancy, and the different canonical plotting have its correspondence containing amino antibiosis, comprises Streptomycin sulphate, gentamicin and sulfamethoxazole containing amino microbiotic.
(4) containing amino antibiotic detection
The food such as chicken, the flesh of fish is purchased from local market.Fish sample is scaled, is removed the peel, and gets muscle along ridge; Chicken class sample is boned, and gets muscle parts.Sample homogenization treatment becomes the sample to be tested of meat gruel shape.Accurately take sample, be placed in polystyrene centrifuge tube, add acidifying acetonitrile, homogenization treatment, whirlpool mixes, ultrasonic extraction, and with the centrifugal 5 ~ 10min of 4000r/min, supernatant liquor is transferred in evaporative flask; Solvent evaporated under reduced pressure, redissolves with PBS damping fluid, obtains, containing amino antibiotic PBS damping fluid, preserving, for subsequent use.
Pretreated thing to be detected is detected as follows:
Be that 0.04mg/ml contains amino microbiotic nanometer molecular imprinting polymer solution 1 and is coated in 96 hole enzyme plates by the concentration in 40 μ L embodiments 1; At room temperature evaporate into dry, with 300 μ L PBS solution washings, add containing 300 μ L containing tensio-active agent Tween 20 mass concentration be 3% and bovine serum albumin BSA mass concentration be the PBS solution of 0.05%, hatching 1h, wash 3 times by 300 μ L PBS solution, the enzyme mark thing solution and the concentration that then add 100 μ L in each hole are 1 degree 1 × 10
2nM containing amino microbiotic standardized solution incubated at room temperature 1h, with 300 μ L contain Tween 20 mass concentration be 3% and BSA mass concentration be 0.05% PBS solution wash 3 times, add 100 μ LTMB reagent react 10min, add 100 μ L, 0.5mol/L H
2sO
4solution termination reaction; Microplate reader reader reads the absorbancy of each micropore solution in 450nm place.
In embodiments of the present invention, the canonical plotting of the antibiotic concentration-absorbancy absorbancy obtained in step (4) and step (3) set up determines the antibiotic concentration corresponding to this absorbancy and type after comparing.In the present invention, calculating containing amino microbiotic detection limit: as the present embodiment enzyme linked immunological competitive adsorption method measures in the making of microbiotic typical curve, not containing under amino microbiotic standardized solution, confidence level 99% time, repeat 5 times, detection limit=3 × standard deviation.According to these detection limit method of calculation, detection limit of the present invention reaches 0.003nM, and rate of accuracy reached is to 95%.
Containing amino antibiotic detection in embodiment 4 milk
(1) synthesis of enzyme mark thing horseradish peroxidase-template molecule HRP-T
5mg horseradish peroxidase is dissolved in the MES damping fluid of 0.7mL, pH6.0, add 0.2mgEDC and 0.3mg NHS again, 15min is reacted under room temperature, with being separated removing damping fluid under micropore ultracentrifugation filtration unit 3500rpm rotating speed, wash with the PBS of 3mL, pH 7.40, collect the horseradish peroxidase after activation, 3h is hatched containing the amino antibiotic PBS liquid of 8mg immediately with 10mL, then wash away with 4.0mL PBS damping fluid the microbiotic do not connected, be separated under rotating speed 3500rpm with micropore ultracentrifugation filtration unit and obtain enzyme mark thing HRP-T solution;
(2) condition optimizing of enzyme linked immunological competitive adsorption method is explored
Bag quilt: getting concentration prepared by 30 μ L embodiments 2 is in nanometer polymer solution 2 to the 96 hole enzyme plate of the amino microbiotic trace of 0.06mg/ml; At room temperature evaporate into dry, with 300 μ L PBS solution washings;
Close: add containing 300 μ L containing tensio-active agent Tween 20 mass concentration be 0.5% and bovine serum albumin BSA mass concentration be the PBS solution of 0.3%, hatching 2h; And then wash by 300 μ L PBS solution;
Application of sample: add 100 μ L enzyme mark thing HRP-T solution, hatching 2h;
Colour developing with measure: containing Tween 20 mass concentration be 0.5% and BSA mass concentration be 0.3% PBS solution 300 μ L wash, dry, add 100 μ LHRP substrate 3,3', 5,5'-tetramethyl benzidine TMB reagent, hatch after 10min, add 0.5mol/L H
2sO
4100 μ L solution color development stopping reactions, then measure its absorbancy at 450nm place by microplate reader, determine enzyme linked immunological competitive adsorption method optimum location parameter.
(3) enzyme linked immunological competitive adsorption method measures microbiotic
By 40 μ L, concentration for being coated in 96 hole enzyme plates containing amino microbiotic nanometer molecular imprinting polymer solution 2 described in 0.06mg/ml, at room temperature evaporate into dry, wash by PBS solution, add containing 200 μ L containing mass concentration be 1% tensio-active agent Tween 20 and mass concentration be the PBS solution of the bovine serum albumin BSA of 0.1%, hatching 2h, wash by PBS solution, then add in each hole the enzyme mark thing HRP-T solution of 60 ~ 100 μ L and different concns containing amino microbiotic standardized solution incubated at room temperature 1.5h, with containing mass concentration be 1% Tween 20 and mass concentration be the BSA of 0.1% PBS solution washing, add 60 μ LTMB reagent react 2min, add 60 μ L, 0.5mol/L H
2sO
4solution termination reaction, microplate reader reader reads the absorbancy of each micropore solution in 450nm place, sets up the canonical plotting of antibiotic concentration-absorbancy.
(4) containing amino antibiotic detection
The pre-treatment of milk: get a certain amount of milk and carry out centrifugation, collect supernatant liquid, supernatant liquor is transferred in evaporative flask; Solvent evaporated under reduced pressure, redissolves with PBS damping fluid, obtains, containing amino antibiotic PBS damping fluid, preserving, for subsequent use.
Pretreated thing to be detected is detected as follows: by 40 μ L, concentration for being coated in 96 hole enzyme plates containing amino microbiotic nanometer molecular imprinting polymer solution 2 described in 0.06mg/ml, at room temperature evaporate into dry, wash by PBS solution, add containing 200 μ L containing mass concentration be 1% tensio-active agent Tween20 and mass concentration be the PBS solution of the bovine serum albumin BSA of 0.1%, hatching 2h, wash by PBS solution, then add in each hole the enzyme mark thing HRP-T solution of 60 ~ 100 μ L and concentration be 1 degree of 0.0001nM containing amino microbiotic standardized solution incubated at room temperature 1.5h, with containing mass concentration be 1% Tween 20 and mass concentration be the BSA of 0.1% PBS solution washing, add 60 μ LTMB reagent react 2min, add 60 μ L, 0.5mol/L H
2sO
4solution termination reaction, microplate reader reader reads the absorbancy of each micropore solution in 450nm place.
The canonical plotting of the antibiotic concentration-absorbancy absorbancy obtained in step (3) and step (2) set up determines the antibiotic concentration changed corresponding to absorbancy after comparing.
Containing amino antibiotic detection in embodiment 5 water sample
The pre-treatment of water sample: water sample takes from the drainageway of Xiang River and plant, with 0.45um filter membrane removing suspended substance, collect supernatant liquid, supernatant liquor is transferred in evaporative flask; Solvent evaporated under reduced pressure, redissolves with PBS damping fluid, obtains, containing amino antibiotic PBS damping fluid, preserving, for subsequent use.
In water sample containing amino antibiotic detection concrete steps with above-described embodiment 3 or 4 in introduce content same or similar, do not repeat them here.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1., containing amino microbiotic nanometer molecular imprinting polymer and preparation method thereof and an application, it is characterized in that, the method comprises the following steps:
(1) solid phase carrier granulated glass sphere introduces microbiotic
Joined by 250 ~ 300g granulated glass sphere in the NaOH aqueous solution of 120 ~ 160mL, 1 ~ 4mol/L, ebuillition of heated 10 ~ 15min, taking-up granulated glass sphere washes pH=8 ~ 9 to washing lotion with water, dries and obtains hydroxylation granulated glass sphere;
Hydroxylation granulated glass sphere is joined in the dry toluene of 150mL, then add 3.3mL N-[3-(trimethoxy is silica-based) propyl group] quadrol, after violent shake, kept at room temperature overnight, filtration, acetone rinsing, obtain silanized glass pearl;
100g silanized glass pearl is joined in the PBS of 50mL, pH7.4, then adds 3.5mL glutaraldehyde, react 2 ~ 3h under room temperature, suction filtration, washing, collect Semi-finished glass pearl;
Mixed with Semi-finished glass pearl containing amino microbiotic by PBS and 25mg of 50mL, pH 7.4, after violent shake, kept at room temperature overnight, filters, washes, and obtains containing the derivative granulated glass sphere of amino microbiotic.
(2) nano print polymer of synthetic antibiotic trace
By 1mmol N, N'-dimethylene bisacrylamide, 25 ~ 30mmol NIPA, 18 ~ 20mmol N tert butyl acrylamide and 0.0025 ~ 0.003mmol vinylformic acid are by mixing and being dissolved in 100mL ultrapure water, logical nitrogen 20 ~ 30 minutes, mixture is poured in the described granulated glass sphere derivative containing amino microbiotic, then the aqueous solution of 40 ~ 60mg ammonium peroxydisulfate and 13 ~ 26 μ L Tetramethyl Ethylene Diamines is added, after violent shake, 1 ~ 1.5h is reacted under room temperature, reaction mixture is poured in solid phase extraction tube, with the room temperature water cleaning glass pearl of 6 ~ 8, use 80 ~ 100mL again, 60 ~ 70 heat DEG C water wash, naturally cool to room temperature, obtain containing amino microbiotic nanometer molecular imprinting polymer solution with the centrifugation of micropore ultracentrifugation filtration unit.
2. according to claim 1ly to detect containing the application in amino microbiotic containing amino microbiotic nanometer molecular imprinting polymer.
3. apply as claimed in claim 2, it is characterized in that, describedly to comprise the following steps containing amino antibiotic detection:
(1) synthesis of enzyme mark thing horseradish peroxidase-template molecule HRP-T
5 ~ 10mg horseradish peroxidase is dissolved in 0.7 ~ 1.0mL, in the MES damping fluid of pH6.0, add 0.2 ~ 0.4mgEDC and 0.3 ~ 0.6mg NHS again, 15min is reacted under room temperature, with being separated removing damping fluid under micropore ultracentrifugation filtration unit 3500rpm rotating speed, with 3 ~ 5mL, the PBS washing of pH 7.40, collect the horseradish peroxidase after activation, 2 ~ 3h is hatched containing the amino antibiotic PBS liquid of 8 ~ 10mg immediately with 10mL, then wash away with 4.0 ~ 5.0mL PBS damping fluid the microbiotic do not connected, be separated under rotating speed 3500rpm with micropore ultracentrifugation filtration unit and obtain enzyme mark thing HRP-T solution,
(2) canonical plotting makes
Measure different concns containing amino antibiotic absorbancy by enzyme linked immunological competitive adsorption method, set up the canonical plotting of antibiotic concentration-absorbancy;
(3) containing amino antibiotic detection
Pretreated thing to be detected is measured containing amino antibiotic absorbancy by enzyme linked immunological competitive adsorption method, according to this absorbancy and canonical plotting comparison, obtains in thing to be detected containing amino antibiotic concentration;
In step (2) and (3), described enzyme linked immunological competitive adsorption method is specially: be coated in 96 hole enzyme plates containing amino microbiotic nanometer molecular imprinting polymer solution described in the claims 1 being 0.04 ~ 0.06mg/ml by 40 μ L, concentration, at room temperature evaporate into dry, wash by PBS solution, add containing 200 ~ 300 μ L containing mass concentration be 1% tensio-active agent Tween 20 and mass concentration be the PBS solution of the bovine serum albumin BSA of 0.1%, hatching 1h, wash by PBS solution, then add the enzyme mark thing HRP-T solution of 60 ~ 100 μ L in each hole and contain amino microbiotic standardized solution incubated at room temperature 1h, with containing mass concentration be 1% Tween 20 and mass concentration be the BSA of 0.1% PBS solution washing, add 60 ~ 100 μ LTMB reagent react 2 ~ 10min, add 0.5mol/L, 60 ~ 100 μ L H
2sO
4solution termination reaction, microplate reader reader reads the absorbancy of each micropore solution in 450nm place.
4. apply as claimed in claim 3, it is characterized in that, before step (2), also comprise the condition optimizing step of enzyme linked immunological competitive adsorption method, the condition optimizing step detailed process of described enzyme linked immunological competitive adsorption method is:
Bag quilt: get 30 ~ 50 μ L, in nanometer polymer solution to 96 hole enzyme plate that concentration is amino microbiotic trace described in 0.04 ~ 0.06mg/ml claims book 1; At room temperature evaporate into dry, wash by PBS solution;
Close: add containing 300 μ L containing tensio-active agent Tween 20 mass concentration be 0.5 ~ 3% and bovine serum albumin BSA mass concentration be the PBS solution of 0.05 ~ 0.3%, hatching 1 ~ 2h; And then wash 3 times by 300 μ L PBS solution;
Application of sample: add 100 μ L enzyme mark thing HRP-T solution, hatching 2h;
Colour developing with measure: with containing mass concentration be 0.5 ~ 3% Tween 20 and mass concentration be that the PBS solution of the BSA of 0.05 ~ 0.3% wash, drying, adds 100 μ LHRP substrates 3,3', 5,5'-tetramethyl benzidine TMB reagent, after hatching 10min, add 0.5mol/L H
2sO
4100 μ L solution color development stopping reactions, then measure its absorbancy at 450nm place by microplate reader, determine optimal detection parameter.
5. apply as claimed in claim 4, it is characterized in that, in step (3), described thing to be detected is meat, and described meat comprises fish, chicken; The pre-treatment of this meat comprises: fish sample is scaled, removed the peel, and gets muscle along ridge; Chicken class sample is boned, and gets muscle parts.Sample homogenization treatment becomes the sample to be tested of meat gruel shape.Accurately take sample, be placed in polystyrene centrifuge tube, add acidifying acetonitrile, homogenization treatment, whirlpool mixes, ultrasonic extraction, and with the centrifugal 5 ~ 10min of 4000r/min, supernatant liquor is transferred in evaporative flask; Solvent evaporated under reduced pressure, redissolves with PBS damping fluid, obtains containing amino antibiotic PBS damping fluid.
6. apply as claimed in claim 4, it is characterized in that, in step (3), described thing to be detected is milk, and the pre-treatment of described milk is: get milk and carry out centrifugation, and collect supernatant liquid, supernatant liquor is transferred in evaporative flask; Solvent evaporated under reduced pressure, redissolves with PBS damping fluid, obtains containing amino antibiotic PBS damping fluid.
7. apply as claimed in claim 4, it is characterized in that, in step (3), described thing to be detected is water, and the pre-treatment of described water is: 0.45um filter membrane removing suspended substance, and collect supernatant liquid, supernatant liquor is transferred in evaporative flask; Solvent evaporated under reduced pressure, redissolves with PBS damping fluid, obtains containing amino antibiotic PBS damping fluid.
8. the application described in any one of claim 2 ~ 7, is characterized in that, describedly comprises Streptomycin sulphate, gentamicin and sulfamethoxazole containing amino microbiotic.
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| CN108084337A (en) * | 2017-12-06 | 2018-05-29 | 华中科技大学 | The double imprinted materials of the paper substrate of Selective recognition protein and preparation method and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108084337A (en) * | 2017-12-06 | 2018-05-29 | 华中科技大学 | The double imprinted materials of the paper substrate of Selective recognition protein and preparation method and application |
| CN108084337B (en) * | 2017-12-06 | 2019-06-07 | 华中科技大学 | The double imprinted materials of the paper base of Selective recognition protein and preparation method and application |
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