CN105713156A - Preparation method and application of carbostyril nanometer molecularly imprinted polymer - Google Patents
Preparation method and application of carbostyril nanometer molecularly imprinted polymer Download PDFInfo
- Publication number
- CN105713156A CN105713156A CN201610088952.2A CN201610088952A CN105713156A CN 105713156 A CN105713156 A CN 105713156A CN 201610088952 A CN201610088952 A CN 201610088952A CN 105713156 A CN105713156 A CN 105713156A
- Authority
- CN
- China
- Prior art keywords
- solution
- antibiotic
- quinolone antibiotic
- pbs
- add
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F292/00—Macromolecular compounds obtained by polymerising monomers on to inorganic materials
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
-
- 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
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2351/00—Characterised by the use of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives of such polymers
- C08J2351/10—Characterised by the use of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives of such polymers grafted on to inorganic materials
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Inorganic Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a preparation method and application of a carbostyril nanometer molecularly imprinted polymer. The method comprises the steps of introducing quinolone antibiotic onto solid phase carrier glass beads, so that glass beads derived from quinolone antibiotic are obtained; then making the glass beads be subjected to a ultraviolet irradiation reaction with a functional monomer, a cross-linking agent and photoinitiator carbamodithioicacid,N,N-diethyl-phenylmethylester, pouring a reaction mixture into a solid phase extraction tube, washing the glass beads with cold acetonitrile, removing the functional monomer and the cross-linking agent which are not involved into the reaction, and conducting leaching with hot acetonitrile, so that the molecularly imprinted polymer is obtained; cooling the molecularly imprinted polymer to be room temperature, and conducting micropore ultracentrifugal filtering and separation, so that a nanometer molecularly imprinted polymer is obtained. The nanometer molecularly imprinted polymer can be applied to microscale carbostyril antibiotic, in particular to detection of carbostyril antibiotic in food and surface water. The preparation method and application of the carbostyril nanometer molecularly imprinted polymer are 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 quinolinones nanometer molecular imprinting polymer and preparation method thereof and application.
Background technology
Antibiotic is a kind of by microorganism self generation or synthetic, micro-organisms can be suppressed under low concentration or kill the organic chemicals of other microorganisms, except the treatment for human and animal's bacterial infection disease, antibiotic is also taken as growth promoter and feed additive and widely applies in intensive animal husbandry and aquaculture, along with a large amount of antibiotic uses, more and more antibiotic that can not be fully absorbed utilization are expelled directly out by human and animal excreta, these can pass through using of organic fertilizer containing antibiotic feces and migrate between entrance farmland and plant, in addition these antibiotic are possibly through infiltration, runoff, the modes such as leaching move in surface water and groundwater, back flow back into environment.
Monitoring method for antibiotic residue detection mainly has microbial method, immunization, gas chromatography, high performance liquid chromatography and Liquid Chromatography-Tandem Mass Spectrometry at present.Microbial method is simple and quick, but detection 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, quick, and 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 prone to inactivation in the presence of a harsh environment, affects testing result.
Additionally, also have the not Lip river quinoline ketone antibiotic such as the ciprofloxacin detecting in human urine with magnetic fluorescence molecular engram silicon nanosphere or norfloxacin that utilize in molecular engram solid phase extraction-high-efficient liquid phase chromatogram technique analysis Carnis Gallus domesticus；Three kinds of fluoroquinolone antibiotics in magnetic molecularly imprinted polymer Solid-Phase Extraction milk, and detect with HPLC.But the molecular engram method of report is all adopt precipitation polymerization method Synthesis of Molecular Imprinting Polymers at present, response time is long, and the polymer synthesized becomes bulk, need to be used for Solid-Phase Extraction through polishing for a long time, screening, molecularly imprinted polymer utilization rate is not high, additionally needing the technology such as coupling HPLC or HPLC-MS, required instrument is valuable.
Summary of the invention
It is an object of the invention to provide a kind of quinolinones nanometer molecular imprinting polymer and preparation method thereof and application, it is intended to solve existing quinolone antibiotic detection technique and be prone to problems such as inactivating, testing result is not good.
The preparation method that the present invention is achieved in that a kind of quinolinones nanometer molecular imprinting polymer, the method comprises the following steps:
(1) solid phase carrier bead introduces antibiotic
250 ~ 300g bead is joined in the NaOH aqueous solution of 120 ~ 160mL, 1 ~ 4mol/L, ebuillition of heated 10 ~ 15 minutes, takes out bead and washes pH=8 ~ 9 to washing liquid with water, dry and obtain hydroxylating bead；
Hydroxylating bead is joined in the dry toluene of 150mL, add 3.3mLN-[3-(trimethoxy is silica-based) propyl group] ethylenediamine, after being aggressively shaken, kept at room temperature overnight, filtration, acetone rinsing, obtain silanized glass pearl；
In 20mL acetonitrile and 80mLpH6.0PBS buffer solution, add 80 ~ 120mg quinolone antibiotic, 80 ~ 100mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 120 ~ 150mgN-N-Hydroxysuccinimide, room temperature is poured in the above-mentioned silanized glass pearl of 100g after placing 10 ~ 15 minutes, the NaOH aqueous solution adding 1mol/L regulates reactant liquor pH to 7.4,2 ~ 3h, sucking filtration, washing is reacted under room temperature, dry, obtain the derivative bead of quinolone antibiotic；
(2) nano print polymer of quinolone antibiotic trace is synthesized
8.0 ~ 10.0mmol methacrylic acid, 4.0 ~ 5.0mmol ethylene glycol dimethacrylate, 3.0 ~ 3.5mmol trimethylol-propane trimethacrylate, 1.0mmol diethyl-dithio aminocarboxylic acid benzyl ester and 0.1 ~ 0.15mmol tetra-(3-mercaptopropionic acid) pentaerythritol ester are dissolved in the acetonitrile of 10g, obtain mixed solution logical nitrogen 20 minutes in described mixed solution；
Take the derivative bead of quinolone antibiotic described in 25 ~ 30g in tumbler, logical nitrogen 20 minutes in beaker；
Described mixed solution is poured in above-mentioned tumbler, ultra-vioket radiation 1 ~ 3 minute, obtain reactant mixture；
Described reactant mixture is transferred in solid phase extraction tube, with the cold acetonitrile wash bead of 6 ~ 8, obtains Semi-finished glass pearl, logical nitrogen 10 minutes；
Take 220 ~ 270mgPEG4000 and be dissolved in 8mL acetonitrile, logical nitrogen is after 5 minutes, pour above-mentioned Semi-finished glass bead surface into, shake mixing, ultra-vioket radiation 1 ~ 3 minute, described reactant mixture is poured in solid phase extraction tube, with the cold acetonitrile wash bead of 6 ~ 8, then with 80 ~ 100mL, 60 ~ 70 DEG C of acetonitrile drip washing, obtain the nanometer molecular imprinting polymer solution of quinolone antibiotic trace, naturally cool to room temperature, be centrifugally separating to obtain quinolone antibiotic nanometer molecular imprinting polymer solution with micropore ultracentrifugation filter (30kDaMWCO).
Invention further provides the application in detection quinolone antibiotic of the above-mentioned quinolinones nanometer molecular imprinting polymer.
Preferably, the detection of described quinolone antibiotic comprises the following steps:
(1) synthesis of enzyme mark thing horseradish peroxidase-template molecule HRP-T
20 ~ 30mg quinolone antibiotic is dissolved in the MES buffer of 0.3mL acetonitrile and 2.0mL, pH6.0, add 0.2 ~ 0.4mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 0.3 ~ 0.6mgN-N-Hydroxysuccinimide, after reacting 15 minutes under room temperature, hatch 2 hours with the 10mL PBS buffer solution containing the pH7.40 of 5 ~ 8mg horseradish peroxidase immediately, then wash away the antibiotic not coupled with 4.0 ~ 5.0mLPBS buffer, separate under rotating speed 3500rpm with micropore ultracentrifugation filter (30kDaMWCO) and obtain enzyme mark thing HRP-T solution；
(2) canonical plotting makes
Measured the absorbance of variable concentrations quinolone antibiotic by enzyme linked immunological competitive Adsorption method, set up the canonical plotting of antibiotic concentration-absorbance；
(3) detection of quinolone antibiotic
Pretreated thing to be detected is measured by enzyme linked immunological competitive Adsorption method the absorbance of quinolone antibiotic, according to this absorbance and canonical plotting comparison, obtains the concentration of quinolone antibiotic in thing to be detected；
In step (2) and (3), described enzyme linked immunological competitive Adsorption method is particularly as follows: be that 0.04 ~ 0.06mg/mL quinolinones nanometer molecular imprinting polymer solution is coated in micropore dish by 40 μ L, concentration；PBS solution is washed, and is subsequently adding the 300 μ L PBS solution containing the Tween20 that mass concentration is 1.5% and BSA that mass concentration is 0.15%；Hatching 1 ~ 1.5h, PBS solution is washed, then each hole adds enzyme mark thing HRP-T solution and the quinolone antibiotic liquid incubated at room temperature 1h to be measured of 100 μ L, wash by the PBS solution containing Tween20 and BSA, add 60 ~ 100 μ LTMB reagent colour developments to react 10 minutes, add 60 ~ 100 μ L0.5mol/LH2SO4Solution terminates reaction；Microplate reader reader reads the absorbance of each micropore solution in 450nm place.
Preferably, before step (2), also including 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:
It is coated: take quinolinones nanometer molecular imprinting polymer solution that concentration in 30 ~ 50 μ L claims (1) is 0.04 ~ 0.06mg/ml to 96 hole ELISA Plate；At room temperature evaporate into dry, with 300 μ LPBS solution washings；
Close: add containing 200 ~ 300 μ L PBS solution containing the surfactant Tween20 that mass concentration is 1 ~ 3% and bovine serum albumin BSA that mass concentration is 0.1 ~ 0.3%, hatch 1 ~ 2h；Then again with 300 ~ 400 μ LPBS solution washings；
Application of sample: add 60 ~ 100 μ L enzyme mark thing HRP-T solution, hatches 1 ~ 2h；
Colour developing and mensuration: add 300 ~ 400 μ L PBS solution containing the Tween20 that mass concentration is 1 ~ 3% and the BSA that mass concentration is 0.1 ~ 0.3% washing, dry, add 60 ~ 100 μ LHRP substrate TMB reagent, addition H after hatching 2 ~ 10 minutes2SO4Solution color development stopping is reacted, and finally measures its absorbance at 450nm place by microplate reader, it is determined that optimal detection parameter.
Preferably, in step (3), described thing to be detected is meat, and described meat includes Fish, Carnis Gallus domesticus；The pretreatment of this meat includes: Fish sample is scaled, removed the peel, and takes muscle along ridge；Carnis Gallus domesticus class sample is boned, and takes muscle parts.Sample homogenizing is processed into the sample to be tested of meat paste shape.Accurately weighing sample, be placed in polystyrene centrifuge tube, add acidifying acetonitrile, homogenizing processes, and whirlpool mixes, ultrasonic extraction, and with 4000r/ minute centrifugal 5 ~ 10 minutes, supernatant was transferred in evaporative flask；Solvent is evaporated under reduced pressure, redissolves with PBS, obtain the PBS of quinolone antibiotic.
Preferably, in step (3), described thing to be detected is milk, and the pretreatment of described milk is: taking milk and be centrifuged separating, collect the supernatant, supernatant is transferred in evaporative flask；Solvent is evaporated under reduced pressure, redissolves with PBS, obtain the PBS of quinolone antibiotic.
Preferably, in step (3), described thing to be detected is water, and the pretreatment of described water is: 0.45um filter membrane removes float, collects the supernatant, and supernatant is transferred in evaporative flask；Solvent is evaporated under reduced pressure, redissolves with PBS, obtain the PBS of quinolone antibiotic.
Preferably, described quinolone antibiotic includes enrofloxacin, ofloxacin, norfloxacin.
Shortcoming and defect compared to prior art, the method have the advantages that the nanometer molecular imprinting polymer of Solid phase synthesis antibiotic trace of the present invention can directly replace antibody, with in bionical enzyme-linked immunosorbent assay water and food (cattle milk, Carnis Gallus domesticus, Carnis Sus domestica, the flesh of fish, egg etc.) in quinolone antibiotic, it is possible not only to avoid the defect of above method, and the pre-treatment of the method sample is simple, it is made without separating, simple and quick, detection specificity is high, detection limit is low, wherein, the detection limit of quinolone antibiotic is reached 0.003nM by nanometer molecular imprinting polymer of the present invention, rate of accuracy reached is to 95%.
Detailed description of the invention
In order to make the purpose 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 is only in order to explain the present invention, is not intended to limit the present invention.
The preparation of embodiment 1 quinolone antibiotic nanometer molecular imprinting polymer solution
Taking bead 300g, add 160mL, 2mol/LNaOH aqueous solution, ebuillition of heated 10 minutes, bead washes the pH=9 to washing liquid with water, dries, obtains hydroxylating bead.The dry toluene of 150mL adds to above-mentioned hydroxylating bead, is subsequently adding 3mL3-amine propyl trimethoxy silicane, is aggressively shaken mixture, kept at room temperature overnight, filters, acetone rinsing, collect to obtain silanized glass pearl.
In 20mL acetonitrile and 80mLpH6.0PBS buffer solution, add 80mg quinolone antibiotic, 80mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 120mgN-N-Hydroxysuccinimide, room temperature is poured in the above-mentioned silanized glass pearl of 100g after placing 10 ~ 15 minutes, the NaOH aqueous solution adding 1mol/L regulates reactant liquor pH to 7.4,2 ~ 3h, sucking filtration, washing is reacted under room temperature, dry, collect the derivative bead of quinolone antibiotic；
(2) nano print polymer of synthetic antibiotic trace
The mixing of 10.0mmol methacrylic acid, 5.0mmol ethylene glycol dimethacrylate, 3.5mmol cross-linking agent trimethylol-propane trimethacrylate, 1.0mmol chain initiator diethyl-dithio aminocarboxylic acid benzyl ester and 0.15mmol chain transfer agents four (3-mercaptopropionic acid) pentaerythritol ester is dissolved in the acetonitrile of 10g, obtain mixed solution, logical nitrogen 20 minutes in mixed solution.
By derivative for quinolone antibiotic described in 30g bead in flat bottom beaker, logical nitrogen 20 minutes in beaker.
Described mixed solution is poured in above-mentioned beaker, irradiation under ultraviolet ray 2 minutes, obtain reactant mixture；
Described reactant mixture is poured in solid phase extraction tube, with the cold acetonitriles of 6 (namely acetonitrile is used from cold room taking-up) cleaning glass pearl, obtains Semi-finished glass pearl, logical nitrogen 10 minutes；
Taking 220mgPEG4000 and be dissolved in 8mL acetonitrile, logical nitrogen, after 5 minutes, pours above-mentioned Semi-finished glass bead surface, shake mixing, irradiation under ultraviolet ray 2 minutes into；
Described reactant mixture is poured in solid phase extraction tube, with the cold acetonitriles of 6 (namely acetonitrile is used from cold room taking-up) cleaning glass pearl, then with 100mL, 60 DEG C of acetonitrile drip washing, obtain the nanometer molecular imprinting polymer solution of quinolone antibiotic trace, naturally cool to room temperature, be centrifugally separating to obtain, with micropore ultracentrifugation filter (30kDaMWCO), the quinolone antibiotic nanometer molecular imprinting polymer solution 1 that concentration is 0.05mg/mL.
The preparation of embodiment 2 quinolone antibiotic nanometer molecular imprinting polymer solution
(1) solid phase carrier bead introduces antibiotic
250g bead is joined in the NaOH aqueous solution of 120mL, 1mol/L, ebuillition of heated 10 minutes, takes out bead and washes the pH=8 to washing liquid with water, dry and obtain hydroxylating bead；
Being joined in the dry toluene of 150mL by above-mentioned hydroxylating bead, add 3mL3-amine propyl trimethoxy silicane, after being aggressively shaken, ambient temperatare puts 16 ~ 20h, filtration, acetone rinsing, obtains silanized glass pearl；
In 20mL acetonitrile and 80mLpH6.0PBS buffer solution, add 120mg quinolone antibiotic, 100mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 150mgN-N-Hydroxysuccinimide, room temperature is poured in the above-mentioned silanized glass pearl of 100g after placing 10 ~ 15 minutes, the NaOH aqueous solution adding 1mol/L regulates reactant liquor pH to 7.4,2 ~ 3h, sucking filtration, washing is reacted under room temperature, dry, collect the derivative bead of quinolone antibiotic；
(2) nano print polymer of synthetic antibiotic trace
8.0mmol methacrylic acid, 4.0mmol ethylene glycol dimethacrylate, 3.0mmol trimethylol-propane trimethacrylate, 1.0mmol diethyl-dithio aminocarboxylic acid benzyl ester and 0.1mmol tetra-(3-mercaptopropionic acid) pentaerythritol ester are dissolved in the acetonitrile of 10g, obtain mixed solution logical nitrogen 20 minutes in described mixed solution；
By derivative for quinolone antibiotic described in 25g bead in tumbler, logical nitrogen 20 minutes in cup.
Described mixed solution is poured in above-mentioned tumbler, irradiation under ultraviolet ray 3 minutes, obtain reactant mixture；
Described reactant mixture is poured in solid phase extraction tube, with the cold acetonitriles of 8 (namely acetonitrile is used from cold room taking-up) cleaning glass pearl, obtains Semi-finished glass pearl, logical nitrogen 10 minutes；
Taking 270mgPEG4000 and be dissolved in 8mL acetonitrile, logical nitrogen, after 5 minutes, pours above-mentioned Semi-finished glass bead surface, shake mixing, irradiation under ultraviolet ray 3 minutes into；
Described reactant mixture is poured in solid phase extraction tube, with the cold acetonitriles of 8 (namely acetonitrile is used from cold room taking-up) cleaning glass pearl, then with 100mL, 70 DEG C of acetonitrile drip washing, obtain the nanometer molecular imprinting polymer solution of quinolone antibiotic trace, naturally cool to room temperature, be centrifugally separating to obtain, with micropore ultracentrifugation filter (30kDaMWCO), the quinolone antibiotic nanometer molecular imprinting polymer solution 2 that concentration is 0.05mg/mL.
The detection of quinolone antibiotic in embodiment 3 Carnis Gallus domesticus, the flesh of fish
(1) synthesis of enzyme mark thing horseradish peroxidase-template molecule HRP-T
30mg quinolone antibiotic is dissolved in the MES buffer of 0.3mL acetonitrile and 2.0mL, pH6.0, add 0.4mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 0.6mgN-N-Hydroxysuccinimide, after reacting 10 minutes under room temperature, hatch 2 hours with the 10mL PBS buffer solution containing the pH7.40 of 8mg horseradish peroxidase immediately, then wash away the antibiotic not coupled with 5.0mLPBS buffer, separate (rotating speed 3500rpm) by micropore ultracentrifugation defecator (30kDaMWCO) ultracentrifugation and obtain enzyme mark thing HRP-T solution.
(2) condition optimizing of enzyme linked immunological competitive Adsorption method
It is coated: take in quinolinones nanometer molecular imprinting polymer solution 1 to the 96 hole ELISA Plate that concentration is 0.05mg/mL that 40 μ L embodiments 1 obtain；At room temperature evaporate into dry, with 300 μ LPBS solution washings；
Close: add containing the 300 μ L PBS solution containing the surfactant Tween20 that mass concentration is 1% and bovine serum albumin BSA that mass concentration is 0.1%, hatch 1h；Then again with 300 μ LPBS solution washings；
Application of sample: add 100 μ LHRP-T, hatches 1h；
Colour developing and mensuration: add the washing of the 300 μ L PBS solution containing the Tween20 that mass concentration is 1% and the BSA that mass concentration is 0.3%, dry, add 100 μ LHRP substrate TMB reagent, add H after hatching 10 minutes2SO4Solution color development stopping is reacted, and finally measures its absorbance at 450nm place by microplate reader, it is determined that enzyme linked immunological competitive Adsorption method optimal detection parameter.
(3) enzyme linked immunological competitive Adsorption method measures the making of antibiotic standard curve
The quinolinones nanometer molecular imprinting polymer solution 1 that 40 μ L, concentration are 0.05mg/mL is coated in micropore dish；At room temperature evaporating into dry, PBS solution is washed, and is subsequently adding containing the PBS solution 300 μ L that Tween20 mass concentration is 1.5% and BSA mass concentration 0.15%；Hatching 1.5h, PBS solution is washed, then enzyme mark thing HRP-T solution and variable concentrations, dissimilar (including enrofloxacin, ofloxacin, norfloxacin) quinolone antibiotic standard solution of adding 100 μ L in each hole at room temperature hatch 1h, wash by the PBS solution containing Tween20 and BSA, add 100 μ LTMB reagent colour developments to react 10 minutes, add 100 μ L, 0.5mol/LH2SO4Solution terminates reaction；Microplate reader reader reads the absorbance of each micropore solution in 450nm place, makes the canonical plotting of antibiotic concentration-absorbance.
(4) detection of quinolone antibiotic
Carnis Gallus domesticus, the flesh of fish pretreatment: Carnis Gallus domesticus, milk, the food such as flesh of fish is purchased from local market.Fish sample is scaled, is removed the peel, and takes muscle along ridge；Carnis Gallus domesticus class sample is boned, and takes muscle parts.Sample homogenizing is processed into the sample to be tested of meat paste shape.Accurately weighing sample, be placed in polystyrene centrifuge tube, add acidifying acetonitrile, homogenizing processes, and whirlpool mixes, ultrasonic extraction, and with 4000r/ minute centrifugal 5 ~ 10 minutes, supernatant was transferred in evaporative flask；Solvent is evaporated under reduced pressure, redissolves with PBS, obtain the PBS of quinolone antibiotic, preserve, standby.
It is that 0.05mg/mL quinolinones nanometer molecular imprinting polymer solution 1 is coated in micropore dish by 40 μ L, concentration；PBS solution is washed, and is subsequently adding containing the PBS solution 300 μ L that Tween20 mass concentration is 1.5% and BSA mass concentration 0.15%；Hatching 1.5h, PBS solution is washed, the PBS of the enzyme mark thing HRP-T solution and quinolone antibiotic that then add 100 μ L in each hole at room temperature hatches 1h, wash by the PBS solution containing Tween20 and BSA, add 100 μ LTMB reagent colour developments to react 10 minutes, add 100 μ L, 0.5mol/LH2SO4Solution terminates reaction；Microplate reader reader reads the absorbance of each micropore solution in 450nm place.After the canonical plotting of the antibiotic concentration-absorbance absorbance obtained in step (4) and step (3) set up is compared, it is determined that the antibiotic concentration corresponding to this absorbance.In embodiments of the present invention, the calculating of detection limit: the enzyme linked immunological competitive Adsorption method such as the present embodiment step (3) measures in the making of antibiotic standard curve, it does not have under quinolone antibiotic standard solution, confidence level 99% time, repeat 5 times, detection limit=3 × standard deviation.According to these detection limit computational methods, detection limit of the present invention reaches 0.003nM, and rate of accuracy reached is to 95%.
The detection of quinolone antibiotic in embodiment 4 milk
(1) synthesis of enzyme mark thing horseradish peroxidase-template molecule HRP-T
20mg quinolone antibiotic is dissolved in the MES buffer of 0.3mL acetonitrile and 2.0mL, pH6.0, add 0.2mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 0.3mgN-N-Hydroxysuccinimide, after reacting 10 minutes under room temperature, hatch 2 hours with the 10mL PBS buffer solution containing the pH7.40 of 5mg horseradish peroxidase immediately, then wash away the antibiotic not coupled with 5.0mLPBS buffer, separate (rotating speed 3500rpm) by micropore ultracentrifugation defecator (30kDaMWCO) ultracentrifugation and obtain enzyme mark thing HRP-T solution.
(2) condition of enzyme linked immunological competitive Adsorption method is excellent
It is coated: take in quinolinones nanometer molecular imprinting polymer solution 2 to the 96 hole ELISA Plate that concentration is 0.04mg/ml that 40 μ L embodiments 2 obtain；At room temperature evaporate into dry, with 300 μ LPBS solution washings；
Close: add containing the 300 μ L PBS solution containing the surfactant Tween20 that mass concentration is 3% and bovine serum albumin BSA that mass concentration is 0.3%, hatch 2h；Then again with 400 μ LPBS solution washings；
Application of sample: add 60 μ L enzyme mark thing HRP-T solution, hatches 2h；
Colour developing and mensuration: add the washing of the 400 μ L PBS solution containing the Tween20 that mass concentration is 3% and the BSA that mass concentration is 0.1%, dry, add 60 μ LHRP substrate TMB reagent, add H after hatching 2 minutes2SO4Solution color development stopping is reacted, and finally measures its absorbance at 450nm place by microplate reader, makes the canonical plotting of antibiotic concentration-absorbance, it is determined that enzyme linked immunological competitive Adsorption method optimal detection parameter.
(3) enzyme linked immunological competitive Adsorption method measures the making of antibiotic standard curve
It is that 0.050mg/mL quinolinones nanometer molecular imprinting polymer solution 2 is coated in micropore dish by 40 μ L, concentration；PBS solution is washed, and is subsequently adding the 300 μ L PBS solution containing the Tween20 that mass concentration is 1.5% and BSA that mass concentration is 0.15%；Hatching 1 ~ 1.5h, PBS solution is washed, then the enzyme mark thing HRP-T solution and variable concentrations, the dissimilar quinolone antibiotic standard solution that add 100 μ L in each hole at room temperature hatch 1h, wash by the PBS solution containing Tween20 and BSA, add 60 μ LTMB reagent colour developments to react 10 minutes, add 60 μ L0.5mol/LH2SO4Solution terminates reaction；Microplate reader reader reads the absorbance of each micropore solution in 450nm place, makes the canonical plotting of antibiotic concentration-absorbance.
(4) enzyme linked immunological competitive Adsorption method measures antibiotic
The pretreatment of milk: taking a certain amount of milk and be centrifuged separating, collect the supernatant, supernatant is transferred in evaporative flask；Solvent is evaporated under reduced pressure, redissolves with PBS, obtain the antibiotic PBS of quinolone containing, preserve, standby.
It is that 0.050mg/mL quinolinones nanometer molecular imprinting polymer solution 2 is coated in micropore dish by 40 μ L, concentration；PBS solution is washed, and is subsequently adding the 300 μ L PBS solution containing the Tween20 that mass concentration is 1.5% and BSA that mass concentration is 0.15%；Hatching 1 ~ 1.5h, PBS solution is washed, then the enzyme mark thing HRP-T solution and variable concentrations, the antibiotic PBS of quinolone containing that add 100 μ L in each hole at room temperature hatch 1h, wash by the PBS solution containing Tween20 and BSA, add 60 μ LTMB reagent colour developments to react 10 minutes, add 60 μ L0.5mol/LH2SO4Solution terminates reaction；Microplate reader reader reads the absorbance of each micropore solution in 450nm place.
After the canonical plotting of the antibiotic concentration-absorbance absorbance obtained in step (4) and step (3) set up is compared, it is determined that the antibiotic concentration corresponding to this absorbance.
The detection of quinolone antibiotic in embodiment 5 water body
The pretreatment of water sample: water sample takes from the drainage of Xiang River and plant, removes float with 0.45um filter membrane, collects the supernatant, and supernatant is transferred in evaporative flask；Solvent is evaporated under reduced pressure, redissolves with PBS, obtain the antibiotic PBS of quinolone containing, preserve, standby.
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 presently preferred embodiments of the present invention, not in order to limit the present invention, all make within the spirit and principles in the present invention any repair this, equivalent replace and this enters, should be included within protection scope of the present invention.
Claims (8)
1. the preparation method of a quinolinones nanometer molecular imprinting polymer, it is characterised in that the method comprises the following steps:
(1) solid phase carrier bead introduces antibiotic
250 ~ 300g bead is joined in the NaOH aqueous solution of 120 ~ 160mL, 1 ~ 4mol/L, ebuillition of heated 10 ~ 15 minutes, takes out bead and washes pH=8 ~ 9 to washing liquid with water, dry and obtain hydroxylating bead；
Being joined by hydroxylating bead in the dry toluene of 150mL, add 3mL3-amine propyl trimethoxy silicane, after being aggressively shaken, ambient temperatare is put 12 ~ 16 hours, filtration, acetone rinsing, obtains silanized glass pearl；
In 20mL acetonitrile and 80mLpH6.0PBS buffer solution, add 80 ~ 120mg quinolone antibiotic, 80 ~ 100mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 120 ~ 150mgN-N-Hydroxysuccinimide, room temperature is poured in the above-mentioned silanized glass pearl of 100g after placing 10 ~ 15 minutes, the NaOH aqueous solution adding 1mol/L regulates reactant liquor pH to 7.4,2 ~ 3h, sucking filtration, washing is reacted under room temperature, dry, collect the derivative bead of quinolone antibiotic；
(2) nano print polymer of quinolone antibiotic trace is synthesized
8.0 ~ 10.0mmol methacrylic acid, 4.0 ~ 5.0mmol ethylene glycol dimethacrylate, 3.0 ~ 3.5mmol trimethylol-propane trimethacrylate, 1.0mmol diethyl-dithio aminocarboxylic acid benzyl ester and 0.1 ~ 0.15mmol tetra-(3-mercaptopropionic acid) pentaerythritol ester are dissolved in the acetonitrile of 10g, obtain mixed solution logical nitrogen 20 minutes in described mixed solution；
Take the derivative bead of quinolone antibiotic described in 25 ~ 30g in tumbler, logical nitrogen 20 minutes in beaker；
Described mixed solution is poured in above-mentioned beaker, ultra-vioket radiation 1 ~ 3 minute, obtain reactant mixture；
Described reactant mixture is poured in solid phase extraction tube, with the cold acetonitrile wash bead of 6 ~ 8, obtains Semi-finished glass pearl, logical nitrogen 10 minutes；
Take 220 ~ 270mgPEG4000 and be dissolved in 8mL acetonitrile, logical nitrogen is after 5 minutes, pour above-mentioned Semi-finished glass bead surface into, shake mixing, ultra-vioket radiation 1 ~ 3 minute, described reactant mixture is poured in solid phase extraction tube, with the cold acetonitrile wash bead of 6 ~ 8, then with 100mL, 60 ~ 70 DEG C of acetonitrile drip washing, obtain the nanometer molecular imprinting polymer solution of quinolone antibiotic trace, naturally cool to room temperature, be centrifugally separating to obtain quinolone antibiotic nanometer molecular imprinting polymer solution with micropore ultracentrifugation filter.
2. the quinolinones nanometer molecular imprinting polymer as claimed in claim 1 application in detection quinolone antibiotic.
3. apply as claimed in claim 2, it is characterised in that the detection of described quinolone antibiotic comprises the following steps:
(1) synthesis of enzyme mark thing horseradish peroxidase-template molecule HRP-T
20 ~ 30mg quinolone antibiotic is dissolved in the MES buffer of 0.3mL acetonitrile and 2.0mL, pH6.0, add 0.2 ~ 0.4mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 0.3 ~ 0.6mgN-N-Hydroxysuccinimide, after reacting 15 minutes under room temperature, hatch 2 hours with the 10mL PBS buffer solution containing the pH7.40 of 5 ~ 8mg horseradish peroxidase immediately, then wash away the antibiotic not coupled with 4.0 ~ 5.0mLPBS buffer, separate under rotating speed 3500rpm with micropore ultracentrifugation filter and obtain enzyme mark thing HRP-T solution；
(2) canonical plotting makes
Measured the absorbance of variable concentrations quinolone antibiotic by enzyme linked immunological competitive Adsorption method, set up the canonical plotting of antibiotic concentration-absorbance；
(3) detection of quinolone antibiotic
Pretreated thing to be detected is measured by enzyme linked immunological competitive Adsorption method the absorbance of quinolone antibiotic, according to this absorbance and canonical plotting comparison, obtains the concentration of quinolone antibiotic in thing to be detected；
In step (2) and (3), described enzyme linked immunological competitive Adsorption method is particularly as follows: be that 0.04 ~ 0.06mg/mL quinolinones nanometer molecular imprinting polymer solution is coated in micropore dish by 40 μ L, concentration；PBS solution is washed, and is subsequently adding the 300 μ L PBS solution containing the Tween20 that mass concentration is 1.5% and BSA that mass concentration is 0.15%；Hatching 1 ~ 1.5h, PBS solution is washed, then each hole adds enzyme mark thing HRP-T solution and the quinolone antibiotic liquid incubated at room temperature 1h to be measured of 100 μ L, wash by the PBS solution containing Tween20 and BSA, add 60 ~ 100 μ LTMB reagent colour developments to react 10 minutes, add 60 ~ 100 μ L0.5mol/LH2SO4Solution terminates reaction；Microplate reader reader reads the absorbance of each micropore solution in 450nm place.
4. applying as claimed in claim 3, it is characterised in that before step (2), also include 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:
It is coated: take quinolinones nanometer molecular imprinting polymer solution that concentration in 30 ~ 50 μ L claims (1) is 0.04 ~ 0.06mg/ml to 96 hole ELISA Plate；At room temperature evaporate into dry, with 300 μ LPBS solution washings；
Close: add containing 200 ~ 300 μ L PBS solution containing the surfactant Tween20 that mass concentration is 1 ~ 3% and bovine serum albumin BSA that mass concentration is 0.1 ~ 0.3%, hatch 1 ~ 2h；Then again with 300 ~ 400 μ LPBS solution washings；
Application of sample: add 60 ~ 100 μ L enzyme mark thing HRP-T solution, hatches 1 ~ 2h；
Colour developing and mensuration: add 300 ~ 400 μ L PBS solution containing the Tween20 that mass concentration is 1 ~ 3% and the BSA that mass concentration is 0.1 ~ 0.3% washing, dry, add 60 ~ 100 μ LHRP substrate TMB reagent, addition H after hatching 2 ~ 10 minutes2SO4Solution color development stopping is reacted, and finally measures its absorbance at 450nm place by microplate reader, it is determined that optimal detection parameter.
5. applying as claimed in claim 4, it is characterised in that in step (3), described thing to be detected is meat, and described meat includes Fish, Carnis Gallus domesticus；The pretreatment of this meat includes: Fish sample is scaled, removed the peel, and takes muscle along ridge；Carnis Gallus domesticus class sample is boned, and takes muscle parts, and sample homogenizing is processed into the sample to be tested of meat paste shape；Accurately weighing sample, be placed in polystyrene centrifuge tube, add acidifying acetonitrile, homogenizing processes, and whirlpool mixes, ultrasonic extraction, and with 4000r/ minute centrifugal 5 ~ 10 minutes, supernatant was transferred in evaporative flask；Solvent is evaporated under reduced pressure, redissolves with PBS, obtain the PBS of quinolone antibiotic.
6. applying as claimed in claim 4, it is characterised in that in step (3), described thing to be detected is milk, and the pretreatment of described milk is: taking milk and be centrifuged separating, collect the supernatant, supernatant is transferred in evaporative flask；Solvent is evaporated under reduced pressure, redissolves with PBS, obtain the PBS of quinolone antibiotic.
7. applying as claimed in claim 4, it is characterised in that in step (3), described thing to be detected is water, and the pretreatment of described water is: 0.45um filter membrane removes float, collects the supernatant, and supernatant is transferred in evaporative flask；Solvent is evaporated under reduced pressure, redissolves with PBS, obtain the PBS of quinolone antibiotic.
8. the application described in any one of claim 2 ~ 7, it is characterised in that described quinolone antibiotic includes enrofloxacin, ofloxacin, norfloxacin.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2015103195487 | 2015-06-11 | ||
| CN201510319548.7A CN104910314A (en) | 2015-06-11 | 2015-06-11 | Quinolone nano molecular imprinting polymer as well as preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105713156A true CN105713156A (en) | 2016-06-29 |
Family
ID=54079797
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510319548.7A Pending CN104910314A (en) | 2015-06-11 | 2015-06-11 | Quinolone nano molecular imprinting polymer as well as preparation method and application thereof |
| CN201610088952.2A Pending CN105713156A (en) | 2015-06-11 | 2016-02-17 | Preparation method and application of carbostyril nanometer molecularly imprinted polymer |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510319548.7A Pending CN104910314A (en) | 2015-06-11 | 2015-06-11 | Quinolone nano molecular imprinting polymer as well as preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN104910314A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107300582A (en) * | 2017-08-16 | 2017-10-27 | 杭州电子科技大学 | Antibiotic detection means and its manufacture method, detection method based on microsensor |
| CN113237874B (en) * | 2021-04-12 | 2024-03-26 | 牛连信 | Test solution for rapidly identifying whether enrofloxacin is contained in eggs and detection method thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104910314A (en) * | 2015-06-11 | 2015-09-16 | 衡阳师范学院 | Quinolone nano molecular imprinting polymer as well as preparation method and application thereof |
| CN106117558B (en) * | 2016-06-30 | 2018-08-28 | 佛山科学技术学院 | A kind of method for separating and detecting of pyrimethamine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103785476A (en) * | 2014-01-15 | 2014-05-14 | 江苏大学 | Preparation method of surface imprinting CdS compound photocatalyst based on magnetic carbon material |
| CN104910314A (en) * | 2015-06-11 | 2015-09-16 | 衡阳师范学院 | Quinolone nano molecular imprinting polymer as well as preparation method and application thereof |
-
2015
- 2015-06-11 CN CN201510319548.7A patent/CN104910314A/en active Pending
-
2016
- 2016-02-17 CN CN201610088952.2A patent/CN105713156A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103785476A (en) * | 2014-01-15 | 2014-05-14 | 江苏大学 | Preparation method of surface imprinting CdS compound photocatalyst based on magnetic carbon material |
| CN104910314A (en) * | 2015-06-11 | 2015-09-16 | 衡阳师范学院 | Quinolone nano molecular imprinting polymer as well as preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 利维亚•尼库、蒂埃里•雷克列著，曹峥译: "《微纳机电生物传感器》", 31 July 2015, 机械工业出版社 * |
| 吴正红: "《纳米药剂学》", 31 July 2011, 江苏美术出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107300582A (en) * | 2017-08-16 | 2017-10-27 | 杭州电子科技大学 | Antibiotic detection means and its manufacture method, detection method based on microsensor |
| CN113237874B (en) * | 2021-04-12 | 2024-03-26 | 牛连信 | Test solution for rapidly identifying whether enrofloxacin is contained in eggs and detection method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104910314A (en) | 2015-09-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105713156A (en) | Preparation method and application of carbostyril nanometer molecularly imprinted polymer | |
| JP4758341B2 (en) | Chromatographic detector, inspection method and kit using the same | |
| Adrian et al. | Preparation of antibodies and development of an enzyme-linked immunosorbent assay (ELISA) for the determination of doxycycline antibiotic in milk samples | |
| CN101261271B (en) | Sudan red 1 immunity-chromatography test paper detection method | |
| CN106932584B (en) | A kind of salbutamol immuno magnetic cell separation enrichment kit and its application | |
| CN105277424B (en) | A kind of Ractopamine immuno magnetic cell separation enrichment kit and its application | |
| CN101799469A (en) | Enzyme linked immunosorbent assay kit for detecting residual clenbuterol hydrochloride and a detection method thereof | |
| CN103116026B (en) | Quick detection method for food-borne pathogens based on immunomagnetic separation of Fe3O4 nano materials | |
| CN105301234A (en) | Clenbuterol immunomagnetic bead separation and enrichment kit and application thereof | |
| CN103275217A (en) | Magnetic bead method for quickly purifying antibody | |
| CN102353775A (en) | Immunochromatographic test strip for detecting malachite green (MG) and preparation process thereof | |
| CN105158459A (en) | Method for high-sensitivity and low-background detection of fumonisin B1 through hydrogel silica photon crystal microsphere chemiluminescence method | |
| CN105153367A (en) | Preparation method of dicyandiamide mesoporous surface molecularly imprinted polymer microspheres | |
| CN1677107A (en) | Immune antibody for testing residual of polyether-like antibiotic and use thereof | |
| CN114113582B (en) | Metal organic framework nanoenzyme biological probe and ELISA kit | |
| CN105334325A (en) | Microfluidic immune chip analysis method based on biotin and streptavidine system | |
| CN107991277B (en) | Serotonin-magnetic particle composite and method for enriching sialylated glycoprotein | |
| CN110007078A (en) | A kind of Sample pretreatment kit of deoxynivalenol enol | |
| CN103364546B (en) | A kind of kit and method detecting Furaxone metabolite | |
| CN102323415A (en) | Immunochromatography test paper for detecting Ciprofloxacin and preparation method thereof | |
| JP2009229320A (en) | Carrier and process for producing it | |
| JP5500791B2 (en) | New inspection method and inspection kit used therefor | |
| CN104987460A (en) | Amino-contained antibiotic nanometer molecularly imprinted polymer and preparation method and application thereof | |
| CN104817639B (en) | Preparation method and application of hexamethylenetetramine complete antigen | |
| CN102928409A (en) | Magnetic particle chemiluminescence kit for detecting lincomycin, and applications thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160629 |