CN109283347A - The method that nano biological sensor based on immunomagnetic beads and fluorescence quantum quickly detects Enrofloxacin in broiler chicken - Google Patents
The method that nano biological sensor based on immunomagnetic beads and fluorescence quantum quickly detects Enrofloxacin in broiler chicken Download PDFInfo
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- G—PHYSICS
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- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/543—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
- G01N33/54326—Magnetic particles
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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
- G01N33/543—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
Abstract
The invention discloses a kind of methods that the nano biological sensor based on immunomagnetic beads and fluorescence quantum quickly detects Enrofloxacin in broiler chicken.Enrofloxacin monoclonal antibody is modified in carboxylated magnetic bead surfaces, is closed with bovine serum albumin(BSA), the immunomagnetic beads solution of enrofloxacin monoclonal antibody modification closed containing bovine serum albumin(BSA) is prepared；Enrofloxacin and bovine serum albumin(BSA) are coupled, Enrofloxacin-bovine serum albumin(BSA) compound is prepared；By obtained BSA-ENR compound and carboxylated quantum point coupling, quantum dot-bovine serum albumin(BSA)-Enrofloxacin compound is prepared；Chicken Tissues sample is handled, chicken meat sample extracting solution is obtained；The concentration of Enrofloxacin in quantitative detection extracting solution.The method of the present invention is simple, quickly, high sensitivity, and do not need complicated sample pretreatment process, have the potentiality of field quick detection Enrofloxacin, there is good development prospect.
Description
Technical field
The present invention relates to a kind of nano biological sensor based on immunomagnetic beads (IMBs) and fluorescence quantum (QDs) is quick
The method for detecting Enrofloxacin in broiler chicken.
Background technique
Enrofloxacin (Enrofloxacin, ENR), also known as Ethyl Ciprofloxacin are a kind of fluoroquinolone antibiotics, energy
By inhibiting the activity of DNA gyrase to have the function that sterilization, with antibacterial range is wide, antibacterial activity is big, tissue penetration is strong
The features such as, it is widely used in the disease treatment of aquatic products, livestock and poultry animal.But the drug can cause potential poison is secondary to make to human body
With, and largely use can induce bacterial drug resistance, indirect hazard human health.Therefore European Union, Japan and China are to grace promise
Maximum residue limit (MRLs) of the Sha Xing in animal-derived food has done clear stipulaties.China provides Enrofloxacin and Ciprofloxacin
Total amount ox, pig, poultry musculature in highest limitation be 100 μ g kg-1.Therefore grace promise in animal-derived food is realized
Quick, the Sensitive Detection of Sha Xing is particularly important.
Sample pre-treatments are to influence one of the significant process of detection effect.It is extracted with Solid Phase Extraction column extracting and organic reagent
Matrix effect can be effectively reduced etc. traditional sample-pretreating method, but it is complicated for operation, and time-consuming, effort, therefore not
Suitable for field quick detection.5-sulphosalicylic acid can effectively remove the protein in sample, reduce the interference of sample substrate,
And acid environmental benefits are in the extraction of Enrofloxacin, therefore are expected in the pretreatment process applied to chicken meat sample.
In the detection field of Enrofloxacin, high performance liquid chromatography (HPLC) is the mainstream side of current detection Enrofloxacin
Method, high sensitivity, accuracy are good, but need complicated sample pretreatment process, and detection time is long, testing cost
Height, therefore it is not suitable for field quick detection.Enzyme linked immunosorbent assay (ELISA) can significantly Simplified analysis process, specifically
Property it is higher and relatively quick, but stability is poor.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to explore one detected for Enrofloxacin in broiler chicken
Nano biological sensor method and chicken meat sample pre-treating method of the kind based on immunomagnetic beads and fluorescence quantum, the inventive method
It is sensitive, quick, specific good and easy to operate, it is easy to accomplish portability does not need complicated sample pretreatment process, tool
The potentiality of standby field quick detection Enrofloxacin are expected to become a kind of field quick detection means, have good development prospect.
Key step of the present invention are as follows: the Enrofloxacin in sample is captured by immunomagnetic beads (IMBs) first and occupies IMBs
The part binding site on surface；After Magneto separate and washing, quantum dot-bovine serum albumin(BSA)-En Nuo as competitor is added
Sha Xing (QDs-BSA-ENR) compound is in conjunction with the remaining site on the surface IMBs；Eventually by detection immunomagnetic beads-En Nuosha
The realization of star-bovine serum albumin(BSA)-quantum dot (IMBs-ENR-BSA-QDs) compound fluorescence signal quantifies Enrofloxacin
Detection.
The technical solution adopted by the present invention is that method the following steps are included:
1) enrofloxacin monoclonal antibody is modified in carboxylated magnetic bead surfaces, is closed with bovine serum albumin(BSA), preparation contains ox
Immunomagnetic beads (IMBs) solution that seralbumin is closed, enrofloxacin monoclonal antibody is modified；
2) Enrofloxacin (ENR) and bovine serum albumin(BSA) (BSA) is coupled, prepares Enrofloxacin-bovine serum albumin(BSA)
(BSA-ENR) compound；
3) BSA-ENR compound obtained in step 2) and carboxylated quantum dot (QDs-COOH) are coupled, prepare quantum
Point-bovine serum albumin(BSA)-Enrofloxacin (QDs-BSA-ENR) compound；
4) Chicken Tissues sample is handled, chicken meat sample extracting solution is obtained, specific as follows:
4.1) chicken meat sample of 1-5g chopping is weighed in homogenizing bag；
4.2) it is added into the homogenizing bag described in step 4.1) containing chicken meat sampleMass concentration isThe 5- sulfosalisylic of 1-5%
Acid, the homogeneous in Patting type homogenizer obtain the mixed liquor containing Chicken Tissues；
4.3) by above-mentioned steps 4.2) 3,000rpm or more is centrifuged 5- at normal temperature for the obtained mixed liquor containing Chicken Tissues
10min obtains supernatant；
4.4) by above-mentioned steps 4.3) filtering with microporous membrane in 0.22-0.45 μm of aperture of obtained supernatant, obtain chicken
The extracting solution of meat tissue；
4.5) by above-mentioned steps 4.4) the obtained extracting solution of Chicken Tissues adjusts with NaOH solution to pH 5-7.4, and 4 DEG C
Under save backup；
5) in the quantitative detection step 4) extracting solution Enrofloxacin concentration, it is specific as follows:
5.1) the standard curve model between Enrofloxacin concentration and fluorescent value is established；
5.1.1 a series of known concentrations 10) are prepared in the phosphate buffer of pH 5-8-2-105ng mL-1En Nuosha
Star solution；
5.1.2) closed containing bovine serum albumin(BSA) described in step 1), enrofloxacin monoclonal antibody is modified immune
Magnetic bead solution is mixed from the solution described in above-mentioned steps 5.1.1) containing different Enrofloxacin concentration, is placed in anti-on rotation blending instrument
It answers, the mixed liquor after reaction is redispersed in phosphate buffer, is obtained with phosphate buffer repeated washing 3 times of pH 5-8
Obtain the immunomagnetic beads (IMBs-ENR) of surface modification Enrofloxacin and magnetic bead (IMBs-Non ENR) structure of unmodified Enrofloxacin
At mixed solution；
5.1.3) take step 5.1.2) obtain mixed liquor, be added step 3) the closed QDs- containing bovine serum albumin(BSA)
The dispersion liquid of BSA-ENR compound is placed on rotation blending instrument and reacts, clear with the phosphate buffer repetition of pH 7.4 after reaction
It washes 3 times, is redispersed in phosphate buffer, detect the fluorescence signal intensity of specific wavelength；
It 5.1.4) will be different known to the resulting fluorescence signal intensity of above-mentioned steps 5.1.3) and corresponding Enrofloxacin
Concentration establishes standard curve model by fitting；
5.2) the Enrofloxacin concentration of standard curve model inspection chicken meat sample to be measured is utilized.
One aspect of the present invention prepares IMBs and QDs-BSA-ENR compound, chicken meat sample is on the other hand handled, then by two
Person is combined the Enrofloxacin that IMBs and QDs-BSA-ENR compound comes in quantitative detection Chicken Tissues.
Step 1) the preparation process is specific as follows:
1.1) 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) and N- hydroxysuccinimidyl acyl are prepared
The mixed solution of imines sulfonate sodium (NHSS)；
1.2) carboxylated magnetic bead is dispersed in 2- (N- morpholine) ethanesulfonic acid (MES) buffer of pH 6.0, it is slow with MES
Fliud flushing washes repeatedly 3 times, is resuspended in the mixed liquor of step 1.1), and rotation mixes reaction 30min, the magnetic activated under room temperature
Pearl solution；
By the magnetic bead solution of activation with borate buffer solution repeated washing 3 times of pH 7.4-8.5, it is then dispersed in boric acid
In salt buffer, the boric acid salt buffer dispersion liquid containing activated magnetic beads is obtained；
1.3) the boric acid salt buffer dispersion liquid containing activated magnetic beads is mixed with enrofloxacin monoclonal antibody solution, Yu Chang
The lower concussion reaction 2.5h of temperature, obtains the boric acid salt buffer dispersion liquid for the immunomagnetic beads modified containing enrofloxacin monoclonal antibody；
1.4) it is 2% containing bovine serum albumin that mass concentration is added into the boric acid salt buffer dispersion liquid that step 1.3) obtains
White solution, room temperature concussion reaction 1h are washed repeatedly 3 times with the phosphate buffer of pH 7.4, are then dispersed in phosphate-buffered
In liquid, phosphate dispersion liquid, as closed containing bovine serum albumin(BSA), enrofloxacin monoclonal antibody modification immune magnetic are obtained
Pearl solution saves backup at 4 DEG C.
Step 1.1) the mixed solution is the MES buffer containing EDC and NHSS, and pH 6.0, EDC and NHSS's is dense
Degree is respectively 10mM and 15mM.
The surface-bound carboxylic content of step 1.2) the carboxylated magnetic bead is 2.7mmol g-1, partial size 130-170nm,
Concentration is 10mg mL-1。
Enrofloxacin monoclonal antibody described in the step 1.3) is source of mouse, and IgG2b is Protein G purified, and purity is greater than
95%, concentration is 1mg mL-1, the dosage of monoclonal antibody is every milligram of magnetic bead of 0.02-0.1mg.
The present invention is in the step 1.1) -1.4) in each buffer distinguishingly added with mass fraction be 0.01%
Tween-20 solution, magnetic bead can be effectively prevent to be sticked on tube wall during modification.
Step 2) the preparation process is specific as follows:
2.1) Enrofloxacin, 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) and N- hydroxyl amber
Amber acid imide sulfonate sodium (NHSS) is dissolved in n,N-Dimethylformamide (DMF), the mass ratio of Enrofloxacin, NHSS and EDC
For 2:1:1.25, concussion reaction is stayed overnight under room temperature, and A liquid is made；
2.2) bovine serum albumin(BSA), which is dissolved in the phosphate buffer of pH 7.4, is made B liquid, and 2.5mg bovine serum albumin(BSA) is every
Milligram Enrofloxacin；
2.3) by above-mentioned steps 2.1) A liquid above-mentioned steps 2.2 are added dropwise) B liquid in, it is stirring while adding, then exist
3h is stirred under room temperature, then fills this blend into bag filter, is dialysed 3 days at 4 DEG C, is changed 3 dialyzates, centrifuging and taking supernatant daily
Liquid obtains the dispersion liquid of the compound containing BSA-ENR, and bovine serum albumin(BSA) concentration is 1mg mL in dispersion liquid-1。
The mass ratio of Enrofloxacin, NHSS and EDC is 2:1:1.25 in the step 2.1).
In the step 2.3) molecular cut off of bag filter be 8,000-14,000D, centrifugal condition 3,000rpm with
On, centrifugation time 5-10min.
Step 3) the preparation process is specific as follows:
3.1) 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) and N- hydroxysuccinimidyl acyl are prepared
The mixed solution of imines sulfonate sodium (NHSS)；
3.2) carboxylated quantum dot (QDs) is dispersed in above-mentioned steps 3.1) obtain mixed solution in, shaken under room temperature
React 40min, the quantum dot solution activated；
By the borate buffer solution repeated washing of the quantum dot solution of activation pH 7.4-8.5, centrifugal ultrafiltration 3 times, then
It is dispersed in borate buffer solution, obtains the boric acid salt buffer dispersion liquid containing activation quantum dot；
3.3) by the dispersion of the boric acid salt buffer dispersion liquid containing activation quantum dot and the compound containing BSA-ENR of step 2)
Liquid mixing, the concussion reaction 2.5h under room temperature obtain the boric acid salt buffer dispersion liquid of the compound containing QDs-BSA-ENR；
3.4) mass concentration is added into the boric acid salt buffer dispersion liquid of the compound containing QDs-BSA-ENR of step 3.3) is
2% contains bovine serum albumin solution, and room temperature concussion reaction 1h is super with the phosphate buffer of pH 7.4 repeated washing, centrifugation
Filter 3 times, is then dispersed in phosphate buffer, obtains point of the closed QDs-BSA-ENR compound containing bovine serum albumin(BSA)
Dispersion liquid saves backup at 4 DEG C.
Step 3.1) the mixed solution is the MES buffer containing EDC and NHSS, the concentration point of pH 6.0, EDC and NHSS
It Wei not 10mM and 15mM.
Carboxylated quantum dot is water-soluble CdSe/ZnS core shell structure quantum dot, emission maximum wave in the step 3.2)
For length between 610-625nm, concentration is 8 μM；The molecular cut off of ultrafiltration membrane be 50kD, centrifugal condition 8,000rpm with
On, centrifugation time is 10min or more.
In the step 3.3), point of the boric acid salt buffer dispersion liquid containing activation quantum dot and the compound containing BSA-ENR
Proportion dosage between dispersion liquid is the every micromole's quantum dot of 0.5g BSA-ENR.
The molecular cut off of ultrafiltration membrane is 100kD in the step 3.4), and centrifugal condition 8,000rpm, centrifugation time is
10min or more.
The step 5.1.3) in fluorescence signal intensity detect in the following manner: using 300-450nm as excitation wavelength
Excitation acquires the fluorescence signal intensity within the scope of 500-700nm.
The present invention is in above-mentioned steps 5.1.1 and 5.1.2) pH 5-8 phosphate buffer in distinguishingly added with contain matter
The Tween-20 solution that score is 0.05% and the bovine serum albumin solution that mass fraction is 0.1% are measured, can be reduced non-targeted
The non-specific adsorption of object.
Above-mentioned steps 5.1.2) in, enrofloxacin monoclonal antibody closed containing the bovine serum albumin(BSA) modification added
The concentration of immunomagnetic beads solution is 1mg mL-1(with the densimeter of magnetic bead), dosage are 2-25 μ L, and the reaction time is no less than 10min.
Above-mentioned steps 5.1.3) in add the closed QDs-BSA-ENR containing bovine serum albumin(BSA) phosphate-buffered dispersion
The concentration of liquid is 0.2 μM (with the densimeter of quantum dot), and dosage is 0.5-10 μ L, and the reaction time is no less than 30min.
The present invention is in the phosphate buffer of the pH 7.4 of above-mentioned steps 5.1.3) distinguishingly added with containing mass fraction
For 0.1% Tween-20 solution, non-specific adsorption can be reduced, while IMBs in reaction process being prevented to be sticked on tube wall.
The step 5.2) is specific as follows: obtaining chicken extracting solution after being handled by step 4) chicken meat sample to be measured, so
Step 5.1.2 is pressed afterwards) -5.1.3) processing acquisition fluorescence signal value, the standard curve model comparison obtained with step 5.1), acquisition
The Enrofloxacin concentration of sample to be tested.
The method of the present invention using 5-sulphosalicylic acid immersions, homogeneous, centrifugation, membrane filtration and etc. handle Chicken Tissues sample
Product obtain sample extracting solution and reduce influence of the sample substrate to detection effect.Constructed nano biological sensor is main
It is related to the IMBs of enrofloxacin monoclonal antibody modification and the quantum of bovine serum albumin(BSA)-Enrofloxacin (BSA-ENR) functionalization
Point, i.e. QDs-BSA-ENR.
The superior optical characteristics for efficiently separating efficiency and quantum dot of the method for the present invention combination immunomagnetic beads, construct one kind
Nano biological sensor realizes to quick, the sensitive and specific detection of Enrofloxacin, and to chicken meat sample pre-treating method into
It has gone research, has simplified operating procedure, and reduce influence of the matrix effect to detection effect.
The method of the present invention is convenient and efficient, does not need complicated instrument and organic reagent, and be expected to and Portable fluorescence instrument
It combines, there are the potentiality of the Enrofloxacin in poultry production chain field quick detection broiler chicken.
The beneficial effects of the present invention are: in conjunction with the superior optical characteristics for efficiently separating efficiency and quantum dot of immunomagnetic beads,
It constructs a kind of nano biological sensor and realizes quick, Sensitive Detection to Enrofloxacin.With existing HPLC and elisa technique phase
Than the major advantage of the method for the present invention is:
(1) complicated sample pretreatment process is not needed, it is easy to operate, it need to only carry out simple 5-sulphosalicylic acid leaching
Sample pretreatment process can be completed in bubble, homogeneous, centrifugation and membrane filtration, effectively shortens the entire detection and analysis time, generally
It can complete to detect in 1h or so；
(2) using immunomagnetic beads as separation and concentration tool, detection sensitivity is improved；
(3) molecule is reported by fluorescence signal of quantum dot, compared with the biomolecule such as traditional enzyme, stability is higher；
(4) entire detection process is simple, quickly, does not need complicated instrument and organic reagent, and be expected to it is portable
Formula luminoscope combines, and has the potentiality of the Enrofloxacin in poultry production chain field quick detection broiler chicken.
Detailed description of the invention
Fig. 1 is the principle of the present invention figure.
Fig. 2 is the proof diagram of IMBs preparation.
Wherein (A): for carboxylated magnetic bead (MBs-COOH) (a), 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimine
The magnetic bead (MBs-EDC/NHSS) (b) and IMBs of hydrochloride (EDC) and n-hydroxysuccinimide sulfonate sodium (NHSS) activation
(c) Fourier transform infrared spectroscopy figure；
Wherein (B): for the Zeta potential figure of MBs-COOH, MBs-EDC/NHSS and IMBs.
Fig. 3 is the uv absorption spectra of Enrofloxacin (ENR), bovine serum albumin(BSA) (BSA) and BSA-ENR compound.
Fig. 4 is the proof diagram of QDs-BSA-ENR preparation；
Wherein (A): the quantum dot (QDs-EDC/ activated for carboxylated quantum dot (QDs-COOH) (a), EDC/NHSS
NHSS) the Fourier transform infrared spectroscopy figure of (b) and QDs-BSA-ENR (c)；
Wherein (B): for the Zeta potential figure of QDs-COOH, QDs-EDC/NHSS and QDs-BSA-ENR；
Fig. 5 is the standard curve model that biosensor detects Enrofloxacin.
Table 1 is the detection parameters and performance that the present invention is applied to 11 concentration embodiments.
Table 2 is recovery of standard addition of the biosensor in chicken meat sample detection.
Specific embodiment
Below in conjunction with drawings and the specific embodiments, the present invention will be described in detail, but is not the limitation present invention.
The embodiment of the present invention is as follows:
Embodiment
1) IMBs of enrofloxacin monoclonal antibody modification is prepared
1.1) mixed solution of the NHSS of the EDC and 15mM of the 10mM of 400 μ L are prepared；
1.2) carboxylated magnetic bead (being purchased from U.S. ocean nanosecond science and technology Co., Ltd) is dispersed in 2- (the N- morphine of pH 6.0
Quinoline) in ethanesulfonic acid (MES) buffer, the processing step of successively Magneto separate and removal washing supernatant is carried out simultaneously with MES buffer
It being repeated 3 times, is resuspended in EDC and NHSS mixed liquor described in the step 1.1) of 400 μ L, rotation mixes reaction 30min under room temperature,
The magnetic bead solution activated；The magnetic bead solution of activation is subjected to successively Magneto separate and removal with the borate buffer solution of pH 7.4
It washs the processing step of supernatant and is repeated 3 times, be then dispersed in the borate buffer solution of 400 μ L, obtain containing activation magnetic
The boric acid salt buffer dispersion liquid of pearl；
1.3) the 400 μ L boric acid salt buffer dispersion liquid for containing 0.1mg activated magnetic beads and 4 μ L are contained into 0.004mg En Nuosha
The solution mixing of star monoclonal antibody (being purchased from Wuhan Sino-American Biotechnology Company), the concussion reaction 2.5h under room temperature are obtained
The boric acid salt buffer dispersion liquid of the IMBs modified to enrofloxacin monoclonal antibody；
1.4) into the boric acid salt buffer dispersion liquid of the IMBs modified described in step 1.3) containing enrofloxacin monoclonal antibody
It is 2% containing bovine serum albumin solution that 400 μ L mass concentrations, which are added, and room temperature concussion reaction 1h is slow with the phosphate of pH 7.4
Fliud flushing carries out the processing step of successively Magneto separate and removal washing supernatant and is repeated 3 times, and it is slow to be then dispersed in 100 μ L phosphate
In fliud flushing, the phosphate dispersion liquid of closed containing bovine serum albumin(BSA), enrofloxacin monoclonal antibody modification IMBs is obtained
(the final concentration of 1mg mL of IMBs-1), it saves backup at 4 DEG C.
Fig. 2 is the Fourier transform infrared spectroscopy figure (A) and Zeta potential of MBs-COOH, MBs-EDC/NHSS and IMBs
(B).By scheming (A) it is found that MBs-COOH (a), MBs-EDC/NHSS (b) and IMBs (c) are in 1650cm-1And 1530cm-1Nearby
There is stronger absorption peak, correspond respectively to the absorption peak of II band of amide Ⅰ and amide, the reason of absorption peak occurs in three
It is that, according to shop instruction, the carboxylated magnetic bead surfaces bought have been coated with bovine serum albumin(BSA) in advance, and bovine serum albumin(BSA)
In contain amido bond.Compared to MBs-COOH and MBs-EDC/NHSS, IMBs is in 1516cm-1Neighbouring absorption peak relative intensity is bright
Aobvious enhancing, this is because forming new amide between the carboxyl and antibody amino groups of magnetic bead surfaces in immunomagnetic beads preparation process
Also contain amido bond in key and antibody.For further illustrate IMBs successful preparation, respectively to MBs-COOH, MBs-EDC/
NHSS and IMBs carries out Zeta potential characterization.By scheming (B) it is found that the Zeta potential of MBs-COOH is -39.1mV, this is because magnetic
The electronegative COO of bead surface-The presence of group；After activating through EDC/NHSS, the negative electrical charge of magnetic bead surfaces is partially neutralized,
Zeta potential is offset to -27.5mV；After coupled antibody, Zeta potential is further offset to -2.67mV, this is because antibody is at this
Under the conditions of it is positively charged, the negative electrical charge of magnetic bead surfaces is further neutralized, comprehensive Fourier transform infrared spectroscopy figure and Zeta potential
It can illustrate the successful preparation of IMBs.
2) preparation of BSA-ENR
2.1) Enrofloxacin, NHSS and EDC (mass ratio of Enrofloxacin, NHSS and EDC is 2:1:1.25) are dissolved in 1mL
N,N-Dimethylformamide (DMF) in, under room temperature concussion reaction stay overnight, A liquid is made；
2.2) bovine serum albumin(BSA) (every milligram of Enrofloxacin of 2.5mg bovine serum albumin(BSA)) is dissolved in the phosphate of pH 7.4
B liquid is made in buffer；
2.3) by above-mentioned steps 2.1) in A liquid above-mentioned steps 2.2 are added dropwise) B liquid in, it is stirring while adding, then
3h is stirred at normal temperature, then fills this blend into bag filter (8,000-14,000D), is dialysed 3 days, is changed daily 3 times at 4 DEG C
Dialyzate, centrifugation (3,000rpm, 5min) take supernatant, obtain dispersion liquid (the 1mg mL of the compound containing BSA-ENR-1, with cow's serum
The densimeter of albumin).
Fig. 3 is the uv absorption spectra of ENR, BSA and BSA-ENR, and the major absorbance peak of ENR is located at 271nm, 322nm
At 334nm, carrier protein BSA has a maximum absorption band at 278nm, and the absorption peak of BSA-ENR is located at 275nm, 322nm and
At 334nm, BSA-ENR has both the ultraviolet characteristic absorption peak of ENR and BSA, and being superimposed due to both BSA and ENR absorption peak
Effect, the characteristic peak for BSA-ENR occur have certain displacement, can be determined that BSA-ENR accordingly compared with ENR and BSA sterling
Successful synthesis.
3) preparation of QDs-BSA-ENR
3.1) mixed solution of 10mM EDC and 15mM NHSS are prepared；
3.2) carboxylated quantum dot is dispersed in 200 μ L above-mentioned steps 3.1) EDC and NHSS mixed solution in, under room temperature
Concussion reaction 40min, the quantum dot solution activated；By the quantum dot solution of activation with the borate buffer solution of pH 7.4 according to
Secondary step repeated washing 3 times for carrying out centrifugal ultrafiltration and removing waste liquid, are then dispersed in 160 μ L borate buffer solutions, obtain
Boric acid salt buffer dispersion liquid containing activation quantum dot；
3.3) 160 μ L are contained 8 × 10-11Mol activates boric acid salt buffer dispersion liquid and the 40 μ L step 2.3) institutes of quantum dot
The dispersion liquid (the every micromole's quantum dot of 0.5g BSA-ENR compound) for the BSA-ENR compound containing 0.04mg stated mixes, in
Concussion reaction 2.5h under room temperature obtains the boric acid salt buffer dispersion liquid containing QDs-BSA-ENR；
3.4) 200 μ L mass concentrations are added into the boric acid salt buffer dispersion liquid described in step 3.3) containing QDs-BSA-ENR
For 2% bovine serum albumin solution that contains, it is super to carry out successively centrifugation with the phosphate buffer of pH 7.4 by room temperature concussion reaction 1h
The processing step of filter and removal waste liquid is simultaneously repeated 3 times, and is then dispersed in 400 μ L phosphate buffers, is obtained closed containing BSA
The phosphate-buffered dispersion liquid (final concentration of 0.2 μM of QDs-BSA-ENR, with the densimeter of quantum dot) of QDs-BSA-ENR, 4
It is saved backup at DEG C.
Fig. 4 be QDs-COOH, QDs-EDC/NHSS and QDs-BSA-ENR Fourier transform infrared spectroscopy figure (A) and
Zeta potential (B).By scheming (A) it is found that QDs-COOH (a) is in 1704cm-1Nearby there is apparent absorption peak, corresponds to QDs-COOH
The stretching vibration of-C (=O)-in the carboxyl on surface, after EDC/NHSS is activated, due to the decline of quantum dot surface carboxyl-content,
QDs-EDC/NHSS (b) is in 1705cm-1Neighbouring absorption peak relative intensity is obviously reduced；Further coupling BSA-ENR is multiple
After closing object, QDs-BSA-ENR (c) is in 1651cm-1, 1601cm-1And 1493cm-1There is new absorption peak in place, wherein 1651cm-1
The absorption peak at place is that the carboxyl on quantum dot is formed by stretching for-C in amido bond (=O)-with the amino on BSA-ENR compound
Contracting vibration, 1601cm-1And 1493cm-1The absorption peak at place is then the flexible vibration of phenyl ring skeleton in the BSA-ENR compound of coupling
It is dynamic, it is possible thereby to illustrate that BSA-ENR is successfully coupled to quantum dot surface.By scheme (B) it is found that QDs-COOH Zeta potential be-
37.6mV, this is because a large amount of electronegative COO of quantum dot surface-The presence of group；After being activated through EDC/NHSS, quantum dot
The negative electrical charge on surface is partially neutralized, and Zeta potential is offset to -30.0mV；After being coupled BSA-ENR, Zeta potential is further deviated
Extremely -27.4mV, comprehensive Fourier transform infrared spectroscopy figure and Zeta potential can illustrate the successful system of QDs-BSA-ENR compound
It is standby.
4) chicken meat sample pre-processes
4.1) chicken meat sample of 1g chopping is weighed in homogenizing bag；
4.2) 2% 5-sulphosalicylic acid is added into the homogenizing bag described in step 4.1) containing chicken meat sample, in Patting type
Homogeneous in homogenizer obtains the mixed liquor containing Chicken Tissues；
4.3) by above-mentioned steps 4.2) 12,000rpm is centrifuged 10min at normal temperature for the obtained mixed liquor containing Chicken Tissues,
Obtain supernatant；
4.4) by above-mentioned steps 4.3) filtering with microporous membrane in 0.22 μm of aperture of obtained supernatant, obtain Chicken Tissues
Extracting solution；
4.5) by above-mentioned steps 4.4) the obtained extracting solution of Chicken Tissues adjusts to pH 6 with NaOH solution, protected at 4 DEG C
It deposits spare.
5) in chicken meat sample Enrofloxacin detection
5.1) the standard curve model between Enrofloxacin concentration and fluorescent value is established；
5.1.1 a series of known concentrations (10) are prepared in the phosphate buffer of pH 6.0-2-105ng mL-1) En Nuo
Husky star solution.
5.1.2) by the phosphoric acid salinity of the closed IMBs of bovine serum albumin(BSA) described in 20 μ L steps 1.4) containing 0.02mg
Dispersion liquid from described in 180 μ L above-mentioned steps 5.1.1) contain different Enrofloxacin concentration (10-2-105ng mL-1) solution mixing, set
In normal-temperature reaction 10min on rotation blending instrument, the mixed liquor after reaction carries out successively magnetic point with the phosphate buffer of pH 6.0
It from the processing step with removal washing supernatant and is repeated 3 times, is redispersed in phosphate buffer, obtains the promise of surface modification grace
The mixed solution that the immunomagnetic beads (IMBs-ENR) of Sha Xing and the magnetic bead (IMBs-Non ENR) of unmodified Enrofloxacin are constituted；
5.1.3) take step 5.1.2) obtain mixed liquor, be added 4 μ L steps 3.4) described in contain 8 × 10-11Mol ox blood
The phosphate-buffered dispersion liquid of the QDs-BSA-ENR of pure protein blocking is placed in normal-temperature reaction 30min on rotation blending instrument, instead
Should after carry out the processing step of successively Magneto separate and removal washing supernatant with the phosphate buffer of pH 7.4 and be repeated 3 times,
It is redispersed in 200 μ L phosphate buffers, detects the fluorescence signal of specific wavelength.Fluorescence signal acquires in the following manner:
It is excited by excitation wavelength of 450nm, acquires the fluorescence signal intensity under 614nm；
5.1.4) by the concentration of Enrofloxacin in the resulting fluorescence signal intensity of above-mentioned steps 5.1.3) and known sample
Standard curve is established by fitting；
Embodiment is as shown in table 1 by Enrofloxacin concentration different known to 11 and the sub- embodiment testing result of progress:
Table 1
Using the logarithm of Enrofloxacin concentration as abscissa, the relative intensity of fluorescence at 614nm is ordinate opening relationships
Curve, the linear detection range of the available biosensor are 1-100ng mL-1, equation of linear regression are as follows: y=-
941log x+2929(r2=0.976), detection is limited to 0.94ng mL-1(such as Fig. 5) thus establishes Enrofloxacin concentration and fluorescence
Standard curve model between value.
5.2) specific example measures chicken meat sample recovery of standard addition
Chicken extracting solution is obtained after being handled by step 4) chicken meat sample to be measured, is added into chicken extracting solution known dense
(Enrofloxacin concentration is respectively as follows: 5ng mL to the Enrofloxacin solution of degree in final chicken extracting solution-1,25ng mL-1And 100ng
mL-1), then press step 5.1.2) -5.1.3) processing acquisition fluorescence signal value, the standard curve model pair obtained with step 5.1)
According to obtaining the Enrofloxacin concentration in the sample of chicken position to be measured.
Table 2
The method of the present invention (the case where rate of recovery is 100% between 93%-107% in the rate of recovery that chicken meat sample detects
To be most accurate, those skilled in the art think to be good between 80%-120%).Thus can illustrate of the invention based on immune magnetic
The nano biological sensor of pearl and fluorescence quantum can preferably be applied to the detection of Enrofloxacin in chicken meat sample.
By above-mentioned two embodiment as it can be seen that one kind proposed by the present invention for Enrofloxacin detection in broiler chicken is based on being immunized
The nano biological sensor combination chicken meat sample pre-treating method of magnetic bead and fluorescence quantum is sensitive, quick, easy to operate, is not required to
Complicated sample pretreatment process is wanted, and is expected in conjunction with Portable fluorescence instrument, has field quick detection Enrofloxacin
Potentiality are expected to become a kind of field quick detection means, have good development prospect.
Claims (9)
1. the side that a kind of nano biological sensor based on immunomagnetic beads and fluorescence quantum quickly detects Enrofloxacin in broiler chicken
Method, it is characterised in that method the following steps are included:
1) enrofloxacin monoclonal antibody is modified in carboxylated magnetic bead surfaces, is closed with bovine serum albumin(BSA), preparation contains cow's serum
Immunomagnetic beads (IMBs) solution that albumin is closed, enrofloxacin monoclonal antibody is modified；
2) Enrofloxacin (ENR) and bovine serum albumin(BSA) (BSA) is coupled, prepares Enrofloxacin-bovine serum albumin(BSA) (BSA-
ENR) compound；
3) BSA-ENR compound obtained in step 2) and carboxylated quantum dot (QDs-COOH) are coupled, prepare quantum dot-ox
Seralbumin-Enrofloxacin (QDs-BSA-ENR) compound；
4) Chicken Tissues sample is handled, chicken meat sample extracting solution is obtained, specific as follows:
4.1) chicken meat sample of 1-5g chopping is weighed in homogenizing bag；
4.2) it is added into the homogenizing bag described in step 4.1) containing chicken meat sampleMass concentration isThe 5-sulphosalicylic acid of 1-5%,
The homogeneous in Patting type homogenizer obtains the mixed liquor containing Chicken Tissues；
4.3) by above-mentioned steps 4.2) 3,000rpm or more is centrifuged 5- at normal temperature for the obtained mixed liquor containing Chicken Tissues
10min obtains supernatant；
4.4) by above-mentioned steps 4.3) filtering with microporous membrane in 0.22-0.45 μm of aperture of obtained supernatant, obtain chicken group
The extracting solution knitted；
4.5) by above-mentioned steps 4.4) the obtained extracting solution of Chicken Tissues adjusts to pH 5-7.4 with NaOH solution, protected at 4 DEG C
It deposits spare；
5) in the quantitative detection step 4) extracting solution Enrofloxacin concentration, it is specific as follows:
5.1) the standard curve model between Enrofloxacin concentration and fluorescent value is established；
5.1.1 a series of known concentrations 10) are prepared in the phosphate buffer of pH 5-8-2-105ng mL-1Enrofloxacin it is molten
Liquid；
5.1.2) the immunomagnetic beads for modifying closed containing bovine serum albumin(BSA) described in step 1), enrofloxacin monoclonal antibody
Solution is mixed from the solution described in above-mentioned steps 5.1.1) containing different Enrofloxacin concentration, is placed on rotation blending instrument and is reacted,
Mixed liquor after reaction is redispersed in phosphate buffer with phosphate buffer repeated washing 3 times of pH 5-8, obtains table
What the magnetic bead (IMBs-Non ENR) of immunomagnetic beads (IMBs-ENR) and unmodified Enrofloxacin that Enrofloxacin is modified in face was constituted
Mixed solution；
5.1.3) take step 5.1.2) obtain mixed liquor, be added step 3) the closed QDs-BSA- containing bovine serum albumin(BSA)
The dispersion liquid of ENR compound is placed on rotation blending instrument and reacts, the phosphate buffer repeated washing 3 of pH 7.4 is used after reaction
It is secondary, it is redispersed in phosphate buffer, detects the fluorescence signal intensity of specific wavelength；
5.1.4) by various concentration known to the resulting fluorescence signal intensity of above-mentioned steps 5.1.3) and corresponding Enrofloxacin
Standard curve model is established by fitting；
5.2) the Enrofloxacin concentration of standard curve model inspection chicken meat sample to be measured is utilized.
2. a kind of nano biological sensor based on immunomagnetic beads and fluorescence quantum according to claim 1 quickly detects
The method of Enrofloxacin in broiler chicken, it is characterised in that: the step 1) preparation process is specific as follows:
1.1) 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) and n-hydroxysuccinimide are prepared
The mixed solution of sulfonate sodium (NHSS)；
1.2) carboxylated magnetic bead is dispersed in 2- (N- morpholine) ethanesulfonic acid (MES) buffer of pH 6.0, with MES buffer
Repeated washing 3 times, is resuspended in the mixed liquor of step 1.1), and rotation mixes reaction 30min under room temperature, and the magnetic bead activated is molten
Liquid；
By the magnetic bead solution of activation with borate buffer solution repeated washing 3 times of pH 7.4-8.5, it is slow to be then dispersed in borate
In fliud flushing, the boric acid salt buffer dispersion liquid containing activated magnetic beads is obtained；
1.3) the boric acid salt buffer dispersion liquid containing activated magnetic beads is mixed with enrofloxacin monoclonal antibody solution, under room temperature
Concussion reaction 2.5h obtains the boric acid salt buffer dispersion liquid for the immunomagnetic beads modified containing enrofloxacin monoclonal antibody；
1.4) into the boric acid salt buffer dispersion liquid that step 1.3) obtains be added mass concentration be 2% containing bovine serum albumin(BSA) it is molten
Liquid, room temperature concussion reaction 1h are washed repeatedly 3 times with the phosphate buffer of pH 7.4, are then dispersed in phosphate buffer,
Phosphate dispersion liquid is obtained, as closed containing bovine serum albumin(BSA), enrofloxacin monoclonal antibody modification immunomagnetic beads are molten
Liquid saves backup at 4 DEG C.
3. a kind of nano biological sensor based on immunomagnetic beads and fluorescence quantum according to claim 1 quickly detects
The method of Enrofloxacin in broiler chicken, it is characterised in that: in the step 1.1) -1.4) in each buffer be added with quality
The Tween-20 solution that score is 0.01%.
4. a kind of nano biological sensor based on immunomagnetic beads and fluorescence quantum according to claim 1 quickly detects
The method of Enrofloxacin in broiler chicken, it is characterised in that: the step 2) preparation process is specific as follows:
2.1) Enrofloxacin, 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) and N- hydroxysuccinimidyl acyl
Imines sulfonate sodium (NHSS) is dissolved in n,N-Dimethylformamide (DMF), and concussion reaction is stayed overnight under room temperature, and A liquid is made；
2.2) bovine serum albumin(BSA), which is dissolved in the phosphate buffer of pH 7.4, is made B liquid；
2.3) by above-mentioned steps 2.1) A liquid above-mentioned steps 2.2 are added dropwise) B liquid in, it is stirring while adding, then in room temperature
Lower stirring 3h, then fills this blend into bag filter, dialyses 3 days at 4 DEG C, changes 3 dialyzates daily, centrifuging and taking supernatant,
Obtain the dispersion liquid of the compound containing BSA-ENR.
5. a kind of nano biological sensor based on immunomagnetic beads and fluorescence quantum according to claim 1 quickly detects
The method of Enrofloxacin in broiler chicken, it is characterised in that: the step 3) preparation process is specific as follows:
3.1) 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) and n-hydroxysuccinimide are prepared
The mixed solution of sulfonate sodium (NHSS)；
3.2) carboxylated quantum dot (QDs) is dispersed in above-mentioned steps 3.1) obtain mixed solution in, concussion reaction under room temperature
40min, the quantum dot solution activated；The quantum dot solution of the activation borate buffer solution of pH 7.4-8.5 is repeated clear
It washes, centrifugal ultrafiltration 3 times, is then dispersed in borate buffer solution, obtain the boric acid salt buffer dispersion liquid containing activation quantum dot；
3.3) dispersion liquid of the boric acid salt buffer dispersion liquid containing activation quantum dot and the compound containing BSA-ENR of step 2) is mixed
It closes, the concussion reaction 2.5h under room temperature obtains the boric acid salt buffer dispersion liquid of the compound containing QDs-BSA-ENR；
3.4) it is 2% that mass concentration is added into the boric acid salt buffer dispersion liquid of the compound containing QDs-BSA-ENR of step 3.3)
Contain bovine serum albumin solution, room temperature concussion reaction 1h, with the phosphate buffer of pH 7.4 repeated washing, centrifugal ultrafiltration 3
It is secondary, it is then dispersed in phosphate buffer, obtains the dispersion of the closed QDs-BSA-ENR compound containing bovine serum albumin(BSA)
Liquid saves backup at 4 DEG C.
6. a kind of nano biological sensor based on immunomagnetic beads and fluorescence quantum according to claim 1 quickly detects
The method of Enrofloxacin in broiler chicken, it is characterised in that: the step 5.1.3) in fluorescence signal intensity examine in the following manner
It surveys: being excited by excitation wavelength of 300-450nm, acquire the fluorescence signal intensity within the scope of 500-700nm.
7. a kind of nano biological sensor based on immunomagnetic beads and fluorescence quantum according to claim 1 quickly detects
The method of Enrofloxacin in broiler chicken, it is characterised in that: in above-mentioned steps 5.1.1 and 5.1.2) pH 5-8 phosphate buffer
In distinguishingly added with containing mass fraction be 0.05% Tween-20 solution and mass fraction be 0.1% bovine serum albumin(BSA)
Solution.
8. a kind of nano biological sensor based on immunomagnetic beads and fluorescence quantum according to claim 1 quickly detects
The method of Enrofloxacin in broiler chicken, it is characterised in that: special in the phosphate buffer of the pH 7.4 of above-mentioned steps 5.1.3)
The Tween-20 solution that it is 0.1% containing mass fraction that ground, which is added with,.
9. a kind of nano biological sensor based on immunomagnetic beads and fluorescence quantum according to claim 1 quickly detects
The method of Enrofloxacin in broiler chicken, it is characterised in that: the step 5.2) is specific as follows: step 4) is pressed to chicken meat sample to be measured
Chicken extracting solution is obtained after processing, then presses step 5.1.2) -5.1.3) processing acquisition fluorescence signal value, it is obtained with step 5.1)
Standard curve model comparison, obtain the Enrofloxacin concentration of sample to be tested.
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