CN107894505A - A kind of method of quick detection beta-lactam antibiotics residue - Google Patents
A kind of method of quick detection beta-lactam antibiotics residue Download PDFInfo
- Publication number
- CN107894505A CN107894505A CN201711095951.1A CN201711095951A CN107894505A CN 107894505 A CN107894505 A CN 107894505A CN 201711095951 A CN201711095951 A CN 201711095951A CN 107894505 A CN107894505 A CN 107894505A
- Authority
- CN
- China
- Prior art keywords
- row
- liquid
- detection
- ampicillin
- beta
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
-
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
A kind of method of quick detection beta-lactam antibiotics residue, belongs to inspection technology field.The present invention modifies detection of the multimode fibre probe realization to fluid sample beta-lactam class antibiotic after being directly coupled with carrier protein as parent using ampicillin, by be configured as based on the electric signal exported from detection unit determine biomaterial concentration signal processor detection and identification testing sample solution in whether there is object and concentration information, be achieved in the detection to object.The detection method is compared with traditional instrument analysis method more simple and fast, and testing cost is relatively low, wherein, the sensitivity of non-marked method detection can reach 1.56 ng/ml or so in 50 ng/ml or so, the sensitivity of golden mark detection method.
Description
Technical field
The invention belongs to technical field of chemical detection, and in particular to a kind of quick detection beta-lactam antibiotic chloramphenicol
The method of residual.
Background technology
Beta-lactam antibiotic (β-lactams) means a major class antibiosis in chemical constitution with beta-lactam nucleus
Element, including clinical the most frequently used penicillin and cynnematin, and the cephamycin-type of new development, thiomycin class, monocyclic β-interior acyl
Other atypia beta-lactam antibiotics such as amine.Such antibiotic has that bactericidal activity is strong, toxicity is low, indication is wide and faces
The eutherapeutic advantage of bed, it is widely used in the preventing and treating of mastitis for milk cows on veterinary clinic.The beta-lactam remained in milk
Antibiotic, especially benzyl penicillin, humans allergic's reaction and the increase of bacterial drug resistance are easily caused, the health of human body is caused
Threaten.With it, clinically application study and toxicologic study are goed deep into, the food-safety problem as caused by its residue problem
Cause the extensive concern of food safety management department of various countries and international organization, therefore establish beta-lactam in the food such as milk
The quick determination method of antibiotic residue turns into current urgent problem to be solved.
At present, for the detection method of belt-lactam antibiotics residues, there are microbial method, ELISA, thin layer color
Spectrometry, gas chromatography, gas chromatographymass spectrum, high performance liquid chromatography, Liquid Chromatography-Mass Spectrometry and Capillary Electrophoresis-matter
Spectrometry etc..Instrumental method needs expensive instrument and equipment and special technical staff and somewhat expensive, detection time length；It is micro-
The shortcomings that detection sensitivity is inadequate be present in bioanalysis；Enzyme-Linked Immunospot such as ELISA, test strips etc. have easy to operate, fast
The features such as speed, high sensitivity, low cost, suitable for the detection of a large amount of samples, the favor of people is enjoyed at present.
The high sensitivity of immunological detection method and specificity are stronger, but the research and development time of corresponding antibodies is long and cost is high,
And repeatability and less stable.Sensor, method is the new detection technique to grow up in recent years, based on Biological Principles
Sensor make identification original paper using bioactive substance as sensor, it is anti-that specificity occurs with the test substance in sample
Should, these reactions (forming compound, color development, luminous etc.) are converted into exporting the signal of detection by appropriate transducer
(voltage, frequency etc.), the qualitative and quantitative detection to determinand can be achieved.
Fibre optical sensor is that one kind produces changes in optical properties in the case where physically or chemically encouraging, and causes and light beam is propagated in optical fiber
Characteristic parameter, such as intensity, wavelength, phase, polarization state change, and the various detection devices to be connected with optical fiber are rung
The device answered.Biomembrane interference technique is based on light absorbs, fluorescence and light reflection principle, is attached to by protein molecular on sensor
Difference, the interference spectrum displacement difference for causing visible ray to be formed on sensor film surface, by detecting between this change in displacement
The reversed concentration for reflecting protein molecular.Therefore, developing the beta-lactam antibiotic detection method based on biomembrane interference technique has
Help belt-lactam antibiotics residues in quick detection animal derived food.
The content of the invention
Present invention aims at provide a kind of method of quick detection beta-lactam antibiotic residual chloromycetin.
To achieve the above object, technical scheme provided by the invention is：A kind of quick detection beta-lactam antibiotic is residual
The method stayed, it is characterised in that：Comprise the following steps：
The first step：Prepare ampicillin-BSA
Ampicillin sodium salt is dissolved in dimethylformamide, stirs to after all dissolving to obtain A liquid；By bovine serum albumin(BSA)
It is dissolved in the PBS that pH value is 7.4, stirring obtains B liquid to after being completely dissolved；Under agitation, A liquid is added to B liquid
In, then thereto dropwise, be uniformly added into the glutaraldehyde water solution that volume fraction is 25%, under stirring condition, lucifuge reaction, institute
Obtain the PBS that product is 7.4 with 0.05M pH value to dialyse, obtain coating antigen, i.e. ampicillin-BSA conjugate；
Second step：Fibre optical sensor antigen coat is modified
APS fibre optical sensors end is submerged in the coating original solution of various concentrations, be stored at room temperature 5min；Optical fiber is given birth to again
Thing transducer tip is submerged in the aqueous sucrose solution that mass fraction is 15%, is stored at room temperature 1min, 2 are placed in after then room temperature is dried
Preserved under~8 DEG C of drying conditions, it is standby；
3rd step：Label-free detection
The PB buffer solutions that ampicillin-BSA conjugate pH value is 7.4 are diluted 20 times, take 200 μ L in microwell plate
In 2nd row；Beta-lactam antibiotic acceptor is diluted 60 times with pasteurize milk, then with the liquid diluting ampicillin
Standard items take 200 μ L in the 4th row of microwell plate respectively to 100,50,25,12.5,6.25,3.12 and 1.56ng/mL；It is past micro-
200 μ L water are added in 1st row of orifice plate, 200 μ L pasteurize milk are added toward the 3rd row；Examined with APS fiber-optic sensor probes
Survey：First row 60s, the 2nd row 60s；3rd row 60s；4th row 300s；
4th step：Gold mark detection
1 μ L 2mg/mL beta-lactam antibiotic acceptor is added into 1mL collaurums, is vortexed and mixes, be stored at room temperature
15min, the mass fraction for adding 100 μ L are 10% bovine serum albumin solution, and room temperature closes 15min, 10000rpm centrifugations
10min, supernatant liquor is discarded, add the PBS that 90 μ L pH value are 7.4, resuspended particle, obtain gold mark beta-lactam
Antibiotic acceptor；
The PB buffer solutions that ampicillin-BSA conjugate pH value is 7.4 are diluted 20 times, take 200 μ L in microwell plate
In 2nd row；Ampicillin standard items are diluted to 50,25,12.5,6.25,3.12 and 1.56ng/ml with pasteurize milk, are divided
Do not take 200 μ L in microwell plate the 4th row in, in addition per hole add 50 μ L reaction solutions and 5 μ L gold mark beta-lactam antibiotic by
Body；200 μ L water are added into the 1st row of microwell plate, the 3rd row toward microwell plate add 200 μ L pasteurizes milk, 50 μ L reactions
Liquid, 5 μ L gold marks BSA；Detected with APS fiber-optic sensor probes：First row 60s, the 2nd row 60s；3rd row 60s；4th row
600s；
Preferably technical scheme is：Also include the regeneration of APS fibre optical sensors, including：
Balance：120s in the liquid of bare substrate bottom is submerged into APS fibre optical sensors end, contained in the bare substrate bottom liquid
The gold mark BSA of 200 μ L bottom liquid, 50 μ L reaction solution and 5 μ L；
Test：Chloramphenicol element is submerged into APS fibre optical sensors end and detects 600s in negative bottom liquid, chloramphenicol element detection
In negative bottom liquid negative sample liquid, 50 μ L reaction solutions and 5 μ L gold mark chloramphenicol element acceptors are detected containing 200 μ L chloramphenicol element；
Regeneration：30s in glycine and dimethylformamide mixed liquor is submerged into optical fiber biosensor probe bottom, it is described
Glycine and dimethylformamide mixed liquor are by 0.1M that pH value is 3 glycine with dimethylformamide according to 4：1 volume
Formed than mixing；
Washing：Optical fiber biosensor probe bottom is washed with the PBS that pH value is 7.4.
Preferably technical scheme is：The reaction solution is the PBS that pH value is 7.4.
Preferably technical scheme is：The bottom liquid is to be made after milk powder is dissolved in water, and its mass fraction is 10%.
Preferably technical scheme is：149mg ampicillin sodium salts are dissolved in 8mL dimethylformamides, stirred to whole
A liquid is obtained after dissolving；It is in 7.4 PBS by the pH value that 204mg bovine serum albumin(BSA)s are dissolved in 16mL, stirs to completely molten
B liquid is obtained after solution.
Brief description of the drawings
Fig. 1 is the ampicillin residual of label-free detection detection gradient dilution.
Fig. 2 is that gold mark repeats detection ampicillin mark-on sample.
Because above-mentioned technical proposal is used, the present invention this have the advantage that compared with prior art：
With ampicillin-BSA or gold mark beta-lactam antibiotic receptor conjugate modification fibre-optical probe surface, then by
Fiber spectrum analytical equipment, which is detected and identified in testing sample, whether there is object and concentration information, realize that chlorine is mould in sample
The quantitative detection of element.This method substantially reduces testing cost and detection time, has good reproduction and automation, high flux
Etc. advantage, it is expected to further improve biology sensor immuno analytical method, it is moved towards quantification, multivariate detection and extensiveization should
With.
Embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation
Content disclosed by book understands other advantages and effect of the present invention easily.
The method for determining animal derived food to optical biosensor with reference to following examples is further described.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment one：A kind of method of quick detection belt-lactam antibiotics residues
Ampicillin-BSA coating antigens prepare as follows：
Ampicillin sodium salt 149mg is weighed, is dissolved in 8mL DMF, magnetic agitation dissolves to obtain A liquid to whole.Weigh
BSA204mg, it is dissolved in 16mL PBS (pH 7.4), stirring is to being completely dissolved to obtain B liquid.Under stirrer stirring, A liquid is added
Into B liquid, immediately thereto dropwise, be uniformly added into the μ L of 25% glutaraldehyde 150, gentle agitation at room temperature, lucifuge reaction 3h.Gained
Product obtains coating antigen with 0.05M pH7.4 PBS 3d.
APS fibre optical sensors antigen coat modifies the coating original solution that APS fibre optical sensors end is submerged to various concentrations
In, it is stored at room temperature 5min.APS optical fiber biosensors are submerged in the sucrose solution that mass fraction is 15%, be stored at room temperature again
1min.Room temperature is dried, and is placed in 5 DEG C of kept dries, is just given birth to APS fibre optical sensors and ForteBio Octet Red before use
Thing interaction of molecules instrument connects.
Label-free detection result by ampicillin-BSA conjugate with 1 × PB (pH 7.4) dilute 20 times, take 200 μ L in
In microwell plate (the 2nd row)；Beta-lactam antibiotic acceptor (2mg/mL) is diluted 60 times with pasteurize milk, and with the liquid
Ampicillin standard items are diluted to 100,50,25,12.5,6.25,3.12 and 1.56ng/mL, take 200 μ L respectively in microwell plate
In (the 4th row)；200 μ L water are added in being arranged toward microwell plate the 1st, 200 μ L pasteurize milk are added toward the 3rd row；With APS Fibre Optical Sensors
Device is detected：First row 60s, the 2nd row 60s；3rd row 60s；4th row 300s.As a result it is as shown in Figure 1.
1. prepared by golden labelled antibody for the detection of gold mark：The beta-lactam that 1 μ L 2mg/mL are added into 1mL collaurums resists
Raw plain acceptor, is vortexed and mixes, and is stored at room temperature 15min, adds 100 μ L 10%BSA, room temperature closing 15min, 10000rpm centrifugation
10min, supernatant liquor is discarded, add 90 μ L PBS (pH 7.4), resuspended particle is standby；2. detect：By ampicillin-BSA
Conjugate dilutes 20 times with 1 × PB (pH7.4), takes 200 μ L (the 2nd row) in microwell plate；It is blue or green with pasteurize milk diluted ammonia benzyl
Mycin standard items take 200 μ L (the 4th row) in microwell plate, in addition respectively to 50,25,12.5,6.25,3.12 and 1.56ng/ml
50 μ L reaction solutions and 5 μ L gold mark beta-lactam antibiotic acceptors are added per hole；200 μ L water are added in being arranged toward microwell plate the 1st, it is past
3rd row add 200 μ L pasteurizes milk, 50 μ L reaction solutions, 5 μ L gold marks BSA；Detected with APS fibre optical sensors：First row
60s, the 2nd row 60s；3rd row 60s；4th row 600s.As shown in Figure 2.
Signal is read by spectrometer, draws curve, determines detection sensitivity, obtains qualitative results and quantitative result.Knot
Fruit is shown in Table 1, and acquired results are better than colloidal gold immuno-chromatography test paper strip method.
Embodiment two：A kind of method of quick detection belt-lactam antibiotics residues
A kind of optics immunoassay that can quickly determine specific objective thing beta-lactam antibiotic in animal derived food
Quick determination method, including beta-lactam antibiotic specific antibody, coated beta-lactam antibiotic and carrier protein
Conjugate and following steps：
(1) step prepares ampicillin-BSA and weighs ampicillin sodium salt 149mg, be dissolved in 8mL DMF, magnetic agitation
A liquid is dissolved to obtain to whole.BSA204mg is weighed, is dissolved in 16mL PBS (pH 7.4), stirring is to being completely dissolved to obtain B liquid.Stirring
Mix under sub- stirring, A liquid be added in B liquid, immediately thereto dropwise, be uniformly added into the μ L of 25% glutaraldehyde 150, at room temperature gently
Stirring, lucifuge reaction 3h.Products therefrom obtains coating antigen with 0.05M pH7.4 PBS 3d.
Step (2) fibre optical sensor antigen coat modification APS fibre optical sensors end is submerged various concentrations coating antigen it is molten
In liquid, 5min is stored at room temperature.Optical fiber biosensor is submerged in 15% sucrose again, is stored at room temperature 1min.Room temperature is dried, and is placed in
2~8 DEG C of kept dries, it is standby.
(3) ampicillin-BSA conjugate is diluted 20 times to step by label-free detection result with 1 × PB (pH 7.4), is taken
200 μ L (the 2nd row) in microwell plate；By 60 times of pasteurize milk dilution of beta-lactam antibiotic acceptor (2mg/mL), and
With the liquid diluting ampicillin standard items to 100,50,25,12.5,6.25,3.12 and 1.56ng/mL, 200 μ L are taken respectively
In microwell plate (the 4th row)；200 μ L water are added in being arranged toward microwell plate the 1st, 200 μ L pasteurize milk are added toward the 3rd row；Use APS
Probe is detected：First row 60s, the 2nd row 60s；3rd row 60s；4th row 300s.The sensitivity of non-marked method detection exists
50ng/ml or so.After testing, the content in pasteurize milk is 0.34 microgram/ml.
(4) 1. prepared by golden labelled antibody for the detection of gold mark for step：1 μ L 2mg/mL β-interior acyl is added into 1mL collaurums
Amine antibiotic acceptor, it is vortexed and mixes, be stored at room temperature 15min, adds 100 μ L 10%BSA, room temperature closing particle 15min,
10000rpm centrifuges 10min, discards supernatant liquor, adds 90 μ L PBS (pH 7.4), and resuspended particle is standby；2. detect：By ammonia
Parasiticin-BSA conjugate dilutes 20 times with 1 × PB (pH7.4), takes 200 μ L (the 2nd row) in microwell plate；Use pasteurize
Milk dilutes ampicillin standard items to 50,25,12.5,6.25,3.12 and 1.56ng/ml, takes 200 μ L respectively in microwell plate
(the 4th row), 50 μ L reaction solutions and 5 μ L gold mark beta-lactam antibiotic acceptors are added per hole in addition；Add in being arranged toward microwell plate the 1st
Enter 200 μ L water, 200 μ L pasteurizes milk, 50 μ L reaction solutions, 5 μ L gold marks BSA are added toward the 3rd row；Detected with APS probes：
First row 60s, the 2nd row 60s；3rd row 60s；4th row 600s.The sensitivity of golden mark detection method can reach 1.56ng/ml
Left and right.After testing, the content in pasteurize milk is 5.32ng/ml.
(5) step is detected and 1. balanced：(the μ L of 200 μ L bottoms liquid+50 are submerged into the liquid of bare substrate bottom in APS fibre optical sensors end
The μ of reaction solution+5 L gold mark BSA) in 120s；2. test：Chloramphenicol element is submerged into APS fibre optical sensors end and detects negative bottom liquid
600s in (200 μ L chloramphenicol element detects the negative μ L of the μ of sample liquid+50 L reaction solutions+5 gold mark chloramphenicol element acceptor)；3. regenerate：By light
Gly/DMF is submerged in fine biosensor probe bottom (the 0.1M glycine (pH 3) of 4 times of volumes mixes with the DMF of 1 times of volume)
30s；4. wash：Probe bottom is washed with PBS (pH 7.4) once.
The object of the detection is beta-lactam antibiotic.
The beta-lactam antibiotic coating antigen is to be coupled realization by parent of ampicillin.
The beta-lactam antibiotic envelope antigen is to carry out coupling in fact with ampicillin sodium salt and carrier protein
Existing.
The carrier protein is bovine serum albumin (BSA).
Described optical biosensor be with reference to step (3) ampicillin-BSA conjugate used, (4) it is used gold mark β-
The fibre-optical probe of lactam antibiotics acceptor coating antigen surface modification.
The fibre-optical probe of beta-lactam antibiotic coating antigen surface modification is marked to actual sample with non-marked and gold respectively
Detected, the signal processor of the concentration by being configured as determining biomaterial from detection unit based on the electric signal exported
It whether there is object and concentration information in detection and identification testing sample solution.
The detection method of a kind of beta-lactam antibiotic content according to claim 1, it is characterised in that described
Beta-lactam antibiotic detected using optics immunoassay quick determination method, non-marked method detection sensitivity
In 50ng/ml or so, the sensitivity of golden mark detection method can reach 1.56ng/ml or so.
Embodiment three：A kind of method of quick detection belt-lactam antibiotics residues
A kind of optical immunoassay of quick detection belt-lactam antibiotics residues, methods described include β-interior acyl
The conjugate of amine antibiotic specific antibody, coated beta-lactam antibiotic and carrier protein prepares and following steps：
(1) step prepares ampicillin-BSA and weighs ampicillin sodium salt 149mg, be dissolved in 8mL DMF, magnetic agitation
A liquid is dissolved to obtain to whole.BSA204mg is weighed, is dissolved in 16mL PBS (pH 7.4), stirring is to being completely dissolved to obtain B liquid.Stirring
Mix under sub- stirring, A liquid be added in B liquid, immediately thereto dropwise, be uniformly added into the μ L of 25% glutaraldehyde 150, at room temperature gently
Stirring, lucifuge reaction 3h.Products therefrom obtains coating antigen with 0.05M pH7.4 PBS 3d.
(2) fibre optical sensor antigen coat thing modifies the coating antigen that APS fibre optical sensors end is submerged to various concentrations to step
In solution, 5min is stored at room temperature.Optical fiber biosensor is submerged in 15% sucrose again, is stored at room temperature 1min.Room temperature is dried, and is put
It is standby in 2~8 DEG C of kept dries.
The optimization of step (3) beta-lactam antibiotic acceptor dosage is by ampicillin-BSA conjugate (6mg/ml) with 1
× PBS (pH 7.4) dilutes 20 times, takes 200 μ L (the 2nd row) in microwell plate；By beta-lactam antibiotic acceptor (2mg/mL)
20,60,180,540 and 1620 times are diluted to pasteurize milk, takes 200 μ L (the 4th row) in microwell plate respectively；Toward microwell plate
200 μ L water are added in 1st row, 200 μ L pasteurized milks are added toward the 3rd row；Detected with APS probes：First row 60s, the 2nd
Arrange 60s；3rd row 60s；4th row 300s.Most suitable acceptor dosage is selected according to testing result, it is determined here that 1:60 (i.e. 100 μ g/
ML) it is used as optimal acceptor dosage.
(4) ampicillin-BSA conjugate is diluted 20 times to step by label-free detection result with 1 × PB (pH 7.4), is taken
200 μ L (the 2nd row) in microwell plate；By 60 times of pasteurize milk dilution of beta-lactam antibiotic acceptor (2mg/mL), and
With the liquid diluting ampicillin standard items to 100,50,25,12.5,6.25,3.12 and 1.56ng/mL, 200 μ L are taken respectively
In microwell plate (the 4th row)；200 μ L water are added in being arranged toward microwell plate the 1st, 200 μ L pasteurize milk are added toward the 3rd row；Use APS
Probe is detected：First row 60s, the 2nd row 60s；3rd row 60s；4th row 300s.The beta-lactam antibiosis of pasteurize milk
Cellulose content is 59.65 microgram ng/ml, and through being contrasted with other methods, the sensitivity of non-marked method detection is in 50ng/ml or so.
(5) 1. prepared by golden labelled antibody for the detection of gold mark for step：1 μ L 2mg/mL β-interior acyl is added into 1mL collaurums
Amine antibiotic acceptor, it is vortexed and mixes, be stored at room temperature 15min, adds 100 μ L 10%BSA, room temperature closing particle 15min,
10000rpm centrifuges 10min, discards supernatant liquor, adds 90 μ L PBS (pH 7.4), and resuspended particle is standby；2. detect：By ammonia
Parasiticin-BSA conjugate dilutes 20 times with 1 × PB (pH7.4), takes 200 μ L (the 2nd row) in microwell plate；Use pasteurize
Milk dilutes ampicillin standard items to 50,25,12.5,6.25,3.12 and 1.56ng/ml, takes 200 μ L respectively in microwell plate
(the 4th row), 50 μ L reaction solutions and 5 μ L gold mark beta-lactam antibiotic acceptors are added per hole in addition；Add in being arranged toward microwell plate the 1st
Enter 200 μ L water, 200 μ L pasteurizes milk, 50 μ L reaction solutions, 5 μ L gold marks BSA are added toward the 3rd row；Detected with APS probes：
First row 60s, the 2nd row 60s；3rd row 60s；4th row 600s.The beta-lactam antibiotic content of pasteurize milk is 10.37
Microgram ng/ml, through being contrasted with other methods, the sensitivity of golden mark detection method can reach 1.56ng/ml or so.
Sensitivity test is with gold mark BLI detections and collaurum added with the standard beta-lactam antibiotic of various concentrations
Milk (is negative) with standard method measure beta-lactam antibiotic in advance.The result of two kinds of detection methods is as shown in table 1.
It is big mould that specificity experiments detect the Aflatoxins M1 that spiked levels are 1000ng/ml, celebrating with the method established
Element, kanamycins, streptomysin, tylosin, chloramphenicol and melamine, testing result are feminine gender.
The influence evaluation of matrix is blue or green with the pasteurize milk doubling dilution ammonia benzyl of 6 portions of former milk, 7 parts from different batches respectively
Mycin standard items, gold mark detection mode is respectively adopted and is detected, sensitivity stably reaches 1.56ng/ml.
Sensor regeneration evaluation specific steps：1. balance：(200 μ L10% are submerged into milk in APS fibre optical sensors end
The μ L of the μ L of milk powder+50 reaction solutions+5 gold mark BSA) in 120s；2. detect：APS fibre optical sensors end is submerged into beta-lactam to resist
The negative milk of raw element detection (the negative μ L of+50 μ L reaction solutions of pasteurize milk+5 gold mark β of 200 μ L beta-lactam antibiotics detection-
Lactam antibiotics acceptor) in 600s.3. regenerate：Gly/DMF (4 times of volumes are submerged into optical fiber biosensor probe bottom
0.1M glycine (pH 3) mixed with the DMF of 1 times of volume) 30s.4. wash：Probe bottom is washed with PBS (pH7.4) once.
Experiment shows, is coated with ampicillin-BSA APS fibre optical sensors pH1.7 renewable 5 times of glycine.
The above-described embodiments merely illustrate the principles and effects of the present invention, not for the limitation present invention.It is any ripe
Know the personage of this technology all can carry out modifications and changes under the spirit and scope without prejudice to the present invention to above-described embodiment.Cause
This, those of ordinary skill in the art is complete without departing from disclosed spirit and institute under technological thought such as
Into all equivalent modifications or change, should by the present invention claim be covered.
Claims (5)
- A kind of 1. method of quick detection belt-lactam antibiotics residues, it is characterised in that：Comprise the following steps：The first step：Prepare ampicillin-BSAAmpicillin sodium salt is dissolved in dimethylformamide, stirs to after all dissolving to obtain A liquid；Bovine serum albumin(BSA) is dissolved in PH value is in 7.4 PBS, and stirring obtains B liquid to after being completely dissolved；Under agitation, A liquid is added in B liquid, Then thereto dropwise, be uniformly added into the glutaraldehyde water solution that volume fraction is 25%, under stirring condition, lucifuge reaction, gained production The PBS that thing is 7.4 with 0.05M pH value is dialysed, and obtains coating antigen, i.e. ampicillin-BSA conjugate；Second step：Fibre optical sensor antigen coat is modifiedAPS fibre optical sensors end is submerged in coating original solution, is stored at room temperature 5 min；Again by APS optical fiber biosensors end End is submerged in the aqueous sucrose solution that mass fraction is 15%, is stored at room temperature 1 min, 2~8 DEG C of dryings are placed in after then room temperature is dried Under the conditions of preserve, it is standby；3rd step：Label-free detectionThe PB buffer solutions that ampicillin-BSA conjugate pH value is 7.4 are diluted 20 times, take 200 μ L in the 2nd of microwell plate In row；Beta-lactam antibiotic acceptor is diluted 60 times with pasteurize milk, then with the liquid diluting ampicillin mark Quasi- product take 200 μ L in the 4th row of microwell plate respectively to 100,50,25,12.5,6.25,3.12 and 1.56 ng/mL；It is past micro- 200 μ L water are added in 1st row of orifice plate, 200 μ L pasteurize milk are added toward the 3rd row；Carried out with APS fiber-optic sensor probes Detection：First row 60 s, the s of the 2nd row 60；The s of 3rd row 60；The s of 4th row 300；4th step：Gold mark detection1 μ L 2mg/mL beta-lactam antibiotic acceptor is added into 1mL collaurums, is vortexed and mixes, be stored at room temperature 15min, The mass fraction for adding 100 μ L is 10% bovine serum albumin solution, and room temperature closing 15min, 10000 rpm centrifuge 10 min, Supernatant liquor is discarded, the PBS that 90 μ L pH values are 7.4 is added, resuspended particle, obtains gold mark beta-lactam antibiotic Acceptor；The PB buffer solutions that ampicillin-BSA conjugate pH value is 7.4 are diluted 20 times, take 200 μ L in the 2nd of microwell plate In row；Ampicillin standard items are diluted to 50,25,12.5,6.25,3.12 and 1.56 ng/ml, difference with pasteurize milk Take 200 μ L in microwell plate the 4th row in, in addition per hole add 50 μ L reaction solutions and 5 μ L gold mark beta-lactam antibiotic by Body；200 μ L water are added into the 1st row of microwell plate, the 3rd 200 μ L pasteurizes milk of row addition, the 50 μ L toward microwell plate are anti- Answer liquid, 5 μ L gold marks BSA；Detected with APS fiber-optic sensor probes：First row 60s, the 2nd row 60s；3rd row 60s；4th row 600s。
- 2. the method for quick detection belt-lactam antibiotics residues according to claim 1, it is characterised in that：Also include The regeneration of APS fibre optical sensors, including：Balance：120s in the liquid of bare substrate bottom is submerged into APS fibre optical sensors end, 200 μ L are contained in the bare substrate bottom liquid Bottom liquid, 50 μ L reaction solution and 5 μ L gold mark BSA；Test：Chloramphenicol element is submerged into APS fibre optical sensors end and detects 600s in negative bottom liquid, chloramphenicol element detection is negative In the liquid of bottom negative sample liquid, 50 μ L reaction solutions and 5 μ L gold mark chloramphenicol element acceptors are detected containing 200 μ L chloramphenicol element；Regeneration：30s in glycine and dimethylformamide mixed liquor, the sweet ammonia are submerged into optical fiber biosensor probe bottom Acid and dimethylformamide mixed liquor by pH value be 3 0.1 M glycine and dimethylformamide according to 4：1 volume ratio Mixing is formed；Washing：Optical fiber biosensor probe bottom is washed with the PBS that pH value is 7.4.
- 3. the method for quick detection belt-lactam antibiotics residues according to claim 1, it is characterised in that：It is described anti- It is the PBS that pH values are 7.4 to answer liquid.
- 4. the method for quick detection belt-lactam antibiotics residues according to claim 2, it is characterised in that：The bottom Liquid is to be made after milk powder is dissolved in water, and its mass fraction is 10%.
- 5. the method for quick detection belt-lactam antibiotics residues according to claim 1, it is characterised in that：By 149 Mg ampicillin sodium salts are dissolved in 8mL dimethylformamides, stir to after all dissolving to obtain A liquid；By 204mg bovine serum albumin(BSA)s The pH value for being dissolved in 16mL is in 7.4 PBS, and stirring obtains B liquid to after being completely dissolved.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711095951.1A CN107894505A (en) | 2017-11-09 | 2017-11-09 | A kind of method of quick detection beta-lactam antibiotics residue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711095951.1A CN107894505A (en) | 2017-11-09 | 2017-11-09 | A kind of method of quick detection beta-lactam antibiotics residue |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107894505A true CN107894505A (en) | 2018-04-10 |
Family
ID=61804744
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711095951.1A Pending CN107894505A (en) | 2017-11-09 | 2017-11-09 | A kind of method of quick detection beta-lactam antibiotics residue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107894505A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109085351A (en) * | 2018-09-05 | 2018-12-25 | 顾娟红 | A kind of method of quick detection enrofloxacin residual |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104132913A (en) * | 2013-04-29 | 2014-11-05 | 帕尔公司 | Detection and/or quantitation of endotoxin |
-
2017
- 2017-11-09 CN CN201711095951.1A patent/CN107894505A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104132913A (en) * | 2013-04-29 | 2014-11-05 | 帕尔公司 | Detection and/or quantitation of endotoxin |
Non-Patent Citations (6)
| Title |
|---|
| C.M.MARAGOS: "Signal amplification using colloidal gold in a biolayer interferometry-based immunosensor for the mycotoxin deoxynivalenol", 《FOOD ADDITIVES AND CONTAMINANTS》 * |
| CHRIS M.MARAGOS: "Detection of deoxynivalenol using biolayer interferometry", 《MYCOTOX RES》 * |
| TERRY F.MCGRATH: "An evaluation of the capability of a biolayer interferometry biosensor to detect low-molecular-weight food contaminants", 《ANAL BIOANAL CHEM》 * |
| 刘小军: "用生物膜干涉技术快速检测牛乳中beta内酰胺类抗生素残留", 《安徽农业科学》 * |
| 李宏: "生物膜干涉技术快速定性检测牛乳中喹诺酮类药物残留", 《安徽农业科学》 * |
| 王炜: "牛奶中掺假物及氨苄青霉素快检试纸条的研制", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109085351A (en) * | 2018-09-05 | 2018-12-25 | 顾娟红 | A kind of method of quick detection enrofloxacin residual |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11255854B2 (en) | Signal amplification in lateral flow and related immunoassays | |
| US10732111B2 (en) | Automated immunoanalyzer system for performing diagnostic assays for allergies and autoimmune diseases | |
| Sternesjo et al. | Determination of sulfamethazine residues in milk by a surface plasmon resonance-based biosensor assay | |
| US4954452A (en) | Non-metal colloidal particle immunoassay | |
| CN105758835A (en) | Homogeneous phase immunoassay POCT detection technique and system using same | |
| US20130084580A1 (en) | Chromatographic kit and chromatography method | |
| US20200333337A1 (en) | Method for re-using test probe and reagents in an immunoassay | |
| CN102135535B (en) | Immune colloidal metal detection technology capable of directly performing semi-quantitative analysis, preparation method and application | |
| CN109444427A (en) | A kind of kit detecting human parvovirus IgM antibody | |
| CN108445239B (en) | Homogeneous phase immunoassay reagent kit for detecting beta human chorionic gonadotropin, and preparation method and application thereof | |
| JPS62110155A (en) | Reagent and method for immunoassay | |
| Li et al. | Ultrafast Electrothermal Flow‐Enhanced Magneto Biosensor for Highly Sensitive Protein Detection in Whole Blood | |
| JPH05223820A (en) | Kit and method for detecting microorganism accompanied by periodontal disease using surfactant mixture as extraction composition | |
| CN107894505A (en) | A kind of method of quick detection beta-lactam antibiotics residue | |
| CN107884569A (en) | A kind of method of quick detection residual chloromycetin | |
| CN107860909A (en) | A kind of method of quick detection tetracycline residue | |
| US20140011190A1 (en) | Method for performing a rapid test | |
| US20120031206A1 (en) | Analysis method, sample analysis tool, method for preventing back-flow of sample solution, and method for preventing increase in background | |
| JP5294257B2 (en) | Hemoglobin measurement method and measurement kit | |
| CN104215755A (en) | Immunoassay reagent kit based on flexible core-shell quantum dot coupling marker and application method of immunoassay reagent kit | |
| CN108827875A (en) | A kind of method of quick measurement liquid veterinary drug residue and illegal additive | |
| CN109085351A (en) | A kind of method of quick detection enrofloxacin residual | |
| CA2137238A1 (en) | Immunoassay method | |
| JPS59197862A (en) | Reagent for immunoassay to measure simultaneously multiple terms | |
| CN109444428A (en) | A kind of kit of quantitative detection human parvovirus IgG antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180410 |
|
| RJ01 | Rejection of invention patent application after publication |