CN107860724A - A kind of salbutamol detection method based on optical biosensor - Google Patents
A kind of salbutamol detection method based on optical biosensor Download PDFInfo
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Abstract
The invention discloses a kind of salbutamol detection method based on optical biosensor, is applicable to the detection of salbutamol content in pork and tissue, including：Prepared by heavy nitrogen amination small molecule antigens, envelope antigen modifies fibre-optical probe and is detected and identified the specific steps that whether there is object and concentration information in biological specimen to be measured using multimode interference effect.It is an advantage of the invention that the salbutamolum residue in pork and tissue accurately can be detected delicately, the pretreatment process of sample is simple, takes less, can detect substantial amounts of sample simultaneously, sample detection cost is far below traditional instrument detection method.
Description
Technical field
The invention belongs to biological immune detection field, and in particular to a kind of salbutamol inspection based on optical biosensor
A kind of survey method, more particularly to using optical biosensor simple in construction easy to make and cheap cost to pork product
The quick determination method of middle object.
Background technology
Biology sensor（Biosensor）It is a kind of high-new biological analysis detection technique gradually to grow up in recent years,
Biology or bionics induction of signal member in tight are coupled to or integrated into sensor-based system by it, by fixing on the support
Sensitive biological materials (i.e. sensing element) are specifically bound with analyte, and the change of one or more physicochemical characteristics occurs
(pH value changes, electronics transfer, mass spectrum change, heat transfer, the intake or release of gas or specific ion), then by transducer
It (is typically electric signal to be converted to secondary available signal）, what the power of this signal or the change of frequency and sensing element were combined
Analyte or analyte chemical group are related, and the analysis that determinand is carried out is determined so as to realize.
Optical fiber biosensor based on light absorbs, fluorescence and light reflex etc.（fiber optic biosensor）
It is the study hotspot of optical fiber immunity biosensor in recent years, it is to be measured with being directed on the affinity membrane of coreless fiber surface by determinand
The immune response of high specific, high-affinity occurs for the acceptor of thing, and the high-order mode propagation constant for making to propagate in coreless fiber is sent out
Raw to change, thus can detect to whether there is in testing liquid has specific binding with the antibody for being fixed on coreless fiber surface
The antigen and its concentration information of characteristic.Because optical fiber biosensor has the characteristics of quick, easy, cheap, and specificity
By force, stability is good, and foreign countries have been used for the primary dcreening operation detection of high flux, lower bound amount sample, but correlative study is started late at home, especially
It is still in conceptual phase in practical field.
With salbutamol（salbutamol）As a kind of short-acting beta-2-adrenoreceptor agonists medicine of selectivity, energy
Effectively suppress the release that histamine etc. causes anaphylaxis material, add micro salbutamol in animal feeding-stuff, livestock can be increased
Cutability and meat change rate, reduce fat, but its toxicity be far above with identical function Ractopamine.With hydrochloric acid gram
The recall rates such as Roc spy, Ractopamine, which have respectively obtained, significantly to be controlled, and equally has promotion growth and excitant work(
The salbutamol frequent detection in detection process in the recent period of energy, thus cause the great attention of national correlation department.Effectively
Detection method be to control one of key of salbutamol abuse, it is therefore necessary to research and develop highly sensitive quick determination method.
Traditional instrument analytical method mainly uses high performance liquid chromatography（HPLC）, Capillary Zone Electrophoresis（CE）It is fixed
Amount, liquid chromatography-mass spectrometry method（LC/MS）Or gas chromatography-mass spectrography analytic approach（GC/MS）Confirmation.With radiation
Immunoassay（RIA）, fluorescence immunoassay（FIA）, chemiluminescent immunoassay（CIA）, solid-phase immunity sensor
(SIS), enzyme-linked immunosorbent assay (ELISA) and colloidal gold immune chromatography experiment determination method (GlCA) are divided for the immune of representative
Analysis method is mainly used to screen and quantified.Light reflection interference detection technique is because it has the advantages that non-marked, can detect in real time, closely
It is widely used in the fields such as food security and drug screening, wherein biomembrane interference technique over year（BLI）And thus derive
Multiple-mode interfence fibre optical sensor with its it is special it is sensitive, quick and precisely, easy to operate, Cheap highly effective, simple pre-treatment the features such as
As the important tool of residue detection, also quickly screen monitoring for pork and tissue veterinary drug residue and provide new selection.
The content of the invention
Present invention aims at provide a kind of multiple-mode interfence optical fiber biosensor modified using carrier protein couplet thing
The method for detecting salbutamol, the Quantitative detection for salbutamol in pork and tissue.
To achieve the above object, technical scheme provided by the invention is：A kind of husky butylamine based on optical biosensor
Alcohol detection method, including the diazotising of p-aminobenzoic acid, salbutamol-BSA and salbutamol-OVA conjugates preparation and
Markless detection.
Comprise the following steps：
The first step：P-aminobenzoic acid is dissolved in hydrochloric acid and obtains p-aminobenzoic acid solution, then by sodium nitrite solution by
It is added dropwise in p-aminobenzoic acid solution, lucifuge reacts to obtain solution A；
Second step：Salbutamol sulfate is dissolved in ice-cold borate buffer solution, then the solution A be added dropwise, lucifuge
Reaction obtains orange solution B；Then add boric acid crystal and adjust pH value that it is pure next to add cationization ox blood to 7.4
Albumen, while add 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride, N-N- HOSu NHSs, room temperature
Stir to obtain orange solution C；The PBS that solution C pH value is 7.4 is dialysed, and obtains coating antigen；Obtained by ultra-violet analysis
Absworption peak confirms that SAL-ABA-cBSA is coupled successfully at 278 nm and 336 nm, constant also shows sand as orange-yellow in dialysis procedure
Butylamine alcohol is already connected on carrier protein.
3rd step：Salbutamol sulfate is dissolved in ice-cold borate buffer solution, then the solution A is added dropwise,
Lucifuge reacts to obtain orange solution D；Then add boric acid crystal and adjust pH value then to add chicken egg white, simultaneously to 7.4
1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride, N-N- HOSu NHSs are added, is stirred at room temperature
Orange solution E, the PBS that solution E pH value is 7.4 are dialysed, and obtain immunogene；Confirm SAL-ABA- through ultra-violet analysis
OVA is coupled successfully.
4th step：Fibre optical sensor end is submerged in 1 mg/L coating original solution, be stored at room temperature it after 5 min again
It is placed in mass fraction and is dried in 15% sucrose solution, to be stored at room temperature to take out after 1 min, preserved under 2~8 DEG C of drying condition,
It is standby；
5th step：Actual pork sample is detected.The muscle parts of pork sample are chosen, grease and manadesma is removed, uses knife
Chop up, rubbed with meat grinder into rotten shape, add the methanol solutions of 20ml 70% strength vibration 3 minutes, with Whatman No.1 filter paper mistakes
Filter, it can be used directly to determine if filtrate is clarified；But the method that can use centrifugation if muddy, 3000rpm, 5min,
Clarified supernatant is taken to determine；Spectral signal is read by fiber spectrum analytical equipment, curve is drawn, determines detection sensitivity,
Sample salbutamol content is determined by standard curve.
Preferably technical scheme is：Also include the drafting of standard curve：Salbutamol sulfate standard items accurately are weighed, use boron
Sand buffer solution is configured to the standard reserving solution that concentration is 1mg/mL, then delays the PBS that salbutamol storing solution pH value is 7.4
It is 81,27,9,3,1,0.3 and 0 ng/mL that fliud flushing, which is diluted to concentration, is submerged with fibre optical sensor and is wherein measured, Ran Houhui
Standard curve processed.
Preferably technical scheme is：By 10mg p-aminobenzoic acid dissolve in 1.1mL concentration be 0.2M hydrochloric acid in must be right
In aminobenzoic acid solution, the sodium nitrite solution is dissolved in 0.35 mL distilled water by 6 mg natrium nitrosum and is made.
Preferably technical scheme is：By salbutamol-BSA conjugate obtained by second step（SAL-ABA-cBSA）Adjust concentration
For 6mg/ml, 20 times then are diluted with 1 × PB buffer solutions that pH value is 7.4,200 μ L are taken in microwell plate, by 2mg/mL sand
Butylamine alcohol antibody-solutions dilute 20 times with bare substrate sample liquid, and obtain series standard with the liquid diluting salbutamol standard items
Solution, spectral signal is read by detecting instrument, standard curve is drawn, determines detection sensitivity.Bare substrate sample liquid with it is to be measured
Sample is just the same, but does not contain object i.e. salbutamol.
Using the multiple-mode interfence fiber-optic sensor probe after modification（Fibre optical sensor i.e. made from the 4th step）Addition is marked
The liquid to be checked of quasi- product carries out markless detection, comprises the following steps that：1. balance：Fibre optical sensor end is submerged negative to be checked
120s in liquid (the 200 μ l μ of liquid+50 l buffer solutions to be checked)；2. detect：Fibre optical sensor end is submerged after system balancing
300s in (the μ g antibody of+50 μ l buffer solutions of 200 μ l testing samples+2) in testing liquid；Finally, signal value is gathered according to foregoing conjunction
Suitable curve fitting technique obtains quantitation curves.Liquid to be checked is bare substrate sample liquid.
Because above-mentioned technical proposal is used, the present invention this have the advantage that compared with prior art：
The quantitative detection of salbutamol in biological sample is realized using non-marked optical fibre interferometric sensor technology, this method has
Good specificity and sensitivity, the Quantitative detection of salbutamol in pork and tissue is applicable to, is advantageous to food prison
Pipe portion door carries out site supervision and quantitative primary dcreening operation to pork, ensures people's lives safety.
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 quickly determining salbutamol in pork to BLI technologies with reference to following examples is described further.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Equipment used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment one：A kind of salbutamol detection method based on optical biosensor
P-aminobenzoic acid it is diazotizing prepare it is as follows：
10 mg p-aminobenzoic acid are weighed, are dissolved in 1.1 mL 0.2M HCl, the natrium nitrosum for then weighing 6mg is dissolved in 0.35
In mL distilled water, 0~4 DEG C of stirring, sodium nitrite solution is added dropwise in p-aminobenzoic acid solution, lucifuge reaction 1
H, it is standby to obtain solution A.
(2) the synthesis step of coating antigen (SAL-ABA-cBSA) is as follows：
Weigh 10mg salbutamol sulfates and be dissolved in the ice-cold 0.05M borate buffer solutions of 10 mL (pH 8.5, NaCl containing 0.15M)
In, 4 DEG C of stirrings, take the above-mentioned solution As of 0.9 mL to be added dropwise, lucifuge obtains orange solution B after reacting 2 h, with a small amount of boric acid
Crystal adjusts pH to 7.4, adds 94 mg Cationic bovine serum albumins (cBSA), while add 80 mg 1- ethyls-(3-
Dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 24 mg N-N- HOSu NHSs, it is stirred at room temperature after 3 h
Orange solution C, finally obtain coating antigen (SAL-ABA-cBSA) with the 4 DEG C of dialysis of PBS (pH 7.4) buffer solution.By ultraviolet spectrometry
Photometer is surveyed, and absworption peak confirms that SAL-ABA-cBSA is coupled successfully at 278 nm and 336 nm, as orange in dialysis procedure
Color is constant also to show that salbutamol is already connected on carrier protein.
(3) the synthesis step of immunogene (SAL-ABA-OVA) is as follows：
Weigh 20 mg salbutamol sulfates and be dissolved in the ice-cold 0.05 M borate buffer solutions (pH 8.5, containing 0.15 M of 10 mL
NaCl in), 4 DEG C of stirrings, the above-mentioned solution As of 0.9 mL are taken to be added dropwise, lucifuge obtains orange solution D after reacting 2 h；Add a small amount of
Boric acid crystal adjusts the pH to 7.4 of solution D, then adds 119.54 mg OVA, while adds 160 mg EDC and 48mg NHS,
3h is stirred at room temperature, obtains orange solution E, is finally immunogene (SAL-ABA-OVA) with 5 d of PBS (pH 7.4) dialysis.Through ultraviolet
SAL-ABA-OVA is analyzed to identify to be coupled successfully.
(4) with step, (2) gained conjugate modification fibre-optical probe is allowed to form affinity membrane step, by APS fibre optical sensors end
End is submerged in 1 mg/L coating original solution, is stored at room temperature after 5 min and is placed on again in 15% sucrose solution, is stored at room temperature 1
Take out and dry after min, it is 2~8 DEG C of kept dries, standby.
(5) salbutamol examination criteria Drawing of Curve：
The configuration of standard reserving solution：Salbutamol sulfate standard items are accurately weighed respectively, with 0.05 M borate buffer solutions (pH
8.5, containing 0.15 M NaCl) it is set to the standard reserving solution that concentration is 1mg/mL.
(6) standard curve configures：Be respectively 81 with the PBS diluted concentration of pH=7.4 by salbutamol storing solution,
27th, 9,3,1,0.3 and 0 ng/mL standard liquid.
With APS optical fiber biosensor experimental provisions, the APS optical fiber biosensors experimental provision includes biomolecule phase
Interaction instrument and step（4）Obtained APS optical fiber biosensors, the liquid to be checked that with the addition of standard items is detected, examined
Survey process is as follows：1. balance：APS fibre optical sensors end is submerged into negative liquid to be checked, and (200 μ l liquid to be checked (do not add
The standard liquid of the sample liquid of standard items, i.e. 0 ng/mL)+50 μ l pH=7.4 PBS) in 120s；3. detect：By Fibre Optical Sensor
(200 μ l testing samples are submerged in testing liquid in device end（The ng/mL of i.e. 81,27,9,3,1,0.3 and 0 standard liquid）+ 50 μ l are buffered
The μ g of liquid+2 antibody) in 300s.APS fibre optical sensors are combined with ForteBio Octet Red bio-molecular interactions instrument, i.e.,
ForteBio Octet Red bio-molecular interaction instrument is sent the signal to after the detection of APS fibre optical sensors to be handled.
(7) specificity and sensitivity detection
Salbutamol-BSA conjugate (6mg/mL) is diluted 20 times with 1 × PB (pH7.4), takes 200 μ l the (the 2nd in microwell plate
Row)；Salbutamol antibody (2mg/mL) is diluted 20 times with bare substrate bottom liquid, the bare substrate bottom liquid is negative pig urine
（After testing, the pig urine of salbutamol is not contained）, and with the liquid diluting salbutamol standard items to 81,27,9,3,1,0.3 and
0 ng/mL, 200 μ l (the 4th row) in microwell plate are taken respectively, and add 50 μ l reaction solutions, mix；Add in being arranged toward microwell plate the 1st
Enter 200 μ l water, 200 μ l pigs urine, 50 μ l reaction solutions are added toward the 3rd row（The PBS of pH=7.4）；Carried out with APS probes
Detection (each row used time is followed successively by 60s, 60s, 60s, 300s).As a result prove the sensitivity of this method survey in 9 ng/mL or so.
Clenobuterol hydrochloride, Ractopamine, the hydrochloric acid for being 1000 ng/mL with the method detection spiked levels established
Dopamine, testing result are feminine gender, show that detection of the above-mentioned several materials to salbutamol is noiseless.
(8) actual sample detects
0.5 ± 0.05g of sample after rubbing is taken in 10mL plastic centrifuge tubes, added into pipe 2mL extract solutions (PH=7.4
PBS cushioning liquid), adding salbutamol standard items makes the target concentration in sample be 4.0 μ g/kg, vortex 1min, ultrasonic
20min, 8000r/min centrifugation 10min are placed in a centrifuge, by abovementioned steps, using ForteBio Octet Red biologies point
Son interaction instrument detects with APS optical fiber biosensors to the liquid to be checked for adding standard items, and target concentration subtracts
Add salbutamol standard concentration.Result of calculation：Husky butanolamine containing 2 μ g in every kilogram of sample.
The present invention modifies the optical fiber of optical biosensor with carrier protein couplet thing it can be seen from embodiment more than
Probe, the detection of salbutamol is realized by multimode interference effect.Optical fiber biosensor used in this method makes simply, is easy to
Obtain and cheap.Biomaterial modification, fixation as probe affinity membrane can use any existing method to carry out.Ability
The professional and technical personnel in domain is aware that thought of the invention can use the other modes beyond above-mentioned embodiment real
It is existing.
Embodiment two：A kind of salbutamol detection method based on optical biosensor
A kind of detection method that salbutamol content in pork and tissue is determined using optical biosensor, including following step
Suddenly：
Step is (1)：Weigh 10 mg p-aminobenzoic acid to dissolve in 1.1 mL 0.2M HCl, then weigh 6 mg natrium nitrosum
It is dissolved in 0.35 mL distilled water, 0~4 DEG C of stirring, sodium nitrite solution is added dropwise in p-aminobenzoic acid solution,
Lucifuge reacts 1 h, and it is standby to obtain solution A；
Step is (2)：Weigh 10 mg salbutamol sulfates and be dissolved in 0.05 ice-cold M borate buffer solutions (pH 8.5, containing 0.15 of 10 mL
M NaCl) in, 4 DEG C of stirrings, the solution A for taking 0.9 mL above-mentioned is added dropwise in the solution, and lucifuge reacts 2 h, obtains orange
Color solution B；Add a small amount of boric acid crystal and adjust pH to 7.4, then add 94 mg Cationic bovine serum albumins
(cBSA), at the same add 80 mg 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride (EDC), 24 mg N-N-
HOSu NHS (NHS), is stirred at room temperature 3 h, obtains orange solution C；Solution C is saturating with 4 DEG C of PBS (pH 7.4) buffer solution
Analysis, obtains coating antigen (SAL-ABA-cBSA)；
Step is (3)：Weigh 20 mg salbutamol sulfates and be dissolved in 0.05 ice-cold M borate buffer solutions (pH 8.5, containing 0.15 of 10 mL
M NaCl) in, 4 DEG C of stirrings, above-mentioned 2 mL solution As are added dropwise in the solution, lucifuge reacts 2 h, and it is molten to obtain orange colour
Liquid D；Add a small amount of boric acid crystal to adjust the pH to 7.4 of solution D, then add 119.54 mg chicken egg whites (OVA), add simultaneously
160 mg EDC and 48mg NHS, are stirred at room temperature 3 h, obtain orange solution E, with PBS (pH 7.4) dialyse 5 d after i.e. exempted from
Epidemic focus (SAL-ABA-OVA).
Step is (4)：With step, (2) gained conjugate modification fibre-optical probe is allowed to form affinity membrane, by fibre optical sensor end
Submerge in 1 mg/L coating original solution, be stored at room temperature after 5 min and be placed on again in 15% sucrose solution, be stored at room temperature 1 min
Take out and dry afterwards, it is 2~8 DEG C of kept dries, standby.
Step is (5)：Actual pork sample is detected, chooses the muscle parts of pork sample, removes grease and manadesma,
Chopped up with knife, rubbed with meat grinder into rotten shape, added the methanol solutions of 20ml 70% strength vibration 3 minutes, filtered with Whatman No 1
Paper filters, and filtrate clarification can be used directly to determine
Spectral signal is read by fiber spectrum analytical equipment, curve is drawn, determines detection sensitivity, sample is determined by standard curve
Product salbutamol content.Husky butanolamine containing 3 micro- g kgs in sample.
The optical biosensor be according to step (1) ~ (4), with carrier protein couplet thing be that coating antigen leads to compound
Cross coating or impregnating modification mode form affinity membrane on fibre-optical probe surface, connected by the joints of optical fibre and light probe,
Detected and identified in pork to be measured and tissue by fiber spectrum analytical equipment again and whether there is object and concentration information.
The coating antigen is to turn to parent coupling with p-aminobenzoic acid diazonium to realize.
The salbutamol immunogene is realized with diazol and carrier protein couplet.
The carrier protein is cation bovine serum albumin (cBSA), ovalbumin (OVA).
Tested sample liquid is examined with the multimode fibre probe of salbutamol coating antigen (SAL-ABA-cBSA) surface modification
Survey, by be configured as based on the electric signal exported from detection unit determine biomaterial concentration signal processor detection and
Identify in pork to be measured and tissue and whether there is object and concentration information.
The detection method of a kind of chloromycetin content according to claim 1, it is characterised in that described object is adopted
Detected with non-marked multiple-mode interfence optical biosensor, sensitivity stably reaches 0.0125 ng/ml.
Embodiment three：A kind of salbutamol detection method based on optical biosensor
The present invention provides a kind of optical biosensor analysis method of quick detection salbutamol, and methods described includes following step
Suddenly：The diazotising of p-aminobenzoic acid, salbutamol-BSA and salbutamol-OVA conjugates preparation and markless detection.
(1) step weighs 10 mg p-aminobenzoic acid and dissolved in 1.1 mL 0.2M HCl, then weigh 6 mg nitrous acid
Sodium is dissolved in 0.35 mL distilled water, 0~4 DEG C of stirring, sodium nitrite solution is added dropwise into p-aminobenzoic acid solution
In, lucifuge reacts 1 h, and it is standby to obtain solution A.
(2) step weighs 10 mg salbutamol sulfates and is dissolved in 0.05 ice-cold M borate buffer solutions of 10 mL (pH 8.5, contains
0.15 M NaCl) in, 4 DEG C of stirrings, the solution A for taking 0.9 mL above-mentioned is added dropwise in the solution, and lucifuge reacts 2 h, obtains
Orange solution B.A small amount of boric acid crystal is added in step (2) resulting solution B and adjusts pH to 7.4, then adds 94 mg sun
Bovine serum albumin(BSA) (cBSA) is ionized, while adds 80 mg 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride
Salt (EDC), 24 mg N-N- HOSu NHSs (NHS), are stirred at room temperature 3 h, obtain orange solution C.Solution C PBS (pH
7.4) 4 DEG C of dialysis of buffer solution, obtain coating antigen (SAL-ABA-cBSA), and 278 nm and 336 nm absorb obtained by ultra-violet analysis
It is successfully (orange-yellow in such as dialysis procedure constant also to show that salbutamol is already connected to carrier egg that peak confirms that SAL-ABA-cBSA is coupled
On white).
(3) step weighs 20 mg salbutamol sulfates and is dissolved in 0.05 ice-cold M borate buffer solutions of 10 mL (pH 8.5, contains
0.15 M NaCl) in, 4 DEG C of stirrings, above-mentioned 2 mL solution As are added dropwise in the solution, lucifuge reacts 2 h, obtains orange
Color solution D.Solution D adds a small amount of boric acid crystal to adjust pH to 7.4, then adds 119.54 mg chicken egg whites (OVA), adds simultaneously
Enter 160 mg EDC and 48mg NHS, 3 h are stirred at room temperature, obtain orange solution E.Solution E PBS (pH 7.4) 5 d of dialysis, are obtained
To immunogene (SAL-ABA-OVA), confirm that SAL-ABA-OVA is coupled successfully through ultra-violet analysis.
Step is (4)：With step, (2) gained conjugate modification fibre-optical probe is allowed to form affinity membrane, by fibre optical sensor end
Submerge in 1 mg/L coating original solution, be stored at room temperature and be placed on again after 5 min in the sucrose solution that mass fraction is 15%,
It is stored at room temperature after 1 min to take out and dries, it is 2~8 DEG C of kept dries, standby.
Step（5）By step, (2) gained salbutamol-BSA conjugate (6mg/ml) dilutes 20 times with 1 × PB (pH7.4),
200 μ L are taken in microwell plate, salbutamol antibody (2mg/mL) is diluted 20 times with bare substrate sample liquid, and with the liquid diluting
Salbutamol standard items obtain series standard solution, read spectral signal by detecting instrument, draw standard curve, it is determined that detection
Sensitivity.
Markless detection is carried out to the liquid to be checked for adding standard items using the multiple-mode interfence fibre-optical probe after modification, specifically
Step is as follows：1. balance：Fibre optical sensor end is submerged into negative liquid to be checked, and (the 200 μ l μ of liquid+50 lpH values to be checked are 7.4
PBS) in 120s；The liquid to be checked is identical with the composition of testing sample, but is detected as feminine gender.2. detect：Treat
(the PBS bufferings that the μ lpH values of 200 μ l testing samples+50 are 7.4 are submerged into testing liquid in fibre optical sensor end after system balancing
The μ g of liquid+2 antibody) in 300s；Finally, gather signal value and obtain quantitation curves according to foregoing suitable curve fitting technique.
(5) step detects to actual pork sample, choose the muscle parts of pork sample, removes grease and manadesma, uses
Knife chops up, and is rubbed with meat grinder into rotten shape, adds the methanol solutions of 20ml 70% strength vibration 3 minutes, with the filter paper of Whatman No 1
Filtering, filtrate is muddy, using the method for centrifugation, 3000rpm, 5min, takes clarified supernatant to be measured；Pass through fiber spectrum point
Analysis apparatus reads spectral signal, draws curve, determines detection sensitivity, determines that sample salbutamol content is 5 by standard curve
Micro- g kg.
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 (3)
- A kind of 1. salbutamol detection method based on optical biosensor, it is characterised in that：Comprise the following steps：The first step：P-aminobenzoic acid is dissolved in hydrochloric acid and obtains p-aminobenzoic acid solution, then by sodium nitrite solution by It is added dropwise in p-aminobenzoic acid solution, lucifuge reacts to obtain solution A；Second step：Salbutamol sulfate is dissolved in ice-cold borate buffer solution, then the solution A be added dropwise, lucifuge Reaction obtains orange solution B；Then add boric acid crystal and adjust pH value that it is pure next to add cationization ox blood to 7.4 Albumen, while add 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride, N-N- HOSu NHSs, room temperature Stir to obtain orange solution C；The PBS that solution C pH value is 7.4 is dialysed, and obtains coating antigen；3rd step：Salbutamol sulfate is dissolved in ice-cold borate buffer solution, then the solution A be added dropwise, lucifuge Reaction obtains orange solution D；Then add boric acid crystal and adjust pH value then to add chicken egg white to 7.4, add simultaneously 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride, N-N- HOSu NHSs are added, is stirred at room temperature orange Color solution E, the PBS that solution E pH value is 7.4 are dialysed, and obtain immunogene；4th step：Fibre optical sensor end is submerged in 1 mg/L coating original solution, be placed on again after being stored at room temperature 5 min Mass fraction dries in 15% sucrose solution, to be stored at room temperature to take out after 1 min, is preserved under 2~8 DEG C of drying condition, be standby With；5th step：The muscle parts of pork sample are chosen, grease and manadesma is removed, is chopped up with knife, are rubbed with meat grinder into rotten shape, Vibrate 3 minutes after adding the methanol solution that volume fraction is 70%, filtered with filter paper, Fibre Optical Sensor can be directly used if filtrate is clarified Device submerges filtrate to determine；If filtrate is muddy, filtrate is centrifuged into 5min with 3000rpm, submerges filtrate with fibre optical sensor Clarified supernatant is measured.
- 2. the salbutamol detection method according to claim 1 based on optical biosensor, it is characterised in that：Also Drafting including standard curve：Salbutamol sulfate standard items accurately are weighed, it is 1mg/mL to be configured to concentration with borate buffer solution Standard reserving solution, then by the PBS that salbutamol storing solution is 7.4 with pH value be diluted to concentration be 81,27,9,3, 1st, 0.3 and 0 ng/mL, is submerged with fibre optical sensor and is wherein measured, and then draws standard curve.
- 3. the salbutamol detection method according to claim 1 based on optical biosensor, it is characterised in that：Will Obtained in the hydrochloric acid that the concentration that 10mg p-aminobenzoic acid dissolves in 1.1mL is 0.2M in p-aminobenzoic acid solution, the nitrous acid Sodium solution is dissolved in 0.35 mL distilled water by 6 mg natrium nitrosum and is made.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109085351A (en) * | 2018-09-05 | 2018-12-25 | 顾娟红 | A kind of method of quick detection enrofloxacin residual |
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