CN112526120B - Method for detecting salbutamol based on SPR technology - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 25
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 title claims abstract description 13
- 229960002052 salbutamol Drugs 0.000 title claims abstract description 13
- 238000005516 engineering process Methods 0.000 title claims abstract description 8
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 39
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 30
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 20
- 239000000427 antigen Substances 0.000 claims description 16
- 102000036639 antigens Human genes 0.000 claims description 16
- 108091007433 antigens Proteins 0.000 claims description 16
- 229910052757 nitrogen Inorganic materials 0.000 claims description 15
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- 239000007788 liquid Substances 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 12
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- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- 238000011010 flushing procedure Methods 0.000 claims description 9
- 235000015277 pork Nutrition 0.000 claims description 8
- 239000006185 dispersion Substances 0.000 claims description 7
- -1 hexafluorophosphate Chemical compound 0.000 claims description 7
- 238000002791 soaking Methods 0.000 claims description 7
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 239000012086 standard solution Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
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- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
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- SRZXCOWFGPICGA-UHFFFAOYSA-N 1,6-Hexanedithiol Chemical compound SCCCCCCS SRZXCOWFGPICGA-UHFFFAOYSA-N 0.000 claims description 3
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- 239000012528 membrane Substances 0.000 claims description 3
- 230000000903 blocking effect Effects 0.000 claims description 2
- RAABOESOVLLHRU-UHFFFAOYSA-N diazene Chemical compound N=N RAABOESOVLLHRU-UHFFFAOYSA-N 0.000 claims description 2
- 229910000071 diazene Inorganic materials 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000002608 ionic liquid Substances 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- ACTRVOBWPAIOHC-XIXRPRMCSA-N succimer Chemical compound OC(=O)[C@@H](S)[C@@H](S)C(O)=O ACTRVOBWPAIOHC-XIXRPRMCSA-N 0.000 claims 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 8
- ACTRVOBWPAIOHC-UHFFFAOYSA-N succimer Chemical compound OC(=O)C(S)C(S)C(O)=O ACTRVOBWPAIOHC-UHFFFAOYSA-N 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 239000013545 self-assembled monolayer Substances 0.000 abstract description 5
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract description 4
- 150000003573 thiols Chemical class 0.000 abstract description 4
- 239000002253 acid Substances 0.000 abstract description 3
- 238000000682 scanning probe acoustic microscopy Methods 0.000 abstract description 3
- 125000000524 functional group Chemical group 0.000 abstract description 2
- 230000002708 enhancing effect Effects 0.000 abstract 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- 239000010931 gold Substances 0.000 description 5
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- 206010063344 microscopic polyangiitis Diseases 0.000 description 5
- 238000012360 testing method Methods 0.000 description 4
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 description 3
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- 238000012986 modification Methods 0.000 description 3
- IZFHEQBZOYJLPK-SSDOTTSWSA-N (R)-dihydrolipoic acid Chemical compound OC(=O)CCCC[C@@H](S)CCS IZFHEQBZOYJLPK-SSDOTTSWSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052961 molybdenite Inorganic materials 0.000 description 2
- CWQXQMHSOZUFJS-UHFFFAOYSA-N molybdenum disulfide Chemical compound S=[Mo]=S CWQXQMHSOZUFJS-UHFFFAOYSA-N 0.000 description 2
- 229910052982 molybdenum disulfide Inorganic materials 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000002094 self assembled monolayer Substances 0.000 description 2
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 108010038083 amyloid fibril protein AS-SAM Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 229960001399 clenbuterol hydrochloride Drugs 0.000 description 1
- OPXKTCUYRHXSBK-UHFFFAOYSA-N clenbuterol hydrochloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 OPXKTCUYRHXSBK-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 description 1
- 229940074095 ractopamine Drugs 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- 229920006395 saturated elastomer Polymers 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/59—Transmissivity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/59—Transmissivity
- G01N2021/5903—Transmissivity using surface plasmon resonance [SPR], e.g. extraordinary optical transmission [EOT]
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Abstract
The invention discloses a method for detecting salbutamol based on SPR technology, which comprises the steps of adjusting the concentration of a surface carboxyl functional group by introducing thiol SAMs with different sulfhydryl groups and carboxyl groups, replacing a traditional connecting molecule with dimercaptosuccinic acid (DMSA) of dimercaptodicarboxylic acid, enhancing SPR signals by MoS 2 and AuNPs, and improving the detection sensitivity of an SPR biosensor.
Description
Technical Field
The invention relates to a method for detecting salbutamol based on an SPR technology, belonging to the field of chemical analysis.
Background
Surface plasmon resonance technology was originally mainly applied to macromolecular interactions through optical refractive index changes, such as examining the affinity between antigen-antibody, receptor-ligand, aptamer-identifier, but is gradually applied to the field of small molecule detection due to its advantages of rapidity, no labeling, and high sensitivity. The SPR immunosensor is the most widely applied, and the detection mode is mainly competition reaction, namely, an antigen or an antibody is immobilized on the surface of an SPR chip through a monomolecular layer, then the antigen/antibody is introduced, and a recognition site on the target object competition antibody is detected, so that the refractive index of the surface of the SPR chip changes along with the concentration of the target object, and a certain metering relation exists between the concentration of the target object and the refractive index, so that the target object is quantified.
In SPR immunosensor development, antigen/antibody immobilization on the SPR chip is a critical step. Thiol molecules are bifunctional molecules containing sulfhydryl groups and carboxyl groups, and form Au-S bonds with gold through the sulfhydryl groups, so that a monomolecular layer (SAMs) is formed on the surface of a gold film, and the nonspecific adsorption of proteins on gold flakes is effectively reduced. Meanwhile, the carboxyl group is covalently coupled with an amino group in the antigen/antibody by a carbodiimide method, thereby binding the antigen/antibody to the surface of the chip having a base gold film. As a connecting molecule, the density of carboxyl groups in the thiol greatly influences antigen/antibody binding, thereby influencing the sensitivity and detection performance of the sensor.
Now commonly used as detection molecules for SPR chip attachment are MPAs of monothio monocarboxylic acids, whose carboxyl groups have a very high density in the use of MPAs as SAMs, far exceeding the active sites required for immobilized antigen, which tends to affect antigen/antibody binding and thus sensor sensitivity and detection performance.
Disclosure of Invention
The invention overcomes the defects of the prior art, and provides a method for detecting salbutamol based on an SPR technology.
A method for detecting salbutamol based on SPR technology, which comprises the following steps:
S1, manufacturing a MoS 2 -AuNPs-DMSA SPR chip;
S2, adding 10-12mL of acetonitrile into 1-1.2g of pork, homogenizing, taking supernatant, drying with nitrogen, dissolving with 2-2.3mL of n-hexane, adding 1-1.2mLPBS solution, fully mixing, incubating in 80-85 ℃ water bath for 2-3min, collecting bottom solution, and filtering with a filter membrane to obtain liquid to be tested;
S3, preparing a standard curve by using SAL-Ab solution and SAL standard solutions with different concentrations;
And S4, taking 240-250 mu L of the liquid to be detected in the step S2, detecting by using an SPR immunosensor provided with a MoS 2 -AuNPs-DMSA SPR chip manufactured in the step S1, and obtaining the salbutamol content in pork according to a standard curve manufactured in the step S3.
Further, the SAL-Ab solution concentration in the step S3 is 75 μg/mL; the SAL standard solutions with different concentrations are respectively 5, 10, 20, 30, 40, 60, 80, 100 and 150ng/mL.
Further, the method for manufacturing the MoS 2 -AuNPs-DMSA SPR chip described in step S1 in the above method includes the following steps:
1) Adding 10-15mg MoS 2 into 1mL of ionic liquid (1-butyl-3-methylimidazole hexafluorophosphate) for dissolution, centrifuging for the first time to retain supernatant A, centrifuging for the second time to retain precipitate, washing the precipitate with N, N-Dimethylformamide (DMF) for 3 times, drying, dispersing in dimethylformamide, and keeping the final concentration of the dispersion liquid to be 1.8-2 mg.mL -1;
2) Taking 190-200mL of the dispersion liquid prepared in the step 1), and coating the dispersion liquid on an SPR chip;
3) Soaking the chip treated in the step 2) in1, 6-Hexanedithiol (HDT) solution, respectively washing the chip once by using ethanol and water after soaking, drying by using nitrogen, putting the treated chip into AuNPs solution overnight, washing the chip cleanly by using deionized water, and drying by using nitrogen;
4) Placing the chips treated in the step 3) into 0.5mmol/L DMSA solution respectively, reacting for 23-24h, after the reaction is finished, respectively flushing the SPR chips with ethanol and deionized water, and drying with nitrogen;
5) Placing the SPR chip treated in the step 4) into a mixed solution of 400 mmol/L1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC) and 100mmol/L N-hydroxysuccinimide (NHS), reacting for 25-30min, and drying by flushing deionized water with nitrogen after finishing;
6) And (3) coating 180-200 mu L of SAL antigen solution on the SPR chip treated in the step (5), blocking the non-blocked unreacted active sites by using an ethanolamine solution, and finally flushing the SPR chip by using a PBS solution, wherein the MoS 2 -AuNPs-DMSA SPR chip is prepared.
Further, the conditions of the primary centrifugation in the step 1) are as follows: the rotation speed is 2000rpm, and the time is 20min; the conditions of the secondary centrifugation are as follows: rotational speed 6000rpm, time 20min.
Further, the concentration of the 1, 6-Hexanedithiol (HDT) solution in the step 3) is 0.01mol/L, and the soaking time is 24 hours.
Further, the AuNPs solution in step 3) is a 25nm AuNPs solution.
Further, the DMSA solution concentration in step 4) was 0.5mmol/L.
Further, the mixing ratio of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in step 5) was 4:1.
Further, the SAL antigen solution in step 6) has a concentration of 1mg/mL.
The beneficial effects are that:
in the research, the concentration of the surface carboxyl functional group is regulated by introducing thiol SAMs with different mercapto groups and carboxyl groups, and the dimercaptosuccinic acid (DMSA) of dimercaptobycarboxylic acid is used for replacing the traditional connecting molecules such as MPA of monothiomonocarboxylic acid, so that the problem of steric hindrance in antigen/antibody combination is solved, and the configuration and the orientation of the antibody are controlled and the sensitivity is improved.
Drawings
FIG. 1 shows the refractive index change of the chip at different SAL-Ab concentrations.
FIG. 2 SPR A standard curve for immunosensor detection of SAL.
FIG. 3 DMSA-SPR immunosensor specificity analysis.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present application will be further described with reference to examples, which are only a part of examples, but not all examples, of the present application, and the present application is not limited by the examples described below.
Example 1 preparation of SPR chip
1. Experimental method
1. Modification of SPR chip by MoS 2: 10mg of MoS2 is taken, the ultrasonic action is carried out for 4 hours at room temperature (22+/-2 ℃) and the centrifugation is carried out for 20 minutes at 2000rpm, and the large particles are removed. The supernatant was centrifuged at 6000rpm for 20min, then washed 3 times with N, N-Dimethylformamide (DMF) 1mL each, the collected precipitate was dried at 50℃and dispersed in dimethylformamide at a concentration of 2 mg.mL-1 and stored at 4 ℃.200 mL of the dispersion was pipetted onto a clean SPR bare chip and dried in air.
2. Modification of SPR chip by AuNPs: firstly, soaking an SPR chip modified by MoS 2 in 0.01 mol/L1, 6-Hexanedithiol (HDT) solution for 24 hours, flushing the surface of a gold film with ethanol and water, and drying with nitrogen; then the SPR chip was placed into a 25nm AuNPs solution overnight, rinsed clean with deionized water, and dried with nitrogen.
3. Modification of SPR chip by self-assembled monolayer: auNPs modified SPR chips were placed in 0.5mM mercaptopropionic acid (MPA), dihydrolipoic acid (DHLA) and DMSA solutions, respectively, and reacted for 24 hours. After the reaction was completed, the SPR chip was rinsed with ethanol and deionized water and dried with nitrogen.
4. Assembling salbutamol antigen on SPR chip: the above SPR chip was placed in a solution of 400mM 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC) and 100mM N-hydroxysuccinimide (NHS) and reacted at room temperature for 30min. After the reaction was completed, the SPR chip was rinsed with deionized water and dried with nitrogen. The SPR chip treated in (2) was coated with 200. Mu.L of SAL antigen (SAL-BSA) solution (1 mg/mL), unreacted active sites were blocked with ethanolamine solution (1.0M, pH 8.5), and finally washed with PBS solution to give MoS 2 -AuNPs-MPA SPR chip, moS 2 -AuNPs-DMSA SPR chip and MoS 2 -AuNPs-DHLA SPR chip, which were stored in a sealed condition at 4 ℃.
5. Selection of optimal SAL monoclonal antibody (SAL-Ab) concentration: SAL-Ab concentrations of different species were optimized with 25ug/mL as the optimal reaction concentration.
6. Selection of the best self-assembled monolayer: the binding efficiency of antigen/antibody binding on MoS 2-AuNPs-MPA、MoS2 -AuNPs-DMSA and MoS 2 -AuNPs-DHLA SPR chips was studied using SPR sensors. The change of SPR signal response value generated by the SAL-Ab with the same concentration flowing through the surface of the SPR chip is taken as a judgment standard.
2. Experimental results
As can be seen from FIG. 1, when SAL-Ab concentration was equal to 75. Mu.g/mL, the concentration of SAL-Ab that the three chips could bind was saturated, and the refractive index difference between MoS 2 -AuNPs-MPA chip (a) and MoS 2 -AuNPs-DMSA chip (b) was 58.8 and 115.6, indicating that the DMSA chip could bind more SAL-Ab under the same experimental conditions.
Example 2 test of salbutamol in sample reliability of the test method
1. Sample treatment: 1g of pork is added with 10mL of acetonitrile, homogenized and extracted for 1min, 1mL of supernatant is taken in a 5mL centrifuge tube, dried by nitrogen, dissolved by 2mL of n-hexane, then 1mL of PBS solution is added, the mixture is fully and uniformly oscillated, incubated for 3min in a water bath at 85 ℃, bottom liquid is collected as test liquid, the test liquid is filtered through a 0.22 mu m filter membrane, 250 mu L of the test liquid is taken for SPR immunosensor detection, and each sample level is repeated for 3 times.
2. And (3) methodological verification: the SAL-Ab was incubated at 37℃for 30min by mixing equal volumes of SAL standard solutions (5, 10, 20, 30,40, 60, 80, 100, 150 ng/mL) of the optimal concentration and SAL standard solutions of different concentrations, each concentration was repeatedly measured 3 times, and the change in refractive index was recorded to prepare a standard curve. The results show that the method has good linear range and detection sensitivity between 5 and 150ng/mL, as shown in FIG. 2. Linear regression equation y=0.008 x 2 -1.95x+114.29 linear correlation coefficient is 0.988, detection sensitivity is 5ng/mL.
To verify the accuracy and reliability of the SPR process, pork was used as a sample matrix and the recovery of the addition at 5, 10 and 20 μg/kg concentrations (n=3) was tested. As shown in Table 1, the recovery rate of SAL detected by the method is 94.9% -108.0%, RSD is 1.30-5.58%, and the method has good consistency compared with the result of UPLC-MS/MS method. The method can be used for rapidly detecting the salbutamol in the actual pork sample.
TABLE 1 DMSA-SPR immunosensor and UPLC/MS/MS determination of salbutamol in pork samples
For the purpose of examining the recognition specificity of the methods herein, ractopamine, clenbuterol hydrochloride, marbuterol hydrochloride and simatrox were selected as structural analogues of SAL. As shown in FIG. 3, at a concentration of 120ng/mL, the inhibition rate of SAL was significantly higher than that of other compounds, and was not much different from that of the mixture. Therefore, the method has good specific recognition performance.
Claims (7)
1. The method for detecting salbutamol based on the SPR technology is characterized by comprising the following steps of:
s1: manufacturing a MoS 2 -AuNPs-DMSA SPR chip; the method comprises the following steps:
1) Adding 10-15mg MoS 2 into 1 mL1-butyl-3-methylimidazole hexafluorophosphate ionic liquid to dissolve, centrifuging for the first time to keep supernatant A, centrifuging for the second time to keep precipitate, washing the precipitate with N, N-dimethylformamide for 3 times, drying, dispersing in dimethylformamide, and keeping the final concentration of the dispersion liquid to be 1.8-2 mg.mL -1; the conditions of the primary centrifugation are as follows: the rotation speed is 2000rpm, and the time is 20min; the conditions of the secondary centrifugation are as follows: rotating at 6000rpm for 20min;
2) Taking 190-200mL of the dispersion liquid prepared in the step 1), and coating the dispersion liquid on an SPR chip;
3) Soaking the chip treated in the step 2) in 1, 6-hexanedithiol solution, respectively flushing once by using ethanol and water after soaking, drying by using nitrogen, putting the treated chip into AuNPs solution overnight, flushing cleanly by using deionized water, and drying by using nitrogen;
4) Placing the chips treated in the step 3) into 0.5 mmol/L DMSA solution respectively, reacting 23-24 h, respectively flushing the SPR chips with ethanol and deionized water after the reaction is finished, and drying with nitrogen;
5) Placing the SPR chip treated in the step 4) into a mixed solution of 400 mmol/L of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride and 100 mmol/L N-hydroxysuccinimide, reacting for 25-30min, and flushing deionized water with nitrogen to blow dry after the reaction is finished;
6) Coating 180-200 mu L of SAL antigen solution on the SPR chip treated in the step 5), blocking the non-blocked unreacted active sites by using ethanolamine solution, and finally flushing with PBS solution to obtain MoS 2 -AuNPs-DMSA SPR chip;
S2: adding 10-12mL of acetonitrile into 1-1.2g of pork, homogenizing, taking supernatant, drying with nitrogen, dissolving with 2-2.3mL of n-hexane, adding 1-1.2mLPBS solution, mixing thoroughly, incubating in 80-85deg.C water bath for 2-3min, collecting bottom solution, and filtering with a filter membrane to obtain liquid to be tested;
S3: making a standard curve by using SAL-Ab solution and SAL standard solutions with different concentrations;
S4: and (3) taking 240-250 mu L of the liquid to be detected in the step S2 for detection by using an SPR immunosensor provided with a MoS 2 -AuNPs-DMSA SPR chip manufactured in the step S1, and obtaining the salbutamol content in pork according to the standard curve manufactured in the step S3.
2. The method of claim 1, wherein the SAL-Ab solution concentration in step S3 is 75 μg/mL; the SAL standard solutions with different concentrations are respectively 5, 10, 20, 30, 40, 60, 80, 100 and 150 ng/mL.
3. The method of claim 1, wherein the concentration of the 1, 6-hexanedithiol solution in step 3) is 0.01mol/L and the soaking time is 24 hours.
4. The method of claim 1 wherein said AuNPs solution in step 3) is a 25nm AuNPs solution.
5. The method of claim 1, wherein the DMSA solution in step 4) has a concentration of 0.5 mmol/L.
6. The method of claim 1, wherein the mixing ratio of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride to N-hydroxysuccinimide in step 5) is 4:1.
7. The method of any one of claims 2-6, wherein the SAL antigen solution in step 6) is at a concentration of 1mg/mL.
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