Application Of SPR Immunosensor Based On Self-assembled Monolayers For Detection Of Salbutamol

Salbutamol(SAL)is a kind of clenbuterol.Adding a small amount of salbutamol to livestock feed can increase lean meat weight and reduce fat.However,its residue can cause adverse symptoms such as muscle tremor,headache and palpitation,so it has been banned in animal feed in the European Union and China.There are many methods for the determination of SAL,such as enzyme-linked immunosorbent assay((ELISA)),gas chromatography-mass spectrometry(GC/MS),high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)and liquid chromatography-tandem mass spectrometry(LC-MS/MS)and so on.However,most of these methods require expensive equipment,skilled technicians and long-time detection and analysis.Therefore,it is urgent to develop a simple,convenient,rapid,sensitive,low-cost and high-specificity analytical method.Surface plasmon resonance((SPR))was initially used to monitor the interaction between macromolecules mainly through the change of refractive index in optics,such as the affinity between antigen-antibody,receptor-ligand and aptamer-recognition,and then it was widely used in the field of small molecule detection.It has the advantages of high speed,no labeling and high sensitivity.Among them,the application of SPR immunosensor is more common,and its detection method is mainly inhibition method.When the quantitative antibody binds to the antigen on the chip surface,it produces a response value on the SPR sensor.Mix this amount of antibody with the substance to be tested,and the substance to be tested will consume a certain amount of antibody.When the mixture reacts with the chip surface,the concentration of the substance to be measured can be calculated by calculating the reduced response value of the SPR sensor.In the development of SPR immunosensor,the immobilization of antigen / antibody on SPR chip is a key step.Mercaptan is a kind of bifunctional molecule containing sulfhydryl group and carboxyl group,which forms Au-S bond with gold through sulfhydryl group,and forms a monolayer on the surface of gold film,which effectively reduces the non-specific adsorption of protein on gold film.At the same time,the carboxyl group is covalently coupled with the amino group in the antigen / antibody by carbodiimide method,thus binding the antigen / antibody to the chip surface with substrate gold film.As a connecting molecule,the density of carboxyl groups in mercaptan greatly affects the antigen / antibody binding,which in turn affects the sensitivity and detection performance of the sensor.In recent years,researchers have introduced mixed mercaptan monolayer SAMs to regulate the concentration of carboxyl functional groups on the surface to solve the problem of steric hindrance in antigen / antibody binding.Gobi changes the space environment through a mixture of carboxyl-free(1-decyl mercaptan)and carboxyl(11-mercaptoundecanoic acid).Tsai also proposed the strategy of mixed linker to achieve a good spatial microenvironment by changing the chain length of compounds(hexanethiol and 16-mercaptohexadecanoic acid).However,although these mixed modes reduce the carboxyl density,the effective mercaptan connecting molecules can not be evenly distributed,which will still lead to uneven antigen / antibody aggregation,thus affecting the sensitivity of the test.Based on the above scientific problems,this paper uses dimercaptosuccinic acid DMSA as a self-assembled monolayer to replace the traditional connecting molecules such as mono-mercaptosuccinic acid MPA,to reduce the steric hindrance and control the configuration and orientation of the antibody,improve the recognition efficiency between the antigen-linked SPR chip and the antibody,and improve the detection performance of the SPR immunosensor.In this paper,DMSA was used as monolayer to connect the antigen on the chip of SPR immunosensor,and SAL was used as the detection model to construct DMSA-SPR immunosensor.The detection effect of DMSA-SPR immunosensor was compared with that of traditional mercaptopropionic acid(MPA)-SPR sensor.The results showed that the recognition efficiency of antigen and antibody of DMSA-SPR immunosensor was nearly doubled at room temperature,and the linear range was 5: 150 ng/m L,the quantitative limit(LOQ)was 5 ng/m L.The recovery rate of this sensor applied to the detection of SAL in pork is 94.9% 108.0%,and has a good correlation with the detection results of ultra-high performance liquid chromatography-tandem mass spectrometer(UPLC-MS/MS).Therefore,this method provides a new idea and method for improving the sensitivity of SPR immunosensor,and can be widely used in the detection of other small molecules.