Abstract
A surface plasmon resonance (SPR) immunoassay with an immobilization of self-assembled molecular identification membrane for the detection of residual Clenbuterol Hydrochloride (CLB) in pork liver was systematically investigated and experimentally validated for its high performance. SPR immunoassay with a regular competitive inhibition assay cannot be directly verified to detect CLB residuals. In this study, the binding of Au film with mercaptopropionic acid was investigated using the known form of the strong S-Au covalent bonds formed by the chemical radical of the mercaptopropionic acid and the Au film. After that, the immunoglobulin IgG of swine (SwIgG-CLB) was bonded with the mercaptopropionic acid by covalent -CO-NH- amide bonding. The modified comprehensive analysis of how the membrane structure works was introduced together with the customized SPR bioanalyzer. In order to evaluate the performance of this biomembrane structure, the concentrations of CLB-contained solutions of 0 ng•mL-1, 10 ng•mL-1, 20 ng•mL-1, 33.3 ng•mL-1, and 40 ng•mL-1 were prepared by adding CLB reagents into the solutions of CLB antibody (Clenbuterol Hydrochloride Antibody, CLB-Ab), successively and then the response unit (RU) was measured individually. Using the data collected from the linear CCD array, the fitting curve was established with the R-Square value of 0.9929. Correspondingly, the recovery rate ranged from 88.48% to 103.21% was experimented and the limit of detection of CLB in 1.26 ng•mL-1 was obtained efficiently. It was concluded that the detection method associated with biomembrane properties is expected to contribute much to the determination of residual CLB in pork liver quantitatively by using the customized SPR bioanalyzer.
Highlights
Clenbuterol Hydrochloride (CLB) is 1-(4-amino-3,5-dichlorophenyl)-2-(tert-butylamino) ethanol hydrochloride
When PBS was injected by peristaltic pump through the microfluidic cell over the Au film surface of the integrated biosensor at the speed of 30μLÁmin-1, the baseline was set to 3000 response unit (RU)
The SwIgG-CLB containing-NH2 groups was immobilized by covalent-CO-NH- amide bonding after the carboxyl groups on Mercaptopropionic acid (MPA) were activated by the mixed solution of Ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC)/NHS with the ΔRU of 1560 RU (See Fig. 3)
Summary
Clenbuterol Hydrochloride (CLB) is 1-(4-amino-3,5-dichlorophenyl)-2-(tert-butylamino) ethanol hydrochloride. Clenbuterol is a β2 agonist with some structural and pharmacological similarities to epinephrine and salbutamol, but its effects are more potent and longer-lasting as a stimulant and thermogenic drug. It causes an increase in aerobic capacity, central nervous system stimulation, and an increase in blood pressure and oxygen transportation by promoting protein synthesis [1,2]. At the end of the 1970s, The United States of America and other developed countries started to study on practical applications of CLB, which was used as an agent of nutrition redistribution and a growth promoting agent [3]. CLB is illegally used as a growth promoter in animal production. CLB had become banned drugs in the worldwide pig breeding
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