A SERS/electrochemical dual-signal readout immunosensor using highly-ordered Au/Ag bimetallic cavity array as the substrate for simultaneous detection of three β-adrenergic agonists.

A surface-enhanced Raman scattering (SERS)/electrochemical dual-signal readout immunosensor was developed for simultaneous detection of β-adrenergic agonists salbutamol (SAL), ractopamine (RAC) and phenylethanolamine A (PA). The highly-ordered gold/silver bimetallic cavity array (BMCA) was prepared by electrodepositing Au/Ag nanoparticles to the interstice of highly ordered close-packed polystyrene templates. After electrochemical and SERS characterization, the BMCA was used as the substrate for constructing SERS/electrochemical dual-signal readout immunosensor. 3,3',5,5'-tetramethylbenzidine (TMB), methylene blue (MB) and Nile blue (NB) were selected as the dual-signal reporters, and hybridization chain reaction (HCR) was used as the signal amplifier. The immunoprobe was prepared by absorption of the antibody (Ab) and constructing HCR system embedded with electro/SERS reporter on Au nanoparticles (NPs). After competitive immuno-reaction between coating antigen and analyte for limited Ab on immunoprobe, the SERS/electrochemical dual-signals on BMCA were measured for quantitatively detecting SAL, RAC and PA simultaneously. SAL, RAC and PA were detected in concentration range of 1 pg mL to 100 ng mL with LOD of 0.8, 0.4, and 1.3 pg mL, respectively. The applicability of the proposed immunosensor in spiked pork liver samples was verified by the recovery of 95.0%-108.5% with RSD of 6.9%-10.7%. It was proven that the immunosensor was able to detect multiple β-adrenergic agonists with high sensitivity, specificity, accuracy and precision. The immunosensor can be used as a platform for the determination of other small molecular compounds in biological, food and environmental analytical fields.