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-1 to 100 ng mL-1 with LOD of 0.8, 0.4, and 1.3 pg mL-1, 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.