Sequential sandwich immunoassay for simultaneous detection in trace samples using single-channel surface plasmon resonance.

To analyze multiple analytes in trace samples, low-dosage and high efficiency are crucial in many common cases. Herein, we developed a facile method using a single-channel surface plasmon resonance (SPR)-based biosensor for the simultaneous detection of gentamicin (GEN) and melamine (MEL) in milk and serum with only one sample injection. Based on a sandwich immunoassay, non-interfering antibodies against GEN from mouse (AbGEN) and against MEL from rabbit (AbMEL) were chosen to capture the analytes. Secondary antibodies against mouse (AbM) and rabbit (AbR) were used to bind with AbGEN and AbMEL to determine the concentrations of GEN and MEL on a single channel of an SPR sensor. All of the detection process could be done in 10 min with 50 μL of sample injection. According to the response shifts of AbM and AbR, two standard curves for GEN and MEL were obtained successively, with the limit of detection (LOD) values at 4.4 ng mL-1 and 1.3 ng mL-1, respectively. Moreover, the feasibility was determined by spiking milk and serum samples with GEN and MEL, with recoveries in the range of 81.6%-118.0%. Importantly, the analytes can be substituted by others for much more applications. This method is also expected to multiply the detection efficiency of multi-channel SPR biosensors with low-dosage samples in the future.