Universal simultaneous multiplex ELISA of small molecules in milk based on dual luciferases

The enzyme-linked immunosorbent assay (ELISA) has become the most important and widely used rapid detection technology for food safety because of its simple operation, fast speed and high sensitivity. Multiplex synchronous detection is the goal of ELISA that is always pursuing for. However, the reported multiplex ELISAs have not truly realized synchronous detection because of the complex signal generation and collection procedures. Here, we developed a dual-luciferases competitive direct bioluminescent immunoassay (DBL-cdELISA) with only one substrate addition step followed immediately by simultaneous signal acquisition. It is the first report of simultaneous multiplex analysis of small molecules based on microtiter plates and enzymes without any additional steps. The IC₅₀ values for norfloxacin (NOR) and sulfamethazine (SMZ) were 0.051 ng mL^-1 and 0.211 ng mL^-1, respectively. The results demonstrated that the application of different luciferases and substrates simplified the signal generation and collection procedures and enabled simultaneous detection of small molecules with a simple procedure, high throughput and fast speed, that will be of great significance for the development of multiple assays.