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.

中文翻译：

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基于双荧光素酶的牛奶中小分子通用同时多重 ELISA

酶联免疫吸附试验(ELISA)以其操作简单、速度快、灵敏度高等优点，已成为食品安全最重要、应用最广泛的快速检测技术。多重同步检测是ELISA一直追求的目标。然而，由于复杂的信号生成和收集程序，已报道的多重ELISA并没有真正实现同步检测。在这里，我们开发了一种双荧光素酶竞争性直接生物发光免疫测定 (DBL-cdELISA)，只需一个底物添加步骤，然后立即同步信号采集。这是基于微量滴定板和酶的小分子同步多重分析的第一份报告，无需任何额外步骤。诺氟沙星 (NOR) 和磺胺二甲嘧啶 (SMZ) 的 IC50 值为 0。分别为 051 ng mL-1 和 0.211 ng mL-1。结果表明，不同荧光素酶和底物的应用简化了信号的产生和收集程序，能够以简单的程序、高通量和快速的速度同时检测小分子，这对多种检测方法的发展具有重要意义。