As the most widely used immunoassay, enzyme-linked immunosorbent assay (ELISA) relies on perishable enzymes and usually provides poor sensitivity and stability, limiting its application in detecting trace analytes in harsh environments. Herein, a new ratiometric fluorescence (RF) sensing platform enhanced by a nanozyme–enzyme cascade reaction composed of MnO₂ nanosheets (MnO₂ NSs) and alkaline phosphatase (ALP) was proposed. In this RF platform, the versatile MnO₂ NSs worked as a robust oxidase-like nanozyme to catalyze nonfluorescent Amplex Red (AR) into fluorescent resorufin and as a quencher to quench the fluorescence of carbon dots (CDs). Given that ALP could catalyze ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA), followed by AA reducing MnO₂ NSs into Mn²⁺, resulting in the fluorescence of AR decrease but fluorescence of CD recovery simultaneously. The regulation of MnO₂ NSs for the fluorescence intensity of AR (E_m at 585 nm) and CDs (E_m at 460 nm) could cascade with ALP-triggered reaction to construct an RF sensing platform based on RF signal (F585/F460) output. The proposed RF sensing platform could achieve a superior limit of detection (LOD) of 0.037 mU mL^–1 for ALP activity quantification. Inspired by the excellent performance of the RF sensing platform, a sensitive and accurate RF ELISA for ractopamine (RAC) detection was established with an LOD of 0.029 ng mL^–1, which was nearly 5.5 times more sensitive than traditional colorimetric ELISA. This RF sensing platform based on robust nanozyme-triggered enzymatic cascade signal amplification and excellent RF signal readout has offered a powerful and universal platform for RAC and other trace target detection.

中文翻译：

---

Nanozyme-酶联反应增强比率荧光免疫传感平台，用于灵敏、准确检测莱克多巴胺


酶联免疫吸附测定 （ELISA） 作为应用最广泛的免疫测定法，依赖于易腐烂的酶，通常灵敏度和稳定性较差，限制了其在恶劣环境中检测痕量分析物的应用。在此，提出了一种新的比率荧光 （RF） 传感平台，该平台由由 MnO₂ 纳米片 （MnO₂ NSs） 和碱性磷酸酶 （ALP） 组成的纳米酶-酶级联反应增强。在这个 RF 平台中，多功能 MnO₂ NS 作为一种强大的氧化酶样纳米酶，催化非荧光 Amplex Red （AR） 转化为荧光试卤灵，并作为淬灭剂淬灭碳点 （CD） 的荧光。鉴于 ALP 可以催化 2-磷酸抗坏血酸 （AAP） 生成抗坏血酸 （AA），然后 AA 将 MnO₂ NSs 还原为 Mn²⁺，导致 AR 的荧光减弱，但 CD 的荧光同时恢复。MnO₂ NSs 对 AR （585 nm 处 E_m 处） 和 CDs （460 nm 处 E_m 处） 荧光强度的调节可以与 ALP 触发反应级联，构建基于 RF 信号 （F585/F460） 输出的 RF 传感平台。所提出的射频传感平台可以实现 0.037 mU ^mL-1 的 ALP 活性定量的卓越检测限 （LOD）。受到射频传感平台优异性能的启发，建立了一种用于莱克多巴胺 （RAC） 检测的灵敏、准确的射频 ELISA，其 LOD 为 0.029 ng mL^–1，比传统比色 ELISA 的灵敏度高出近 5.5 倍。 这种射频传感平台基于稳定的纳米酶触发的酶级联信号放大和出色的射频信号读出，为 RAC 和其他痕量靶标检测提供了一个强大的通用平台。