As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection posed a significant threat to public health and the global economy, in vitro diagnosis of the SARS-CoV-2 nucleocapsid protein proved to be an effective way for SARS-CoV-2 infection control in the past years. Tyramide signal amplification (TSA) has been extensively utilized in tissue imaging and pathological diagnosis owing to the powerful signal enhancement. However, the elevated “ALWAYS ON” fluorescence background limited the accuracy and sensitivity of the conventional TSA assay. To achieve an activated “TURN ON” signal, herein, a small molecule, termed dichlorodihydrofluorescein tyramide (T-DCFH), was synthesized for activatable TSA. Under the catalysis of horseradish peroxidase (HRP) with hydrogen peroxide (H₂O₂), this T-DCFH facilitates the “TURN ON” fluorescence signal. Additionally, as a recognition tool, DNA aptamer has been used for developing in vitro diagnostic approaches. Hence, based on HRP-labeled aptamers binding with SARS-CoV-2 nucleocapsid protein, we achieved aptamers-based activatable TSA detection with a higher signal-to-noise ratio than that of fluorescent dye (FITC)-labeled aptamers, while showing lower background than traditional fluorescein tyramide with “ALWAYS ON”. The results demonstrated that the activated T-DCFH significantly enhances the fluorescence signal while diminishing the background noise. By employing multiple aptamers targeting, we offered a timely and accurate in vitro diagnostic approach for future emergent infectious diseases.

中文翻译：

---

基于适配子的可激活酪胺信号放大，用于 SARS-CoV-2 核衣壳蛋白的低背景检测


由于严重急性呼吸系统综合症冠状病毒 2 （SARS-CoV-2） 感染对公共卫生和全球经济构成重大威胁，因此在过去几年中，SARS-CoV-2 核衣壳蛋白的体外诊断被证明是控制 SARS-CoV-2 感染的有效方法。Tyramide 信号放大 （TSA） 具有强大的信号增强功能，已被广泛用于组织成像和病理诊断。然而，升高的“ALWAYS ON”荧光背景限制了传统 TSA 检测的准确性和灵敏度。为了获得激活的“TURN ON”信号，本文合成了一种称为二氯二氢荧光素酪胺 （T-DCFH） 的小分子，用于可激活的 TSA。在辣根过氧化物酶 （HRP） 与过氧化氢 （H₂O₂） 的催化下，该 T-DCFH 促进了“TURN ON”荧光信号。此外，作为一种识别工具，DNA 适配体已用于开发体外诊断方法。因此，基于 HRP 标记的适配体与 SARS-CoV-2 核衣壳蛋白的结合，我们实现了基于适配体的可激活 TSA 检测，其信噪比高于荧光染料 （FITC） 标记的适配体，同时显示出比传统荧光素酪胺更低的背景“ALWAYS ON”。结果表明，激活的 T-DCFH 显着增强了荧光信号，同时降低了背景噪声。通过采用多种适配子靶向，我们为未来的紧急传染病提供了一种及时准确的体外诊断方法。