The combination of horseradish peroxidase (HRP) and a fluorescence substrate has been attracting great interests in developing sensitive biochemical analysis and immunoassays. 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP or Amplex red) is the most sensitive fluorogenic substrate known for HRP in current market, however, it suffers from some drawbacks, such as non-specific reactivity to carboxylesterase and limited fluorescence stability. In the present study, a novel HRP substrate 10-cyclopropylcarbonyl-dichloro-dihydroxyphenoxazine (AR-2), has been prepared, which exhibited improved sensitivity than ADHP in sensing HRP. Moreover, the fluorescence of AR-2/HRP demonstrated improved tolerance to physiological relevant pH fluctuation as compared to ADHP/HRP. Successful detection of uric acid/urate oxidase reaction indicated excellent application prospect of AR-2/HRP for monitoring HO-generating biochemical reactions. More interestingly, an enzyme-linked immunosorbent assay (ELISA) using AR-2 as the fluorescence reporter has been successfully used in detecting IgG against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from human serum samples. Overall, AR-2 exhibits improved performances over the commercial ADHP, which will be an ideal alternative to ADHP in HRP-based fluorescence biochemical analysis and immunoassays.

中文翻译：

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开发用于基于 HRP 的诊断测定的荧光底物，其优于商业 ADHP

辣根过氧化物酶 (HRP) 和荧光底物的组合在开发灵敏的生化分析和免疫分析方面引起了人们的极大兴趣。 10-乙酰基-3,7-二羟基吩恶嗪（ADHP 或 Amplex red）是目前市场上已知的 HRP 最敏感的荧光底物，但它也存在一些缺点，例如对羧酸酯酶的非特异性反应性和有限的荧光稳定性。在本研究中，制备了一种新型 HRP 底物 10-环丙基羰基-二氯-二羟基吩恶嗪 (AR-2)，其在检测 HRP 方面表现出比 ADHP 更高的灵敏度。此外，与 ADHP/HRP 相比，AR-2/HRP 的荧光表现出对生理相关 pH 波动的耐受性有所提高。尿酸/尿酸氧化酶反应的成功检测表明AR-2/HRP在监测HO生成生化反应方面具有良好的应用前景。更有趣的是，使用 AR-2 作为荧光报告基因的酶联免疫吸附测定（ELISA）已成功用于检测人血清样本中针对严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的 IgG。总体而言，AR-2 的性能优于商用 ADHP，这将成为基于 HRP 的荧光生化分析和免疫分析中 ADHP 的理想替代品。