Center for Biomicrosystems, Brain Research Institute, Korea Institute of Science and Technology, 5 Hwarang-ro, 14-gil, Seongbuk-gu, Seoul, 02792, Republic of Korea.
Department of Biomedical Engineering, Sogang University, Seoul, Republic of Korea.
Sci Rep. 2021 Mar 12;11(1):5831. doi: 10.1038/s41598-021-85107-7.
This report suggests a method of enhancing the sensitivity of chemifluorescence-based ELISA, using photooxidation-induced fluorescence amplification (PIFA). The PIFA utilized autocatalytic photooxidation of the chemifluorescent substrate, 10-acetyl 3,7-dihydroxyphenoxazine (ADHP, Amplex Red) to amplify the fluorescent product resorufin, initially oxidized by horse radish peroxidase (HRP). As the amplification rate is proportional to the initial level of resorufin, the level of antigen labeled by HRP is quantified by analyzing the profile of fluorescence intensity. The normalized profile was interpolated into an autocatalysis model, and the rate of increase at half-maximum time was quantified by the use of an amplification index (AI). The lower limit of detection, for resorufin or HRP, was less than one-tenth that of the plate reader. It requires only slight modification of the fluorescence reader and is fully compatible with conventional or commercial ELISA. When it is applied to a commercial ELISA kit for the detection of amyloid beta, it is verified that the PIFA assay enhanced the detection sensitivity by more than a factor of 10 and was compatible with a conventional 96-well ELISA assay kit. We anticipate this PIFA assay to be used in research for the detection of low levels of proteins and for the early diagnosis of various diseases with rare protein biomarkers, at ultra-low (pg/mL) concentrations.
本报告提出了一种利用光氧化诱导荧光放大（PIFA）提高基于化学荧光的 ELISA 灵敏度的方法。PIFA 利用化学荧光底物 10-乙酰基 3,7-二羟基苯并恶嗪（ADHP，Amplex Red）的自催化光氧化来放大最初由辣根过氧化物酶（HRP）氧化的荧光产物 Resorufin。由于放大率与 Resorufin 的初始水平成正比，因此通过分析荧光强度的分布来定量标记 HRP 的抗原水平。将归一化分布插值到自催化模型中，并通过使用放大指数（AI）来量化半最大值时间的增长率。Resorufin 或 HRP 的检测下限小于荧光读取器的十分之一。它只需对荧光读取器进行微小修改，并且与传统或商业 ELISA 完全兼容。当将其应用于用于检测淀粉样蛋白 β 的商业 ELISA 试剂盒时，验证了 PIFA 测定法将检测灵敏度提高了 10 倍以上，并且与传统的 96 孔 ELISA 测定试剂盒兼容。我们预计这种 PIFA 测定法将用于研究中，以检测低水平的蛋白质，并在超低位（pg/mL）浓度下对具有稀有蛋白质生物标志物的各种疾病进行早期诊断。
