Photooxidation-induced fluorescence amplification system for an ultra-sensitive enzyme-linked immunosorbent assay (ELISA)
- Photooxidation-induced fluorescence amplification system for an ultra-sensitive enzyme-linked immunosorbent assay (ELISA)
- 강지윤; 박민철; 허유희; 신관우
- Issue Date
- Scientific Reports
- VOL 11, NO 1-10
- This report suggests a method of enhancing the sensitivity of chemifuorescence-based ELISA, using photooxidation-induced fuorescence amplifcation (PIFA). The PIFA utilized autocatalytic photooxidation of the chemifuorescent substrate, 10-acetyl 3,7-dihydroxyphenoxazine (ADHP, Amplex Red) to amplify the fuorescent product resorufn, initially oxidized by horse radish peroxidase (HRP). As the amplifcation rate is proportional to the initial level of resorufn, the level of antigen labeled by HRP is quantifed by analyzing the profle of fuorescence intensity. The normalized profle was interpolated into an autocatalysis model, and the rate of increase at half-maximum time was quantifed by the use of an amplifcation index (AI). The lower limit of detection, for resorufn or HRP, was less than one-tenth that of the plate reader. It requires only slight modifcation of the fuorescence 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 verifed 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.
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