Related Experiment Video
Updated: Jun 13, 2025
Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency
Published on: March 1, 2024
An ultrasensitive terminal protection-based real-time fluorescence approach for protein detection via an isothermal
Yajing Zhang1, Mengqi Wang1, Yanrui Xie1
1Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Materials Science, Institute of Life Science and Green Development, Hebei University, Baoding, 071002, China. jhx@hbu.edu.cn.
Abstract:
In this assay, based on the terminal protection of small-molecule-linked DNA, a new ultrasensitive real-time fluorescence strategy combined with an isothermal exponential amplification reaction (IEXPAR) has been established for protein assay. By the clever design of DNA, terminal protection is combined with efficient IEXPAR. The target protein explicitly binds to small molecules attached to the template DNA, protecting the template DNA from exonuclease I (Exo I) degradation. The added DNA primer hybridizes with the protected template DNA and triggers the following IEXPAR. IEXPAR has a super amplification efficiency of 106-109 times. The IEXPAR yields numerous double-stranded DNA (dsDNA) molecules. The fluorescence dye SYBR Green I (SG), which is sensitive to dsDNA, is used to determine the real-time fluorescence of the IEXPAR. Conversely, without the target protein, the template DNA is hydrolyzed by Exo I, failing to trigger the IEXPAR. The intriguing combination of IEXPAR and terminal protection realizes the ultrasensitive detection of protein. As low as 100 fmol L-1 SA and 200 pg mL-1 folic acid (FR) are accurately detected.
