Ultrasensitive and colorimetric small extracellular vesicles (sEVs) analysis via dual-cycle signal tool

Abstract

For the clinical diagnosis of diseases and for basic biological research, it is crucial to develop a trustworthy and efficient method for detecting small extracellular vesicles (sEVs) in multiple experimental conditions. Here, we create a colorimetric assay that enables sensitive and precise sEVs identification without the need for pricey equipment. In this assay, the exonuclease III (Exo III)-assisted signal recycle is activated by the released single-strand DNA (ssDNA) from SMBs (streptavidin magnetic beads)-aptamer-ssDNA complex after identification of sEVs. By integrating with the strand displacement amplification (SDA) process, a significant amount of double-strand DNA products with G-rich tails is produced. The G-rich tails fold to G-quadruplex under the assistance of hemin to catalyze the oxidation of TMB, yielding a color change. The approach offers a broad detection range of 5 orders of magnitudes based on the signal recycles and SDA. In addition, single-stranded DNA binding protein (SSB) is exploited in this method to minimize the background signal from non-specific digestion of Exo-III, making the method a robust tool for sEVs detection and disease diagnosis.