A novel specific and ultrasensitive method detecting extracellular vesicles secreted from lung cancer by padlock probe-based exponential rolling circle amplification

Abstract Lung cancer is one of the most common cancers in the world. Early diagnosis of lung cancer usually shows promising rate of survival. However, the detection of lung cancer faces great challenges. Transthoracic needle biopsy combined with image-guidance will make patients suffer from some damages. Liquid biopsy can be used as an auxiliary method for early diagnosis and prognosis of cancers owing to its minimally invasive performance. Proteins on the surface of Extracellular vesicles (EVs) derived from lung cancer are used as biomarkers for cancers. Here, a highly specific and sensitive fluorescence scheme based on padlock probe-based exponential rolling circle amplification (P-ERCA) assay for detecting extracellular vesicles was reported. The aptamer against extracellular vesicles derived from lung cancer cells was linked to a primer sequence (aptamer-primer complex) that was complementary to padlock probe. The padlock probe was composed of a nicking site for nicking endonuclease. At the presence of target extracellular vesicles, aptamer-primer complex could trigger linear rolling circle amplification (LRCA) under isothermal conditions. After multiple nicking reactions, many copies of single-stranded DNAs (trigger DNA) were produced and leading to another circle amplification. The exponential fluorescence signal of P-ERCA product dyed with SYBR Green II was detected. The method exhibits high specific and sensitivity to lung cancer extracellular vesicles with low detection limit of 4.222 × 104 particles/mL, and the linear range is 5 × 104 particles/mL-1.2 × 106 particles/mL. Furthermore, this method could successfully distinguish the signal of extracellular vesicles in real human serum sample between 17 lung cancer patients and 17 healthy persons (P