Label-free detection of cancer related gene based on target recycling and palindrome-mediated strand displacement amplification

摘要

STAT3 plays an important role in regulating gene expression and is closely related with cancer. Thus, the sensitive and specific detection of the STAT3 biomarker is of great importance for disease diagnosis and therapeutics. In this study, by combining the target recycling amplification (TRA) with strand displacement amplification (SDA), we have developed a label-free and highly sensitive method for the dual-amplified detection of STAT3. The assay system consists of polymerization primer and label-free hairpin probe (HP) containing palindromic fragment and nicking site. In the presence of STAT3, the stem of the HP is opened, followed by the primer binding to initiate TRA and SDA with the help of Klenow Fragment (KF) and nickase. After multiple replication, nicking, and strand displacement, STAT3 was released and initiated the next round of reactions, generating a large number of terminal palindrome-contained fragments. Subsequently, the intermolecular hybridization between palindromic fragments occurred and the bidirectional extension by polymerase takes place, forming the dsDNAs. The double-stranded DNA products can be quantified by measuring the fluorescence intensity of SYBR Green I. The proposed strategy shows the excellent specificity and high sensitivity with a detection limit as low as 50 pM. In addition, this designed protocol can be successfully applied to detect the STAT3 in human serum, indicating great potential for the practical application in early diagnosis and prognosis.