Fluorescent biosensor for ultra-stability detection of Pax-5a based on a double cascade amplification strategy

The development of B-lymphoblastic leukemia is tightly associated with aberrant expression of Pax-5a. This work presented a novel dual signal amplification strategy-based Pax-5a detection method by combining the rolling circle amplification reaction (RCA) and the Entropy-driven toehold-mediated strand displacement (ETSD). Particularly noteworthy is the employed ETSD, which effectively improves the rate and stability of the reaction due to its unique entropy-driven principle. The uniqueness of this method is the combination of two amplification techniques, each utilizing its own strengths to achieve our intended purpose. This sensing method has been effectively used to determine the Pax-5a gene which with a reliable linear correlation for detection within a range and achieving a detection limit of 3.34 pM, calculated using the formula (3σ/S). Furthermore, even in 1 % of human serum samples, the biosensor can identify the target gene with exceptional sensitivity. The recovery rates fall within the range of 96.68-101.76 %, with a relative standard deviation (RSD) of 5.47 %. The method has a strong specificity based on sequence-specific hybridization of nucleic acids, thereby effectively preventing potential false-positive results. This fluorescent biosensor has a high detection capability for Pax-5a, and offers stable results. It provides a new way for early clinical diagnosis of acute lymphoblastic leukemia.