A logic dual-channel detection of Hox transcript antisense intergenic RNA using graphene switch and padlock probe-based exponential rolling circle amplification assay

Sensitive and simultaneous detection of multiple low-abundance long noncoding RNA (lncRNA) biomarkers promises an early diagnosis of lung cancer. In this work, we demonstrate a quadruple logic-mode (YES, NOT, AND and OR) sensing platform for dual specific sequences (T1 and T2) of Hox transcript antisense intergenic RNA (HOTAIR) based on padlock probe-based exponential rolling circle amplification (P-ERCA) assay, producing dual-channel electrochemical and fluorescence (FL) signals simultaneously (AND/OR mode). T1/T2 acts as a key unlocking the P-ERCA amplification strategy, combining with fluorescein Cy3/Cy5 and Pb2+/Cd2+ labeled polydopamine nanospheres (PDA-Pb2+/PDA-Cd2+) give out the dual-channel signals, realizing the quantitative detection of T1 and T2. The dual-channel assay exhibits a wide linear range of 1 fM to 100 pM with low detection limits of 0.25 fM and 0.3 fM for these two targets, respectively. Furthermore, the proposed genosensor possesses high sensitivity, selectivity, and anti-interference ability. With standard addition method, our work shows the recovery experimental T1 value of 86-114.6 % and T2 of 82.2-159 % in diluted serum samples. Moreover, the proposed genosensor obtained comparable detection results of HOTAIR content in whole RNA extracted from the real blood samples with those performed by standard qRT-PCR technique. Results in this study confirmed the feasibility of using the HOTAIR genosensing strategy in bioanalysis.

Automated summary

This study presents a sensitive method for the early diagnosis of lung cancer by simultaneously detecting multiple lncRNA biomarkers, using a dual-channel fluorescence and electrochemical signals to achieve efficient quantitative detection with a wide linear range and low detection limits.