A Cathode-Anode Ratiometric Internal Standard Electrochemiluminescence Recognition Strategy Based on Hexagonal AgNCs and ZnCdSe QDs Dual-Labeled Exon Conservative Motif Aggregation for LncRNA Assay

A cathode-anode bipolar electrochemiluminescence (ECL) strategy on multisite recognition for highly conservative gene motifs is anticipated to anchor the >200 nt-long polynucleotide chain for precise quantification. Herein, lncRNA-HOTAIR sequences in the NCBI database, derived from four different species (Homo sapiens, Mus musculus, Pan troglodytes, Canis lupus familiaris), were blasted online to confirm three-segment conservative exon motif texts (abbreviated as motif 1, motif 2 and motif 3), whose complementary gene sequences were artfully designed as capture probe (P1, complementary with motif 1) and signal probes (P2, P3, complementary with motif 2 and motif 3 in a controllable two-base interval). By exploiting the anode potential dominant sulfate radical and reactive oxide radical reaction, a new couple of ECL signals at E_pc of -1.8 V and E_pa of 3.5 V (vs Ag/AgCl) was revealed in hexagonal silver nanocrystal (H-AgNC) coreaction-accelerated graphene oxide/persulfate (GO/S₂O₈^2-) system. While the rolling circle amplification for the adjacent sequences of motif 2/motif 3 followed by motif 1 capture on composite film, H-AgNCs and ZnCdSe QDs dual-labeled probe aggregation on the amplified motifs induced a couple of cathode-anode quenched ECL signals. In proportion to the fairly stronger internal standard signal formed by a single H-AgNC-labeled probe, the dual-label quenched ECL signal ratio (ΔI_pc/ΔI_pa) was linearly fitting as Y = 2.34X + 8.32 to reflect lncRNA HOTAIR level with a limit-of-detection (LOD) of 0.01 pg/L. The analysis performance on bipolar ECL behavior was compared upon different anode final potential to objectively assess the impact of anode polarization and the possibility in trace clinical lncRNA assay.