A facile thermometer-like electrophoresis titration biosensor for alternative miRNA assay via moving reaction boundary chip.

Herein, a facile thermometer-like model of electrophoresis titration (ET) biosensor was proposed as an alternative tool for miRNA assay via moving reaction boundary (MRB) chip. For proof-of-concept demonstration, miRNA-122 and catalyzed hairpin assembly (CHA) were chosen as the model analyte and amplification, respectively. In the developed ET system, miRNA triggered the CHA with two hairpin probes (H1, H2) to yield H1-H2 duplexes with negative charges. Under an electric field, the duplexes moved into ET channel, and neutralized the acidic TAE buffer creating an MRB indicated by SYBR Green I (SGI). The model revealed that the MRB distance was as a function of logarithmic miRNA-122 content, indicating a facile sensing model. The relevant experiments were conducted and systemically validated the model of miRNA ET. Under the optimized conditions, the linear range of ET sensor was from 20 fM to 1 nM and the limit of detection (LOD) was 10 fM, showing a more than 100-fold sensitive increase in contrast to the one with a single CHA amplification. The mechanism of sensitive increase was well unveiled by the designed experiments. In addition, the ET biosensor had good selectivity, stability (less than 5% for intra-day and inter-day) and recovery (96%-110%), and was successfully applied for the assay of miRNA-122 and miRNA let-7a in real bio-fluids of serum and cancer cell lysate. Evidently, the proposed biosensor might be used as an alternative assay tool after nucleic acid amplification due to its high simplicity, sensitivity, specificity, linearity, stability and recovery.