Reusable Bioluminescent Sensor for Ultrasensitive MicroRNA Detection Based on a Target-Introducing “Fuel-Loading” Mechanism

As a kind of important potential biomarkers, the expression level of some microRNAs (miRNAs) is closely related to cancer development and progression. Herein, a reusable ultra-sensitive "fuel-loadings" bioluminescent sensor was constructed to detect the trace miRNA based on the cascading signal amplification, which combined the target-introducing "fuel-loading" mechanism and cyclic bioluminescence assay. In this sensor, magnetic beads labeled with hairpin DNA probes (hDNA) could specifically hybridize with the target miRNA and isolate these targets from samples. Then, the target-introducing "fuel loading" mechanism worked because the poly(A) polymerase can catalyze the template-independent sequential addition of adenosine monophosphate (AMP) to the 3' ends of the miRNA targets to produce long poly(A) tails. The long poly(A) tails provided lots of 5'AMPs (cleaved by Exonuclease T), which further as fuels were converted into adenosine-triphosphate (ATP) to generate an enhanced bioluminescent signal by cyclic AMP pyrophosphorylation-ATP dephosphorylation. The "fuel-loadings" bioluminescent sensor realized a high sensitivity with a limit-of-detection of about 22.6 aM for miRNA 21. Moreover, this "fuel-loadings" bioluminescent sensor not only achieved regenerable and reusable measurement in the same microwell to decrease the analysis costs, but also could directly detect miRNA 21 in the serum without complicated extraction procedures. It showed excellent coherence with quantitative reverse transcription polymerase chain reaction for miRNA 21 detection of cancer patients' samples, indicating clinical translation potential for miRNA detection.