Ultrasensitive electrochemical detection of alternative cleavage and polyadenylation of CCND2 gene at the single-cell level

Alternative polyadenylation (APA) is a novel mechanism in the expressional regulation of most human oncogenes. This process also is involved in cancer progression and prognosis. The potential of APA events as novel prognostic biomarkers has been suggested. CCND2 gene, which is overexpressed in various cancer tissues, tends to utilize the proximal APA site and produces a more prominent mRNA isoform with shorter 3' untranslated region in the cancer cells. In this study, an ultrasensitive detection for CCND2 APA was successfully achieved using electrochemical biosensors constructed by gold nanoparticles-Fe3O4 nanocomposite and p-sulfonated calix [8]arene functionalized reduced graphene oxide as nanocarriers. The detection ranges of CCND2-S and CCND2-L by the constructed biosensors were 10(-18)-10(-11) and 10(-17)-10(-11) M, respectively. The limit of detection (LOD) of CCND2-S was 0.176 aM (the RNA concentration at the single-cell level). To the best of our knowledge, this value was the lowest LOD among the reported non-coding RNA detection. In real samples, the results of the electrochemical detection using the constructed biosensors showed that the lung cancer cell H292 expressed considerably higher CCND2-S than CCND2-L. This result was in agreement with those obtained by RT-qPCR method. The electrochemical method had showed simpler operation, accurate quantitation, faster detection, and cheaper supplies compared with traditional methods. Therefore, this technology not only provides an ultrasensitive and feasible method for APA detection, but can also be applied in the prognosis and diagnosis of cancer progression.