Attomolar sensitivity microRNA detection using real-time digital microarrays

MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. DNA microarray technology is a powerful and high throughput tool for nucleic acid detection and quantification, however, traditional methods do not meet the demands in sensitivity and specificity presenting significant challenges for the adaptation of miRNA detection for diagnostic applica-tions. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA-451a (miR-451) and miRNA-223-3p (miR-223). We demonstrated improvements in sensitivity and specificity by implementing real-time tracking of the target molecules binding to multiplexed probes on the sensor surface, in comparison to conventional end-point assays that employ capture on solid phase support. Real-time particle tracking overcomes the sensitivity limitations for detection of low-abundance biomarkers in the presence of low-affinity but high-abundance background molecules, where endpoint assays fall short. The limit of detection (LOD) calculations with end-point assay format yielded 2.22 fM for miR-223 and 228 aM for miR-451 after a total of 10hr incubation time. The implementation of dynamic tracking improved the LODs in real-time experiments to 13 aM (less than 1600 copies in 0.2ml of sample) for miR-223 and 6.2 aM (less than 800 copies in 0.2ml of sample) for miR-451 after a total incubation time of 35 minutes.