Sample-to-answer detection of miRNA from whole blood using thermally responsive alkane partitions

Circulating miRNA offers a tremendous opportunity as a biomarker paradigm for many applications in disease diagnostics, including point-of-care diagnostics for global health needs. However, despite the numerous miRNA detection schemes reported, there still does not exist a solution for highly sensitive sample-to-answer detection of miRNA directly from complex samples, such as whole blood. We recently developed thermally responsive alkane partitions (TRAPs), which - when combined with magnetic microbeads - enable the complete assay automation from whole blood. Here we apply TRAPs with ligation-LAMP to automate the detection of miRNA in whole blood samples. MBs and a TRAP enable the automated purification of miRNA from blood, while a novel displacement-ligation method is utilized to trigger the ligation-LAMP reaction, which is streamlined into one step by a second TRAP. Using easily manufacturable TRAP-enabled assay cassettes and a custom low-cost handheld instrument, we report the specific detection of miR-155 at concentrations as low as 15 fM in whole blood with no intermediate steps by the user. This new approach creates the opportunity for point-of-care miRNA-based diagnostics with global health applications.