Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. MicroRNA-122 (miR-122) is a sensitive and specific biomarker for hepatocyte injury that has received formal regulatory support. A major roadblock to its widespread adoption is a lack of a fit-for-purpose, scalable assay that can provide quantification of circulating miR-122 with appropriate accuracy and time-to-result. We have developed a PCR-free, sensitive and highly specific assay for measuring miR-122. The assay is based on the hybridization of miR-122 to a peptide nucleic acid (PNA) probe attached to superparamagnetic beads, followed by the specific incorporation of a biotinylated nucleobase. The biotin labels are then labeled with an enzyme, and single enzymes are detected using single molecule arrays (Simoa).

The miR-122 single molecule assay had a limit of detection (LOD) of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay. The specificity of the assay to a single base mismatch in the microRNA sequence was >107-fold ((highest concentration of mismatched target with signal below LOD)/(LOD)). As measured by our single molecule assay, all patients with DILI had higher concentrations of miR-122 in their serum compared to healthy controls, and the concentrations of miR-122 measured correlated closely with those determined using the gold standard PCR (r2 = 0.93; slope = 0.64).

This novel single molecule assay enabled the accurate measurement of miR-122 in the serum of patients with DILI. This assay could have utility in clinical practice as it has the potential to deliver the user-independent sensitivity and time-to-result that are needed to inform pre-clinical and clinical decision making.