Circulating microRNA detection using electrochemical biosensor for rapid diagnosis of liver disease in dogs

Abstract

Liver disease in dogs is a major cause of morbidity and mortality. Non-invasive diagnosis of liver disease in dogs is a clinical challenge and improved tests which could done at point-of-care are highly desirable. Liver-specific circulating microRNAs have emerged as promising biomarkers for liver injury across many vertebrate species including dogs. MicroRNA-122 (miR-122), originating from the damaged hepatocytes, provides high specificity and sensitivity in detecting liver disease, compared to the traditional biomarkers. In this study, we present the development of a point-of-care compatible electrochemical biosensor for rapid, early diagnosis of liver disease in dogs by detecting miR-122 in clinical samples. Building on our prior work utilising electrochemical impedance spectroscopy (EIS) for direct and amplification-free detection of miR-122 in human drug-induced liver injury, we have used a miR-122 target-specific short probe and implemented target overhang formation during hybridisation in a flow-based sample cycling setup for enhanced detection performance and demonstrated its performance in real clinical dog samples for the first time. We determined the hybridisation performance by analysing miR-122 specificity and sensitivity achieving a limit of detection (LOD) and limit of quantification (LOQ) of 10 pM and 100 pM, respectively, and high specificity over a nearly-complementary sequence of a miR-122 precursor. Using conventional sample preparation, the developed EIS assay was used to analyse serum samples from dogs with liver disease which were identified based on an increased serum alanine aminotransferase concentration. The test successfully distinguished samples from dogs with and without liver disease in comparable performance to the gold-standard real-time polymerase chain reaction (qPCR) detection. We will further focus on developing sample-to-answer test by combining our miR-122 EIS biosensor with a compatible sample preparation to measure miR-122 from dog blood at point of care.