MicroRNAs (miRNAs) are a class of small, single-stranded non-coding RNAs which have critical functions in various biological processes. Increasing evidence suggested that abnormal miRNA expression was closely related to many human diseases, and they are projected to be very promising biomarkers for non-invasive diagnosis. Multiplex detection of aberrant miRNAs has great advantages including improved detection efficiency and enhanced diagnostic precision. Traditional miRNA detection methods do not meet the requirements of high sensitivity or multiplexing. Some new techniques have opened novel paths to solve analytical challenges of multiple miRNA detection. Herein, we give a critical overview of the current multiplex strategies for the simultaneous detection of miRNAs from the perspective of two different signal differentiation models, including label differentiation and space differentiation. Meanwhile, recent advances of signal amplification strategies integrated into multiplex miRNA methods are also discussed. We hope this review provides the reader with future perspectives on multiplex miRNA strategies in biochemical research and clinical diagnostics.

Cardiovascular diseases (CVDs) are the number one cause of death worldwide, taking 17.9 million lives each year. The rapid, sensitive, and accurate determination of cardiac biomarkers is vital for the timely diagnosis of CVDs. For accurate diagnosis, dependence on a single biomarker is unreliable because each one has also been linked to other diseases. To overcome this problem, the multiplexed determination of two or more markers has emerged as a promising alternative to single-marker analysis. Over the last 5 years, research interest in the development of biosensors for targeting multiple cardiac markers has increased. In this study, we critically reviewed the various multiplexed biosensing approaches reported during the last 5 years, categorizing them by signal readouts. Prospective detection configurations, capture probes, electrode design strategies, electrode types, nanomaterials, reporter tags, and assay types were reviewed, tabulated, and critically discussed. Then, their advantages and limitations were highlighted. For each category, we provided our perspective as well as the overall critical discussion. Lastly, we summarized potential commercial multiplexed cardiac biosensors and commented on the challenges and future prospects for such sensors.