ABSTRACT:

Accurate estimation of post-mortem interval is crucial in forensic investigations; however, current techniques are mostly limited to the early hours or days after death and are strongly influenced by environmental and individual factors. Recently, microRNAs have emerged as promising biomarkers due to their relative stability against the enzymatic degradation and decomposition process. RNAzymes, catalytic RNA systems, can be fine-tuned to bind specific microRNA sequences and produce a colorimetric signal through 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation, enabling specific detection and quantification. In this work, we present a paper-based device for the detection and quantification of microRNA-206 using an RNAzyme system. MicroRNA-206 is a post-mortem interval biomarker specifically expressed in cardiac tissue, whose levels decrease progressively after death. A solution-based sensor was first developed using specific sensing probes designed to hybridise with the target. This system was subsequently adapted into two paper-based formats: a wax ring delimited paper sensor and a strip-shaped paper device. The system exhibited a measurable increase in colorimetric intensity upon target recognition with a dynamic range of 1040-5760 ng in solution (22.5 min) and 82.56-1440/1040-5760 ng on paper (400 s for circles and 480 s for strips). Notably, the system demonstrated high specificity, not showing signal intensity increasing when exposed to non-complementary microRNAs used as negative controls (microRNA-142-5p, microRNA-320, and microRNA-195). These findings represent the first RNAzyme-based paper sensor for post-mortem interval estimation, offering a low-cost and rapid alternative to traditional forensic techniques