Development of a CRISPR/dCas13a-Based electrochemical miRNA biosensor for the diagnosis and monitoring of epileptic seizures

Abstract

Epilepsy is a serious neurological disorder characterized by recurrent, unprovoked seizures of undetermined etiology, profoundly affecting patients’ quality of life, cognitive function, and overall health. Early detection and continuous monitoring of epileptic seizures are critical for timely intervention, effective treatment management, and prevention of potential neurodegenerative damage to the central nervous system. However, conventional diagnostic approaches, such as electroencephalography (EEG) and neuroimaging, are often limited by high costs, the need for specialized expertise, and restricted accessibility, underscoring the urgent need for low-cost, highly sensitive, and portable biomarker-based diagnostic tools. In recent years, microRNAs (miRNAs) have emerged as promising post-transcriptional gene regulators implicated in epilepsy pathophysiology and as potential circulating biomarkers. Notably, miR-143-3p and miR-145-5p have shown significant dysregulation in plasma and serum samples from epilepsy patients. Studies indicate that their blood levels are inversely correlated with total seizure duration, with downregulation particularly observed in refractory epilepsy and mesial temporal lobe epilepsy (mTLE), often associated with earlier disease onset and higher seizure frequency.This thesis presents the design and development of a novel CRISPR-based electrochemical biosensor platform for the specific, label-free, and point-of-care detection of epilepsy-associated miR-143-3p and miR-145-5p biomarkers. The platform utilizes chemically modified screen-printed gold electrodes (SPGE) functionalized with catalytically inactive Cas13a (dCas13a) complexed with synthetic guide RNA (sgRNA). This configuration enables direct, cleavage-independent recognition of target miRNAs with high-specificity, facilitating quantitative detection through signal modulation without reliance on collateral cleavage activity or target amplification.