Point-of-Care Nucleic Acid Detection: From Molecular Design to Clinical Reality.

Point-of-Care (PoC) diagnostics are transforming healthcare by enabling rapid and accessible disease detection right at the patient's bedside. This comprehensive review examines recent advances in nucleic acid (NA)-based PoC testing, revealing how these technologies are revolutionizing molecular diagnostics. Here, we critically analyze the three key components of NA-based PoC development: (i) probe design strategies, (ii) immobilization techniques, and (iii) detection methodologies. Our analysis uncovers the complex relationship between probe density, hybridization conditions, and detection sensitivity, challenging the conventional trial-and-error approaches currently dominating the field. The review introduces a novel classification of detection methods based on equipment requirements, offering valuable insights for developing truly accessible diagnostic solutions. Notably, we highlight that colorimetric and electrochemical detection methods show superior potential in meeting the REASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, Real-time connectivity, Ease of specimen collection, and Deliverable to end-users). These criteria are essential for global healthcare implementation. Despite major advances, significant gaps remain between laboratory innovations and practical, affordable diagnostic products. We suggest that prioritizing equipment-free detection methods and enhancing standardization could accelerate the translation of these PoC technologies into clinical practice. This review outlines a strategic roadmap for advancing NA-based PoC diagnostics, emphasizing the potential to transform healthcare delivery worldwide through accessible, rapid, and reliable molecular testing.