Integration of isothermal nucleic acid amplification with label-free strategies in Biosensors: Design principles and recent advances

Driven by the increasing demand for point-of-care testing in medical diagnostics, food safety, and environmental monitoring, biosensors with high sensitivity, portability, low cost, and operational simplicity have become a major focus of research. The integration of isothermal nucleic acid amplification technologies (INAATs) with label-free strategies provides a promising approach for the sensitive detection of trace targets in complex samples. INAATs enable nucleic acid amplification at a constant temperature, eliminating the need for expensive thermocyclers and thereby supporting on-site analysis in resource-limited settings. When coupled with label-free signal transduction, INAAT-based biosensors can further reduce assay complexity and show considerable potential for translation into point-of-care testing platforms. Accordingly, label-free INAAT-based biosensors have attracted increasing attention in recent years. In this review, we summarize studies on label-free biosensors integrated with INAATs published between January 2020 and June 2026. Four major label-free strategies integrated with INAATs are discussed, including those based on DNA-binding molecules, functional nucleic acids, nanomaterials, and amplification by-products. Their design principles, analytical advantages, technical challenges, and strategies for multiplex detection are comprehensively summarized. We further assess their potential for translation into point-of-care testing, together with future perspectives on their further development.