Integration of isothermal amplification methods in DNA hydrogels for biosensing

Abstract

DNA hydrogels provide molecular-level programmable recognition and excellent biocompatibility for biosensing but are plagued by serious drawbacks like mechanical instability, reduced sensitivity, expensive fabrication processes, and poor single-target detection. These intrinsic limitations have driven the search for new approaches that can concurrently boost and maintain the advantageous properties of hydrogel-based systems. Isothermal nucleic acid amplification techniques (INAATs) have transformed molecular diagnostics by eliminating thermal cycling requirements through enzyme-dependent strategies (LAMP, RPA, RCA, SDA, and EXPAR) and enzyme-independent strategies (HCR, CHA, and EDC) with high sensitivity and rapid amplification capabilities. However, INAATs typically depend on complex instrumentation for signal readout and interpretation. The strategic coupling of INAATs with DNA hydrogels overcomes the limitations of both approaches by synergistic means. This hybrid platform enhances hydrogel structural properties, reduces equipment dependency, extends detection capabilities, and uses INAAT amplification properties to improve biosensing performance through increased sensitivity. This review describes INAAT integration within DNA hydrogels for high-performance biosensing applications. We introduce the most recent diverse applications in this newly developed area and elucidate the ensuing combination mechanisms and effects of integrating each INAAT strategy into hydrogel systems, comparing their standalone performances and competencies. We demonstrate how the integration of cutting-edge biotools in these platforms, particularly CRISPR-based detection platforms and catalytic/enzymatic amplification methods, enables higher specificity and signal amplification functionalities. Finally, we identify the key challenges and set the top development priorities to propel clinical translation.