Overcoming Liquid Biopsy Barriers: Nucleic Acid Biosensors Integrating DNA Nanotechnology and CRISPR-Cas System for...

Liquid biopsy holds immense potential for the early detection of cancer, yet its clinical utility is hindered not by the lack of available tumor-associated biomarkers but by the inadequate sensitivity and clinical robustness of current molecular diagnostic tools. Nucleic acid-based biosensors have emerged as highly programmable platforms, enabling the detection of low-abundance cancer biomarkers such as microRNAs (miRNAs), circulating tumor DNA (ctDNA), and messenger RNAs (mRNAs) in complex biological fluids. Leveraging advances in DNA nanotechnology, CRISPR-Cas-mediated RNA sensing, and chemically engineered nucleic acid analogues, these biosensors achieve attomolar-level detection through nanoscale spatial confinement and enzyme-assisted signal amplification strategies. However, their clinical translation is hindered by biological sample variability, nonspecific amplification, probe degradation, and poor reproducibility. This review analyzes the core design principles of three major biosensor categories: functional DNA nanostructures, CRISPR-Cas-based sensing systems, and synthetic analogues (PNAs, SNAs). It elucidates their structural and enzymatic optimization mechanisms, distinguishes analytical from clinical sensitivity, and addresses key liquid biopsy challenges. Finally, it outlines promising strategies for clinical translation, including microfluidic integration, artificial intelligence-assisted data analysis, and theranostic nanostructures combining diagnosis with targeted therapy. This review provides a comprehensive theoretical and technical framework for the rational design of next-generation nucleic acid biosensors and offers critical insights to bridge the gap between nanoscale engineering innovation and clinical translation, ultimately advancing the development of minimally invasive and precise cancer theranostics in precision oncology.