Microfluidic biosensors integrate microfluidic and biosensing technologies to achieve the miniaturization, integration, and automation of disease diagnosis, and show great potential for application in the fields of cancer liquid biopsy, pathogenic bacteria detection, and POCT. This paper reviews the recent advances related to microfluidic biosensors in the field of laboratory medicine, focusing on their applications in the above three areas. In cancer liquid biopsy, microfluidic biosensors facilitate the isolation, enrichment, and detection of tumor markers such as CTCs, ctDNA, miRNA, exosomes, and so on, providing support for early diagnosis, precise treatment, and prognostic assessment. In terms of pathogenic bacteria detection, microfluidic biosensors can achieve the rapid, highly sensitive, and highly specific detection of a variety of pathogenic bacteria, helping disease prevention and control as well as public health safety. Pertaining to the realm of POCT, microfluidic biosensors bring the convenient detection of a variety of diseases, such as tumors, infectious diseases, and chronic diseases, to primary health care. Future microfluidic biosensor research will focus on enhancing detection throughput, lowering costs, innovating new recognition elements and signal transduction methods, integrating artificial intelligence, and broadening applications to include home health care, drug discovery, food safety, and so on.

The CRISPR/Cas system has attracted increasing attention in accurate nucleic acid detection. Herein, we reported a CRISPR/Cas12a-chemiluminescence cascaded bioassay (CCCB) for the amplification-free and sensitive detection of human papillomavirus type 16 (HPV-16) and parvovirus B19 (PB-19). A magnetic bead (MB)-linking single-stranded DNA (LssDNA)-alkaline phosphatase (ALP) complex was constructed as the core component of the bioassay. During the detection process, the single-stranded target DNA was captured and enriched by LssDNA and then activated the trans-cleavage activity of Cas12a. Due to the Cas12a-mediated cleavage of LssDNA, ALP was released from the MB, subsequently catalyzing the substrate to generate a chemiluminescence (CL) signal. Given the cascade combination of CRISPR/Cas12a with the CL technique, the limits of detection for HPV-16 and PB-19 DNA were determined as 0.14 pM and 0.37 pM, respectively, and the whole detection could be completed within 60 min. The practicality and reliability of the platform were validated through target-spiked clinical specimens, and the recovery rate was 93.4–103.5%. This dual-amplification strategy—operating without target pre-amplification—featured high specificity, low contamination risk, facile preparation, and robust stability. It provides a novel approach for sensitive nucleic acid detection, with the potential for rapid extension to the diagnosis of various infectious diseases.