This review summarizes the recent advances and the main strategies to improve the sensitivity of amplification-free CRISPR/Cas-based detection techniques.

The cutting‐edge CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR‐associated proteins) system, as an emerging molecular diagnostic technique, is driving revolutionary developments in the detection field due to its high specificity and efficiency. However, the CRISPR‐based assays typically require the combination with an additional pre‐amplification step based on isothermal nucleic acid amplification to meet the requirements of clinical diagnosis, which brings issues including complicated operation and the risk of aerosol contamination. To address these challenges, one‐pot CRISPR platforms are emerging as an attractive solution to streamline workflows, enabling rapid, cost‐effective, and high‐sensitivity diagnostics. This review outlines the current status, challenges, and three key strategies to realize highly efficient one‐pot CRISPR‐based detection. In addition, further perspectives are outlined that will inspire new exploration and promote one‐pot CRISPR/Cas detection as the next generation of diagnostic tools.

Infectious diseases contribute significantly to the global disease burden. Sensitive and accurate screening methods are some of the most effective means of identifying sources of infection and controlling infectivity. Conventional detecting strategies such as quantitative polymerase chain reaction (qPCR), DNA sequencing, and mass spectrometry typically require bulky equipment and well-trained personnel. Therefore, mass screening of a large population using conventional strategies during pandemic periods often requires additional manpower, resources, and time, which cannot be guaranteed in resource-limited settings. Recently, emerging microfluidic technologies have shown the potential to replace conventional methods in performing point-of-care detection because they are automated, miniaturized, and integrated. By exploiting the spatial separation of detection sites, microfluidic platforms can enable the multiplex detection of infectious diseases to reduce the possibility of misdiagnosis and incomplete diagnosis of infectious diseases with similar symptoms. This review presents the recent advances in microfluidic platforms used for multiplex detection of infectious diseases, including microfluidic immunosensors and microfluidic nucleic acid sensors. As representative microfluidic platforms, lateral flow immunoassay (LFIA) platforms, polymer-based chips, paper-based devices, and droplet-based devices will be discussed in detail. In addition, the current challenges, commercialization, and prospects are proposed to promote the application of microfluidic platforms in infectious disease detection.

Mitigating the spread of global infectious diseases requires rapid and accurate diagnostic tools. Conventional diagnostic techniques for infectious diseases typically require sophisticated equipment and are time consuming. Emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated proteins (Cas) detection systems have shown remarkable potential as next‐generation diagnostic tools to achieve rapid, sensitive, specific, and field‐deployable diagnoses of infectious diseases, based on state‐of‐the‐art microfluidic platforms. Therefore, a review of recent advances in CRISPR‐based microfluidic systems for infectious diseases diagnosis is urgently required. This review highlights the mechanisms of CRISPR/Cas biosensing and cutting‐edge microfluidic devices including paper, digital, and integrated wearable platforms. Strategies to simplify sample pretreatment, improve diagnostic performance, and achieve integrated detection are discussed. Current challenges and future perspectives contributing to the development of more effective CRISPR‐based microfluidic diagnostic systems are also proposed.