Clustered Regularly Interspaced short palindromic repeats‐Based Microfluidic System in Infectious Diseases Diagnosis: Current Status, Challenges, and Perspectives

Abstract

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.

Multiplex Detection of Infectious Diseases on Microfluidic Platforms

“…PCR is typically considered as the gold standard detection method of nucleic acid-based diagnosis, but involves costly/advanced equipment and skilled personnel, so it cannot be easily combined with microfluidic technology [ 53 ]. Therefore, isothermal amplification and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR/Cas) system, which have the advantage of low-cost, reliability, and do not require for bulky equipment, can be compatible with the microfluidic platforms for nucleic acid detection [ 20 , 54 ]. Based on the spatial separation of detection sites on microfluidic platforms, the multiplex detection of different nucleic acids can be realized [ 14 ].…”

“…Fabrication of microfluidic devices is an important step in integrated automated nucleic acid sensing. In this regard, polymers (e.g., polydimethylsiloxane, PDMS) are one of the most common materials, due to their advantages such as cost-effectiveness, good biocompatibility, and simple fabrication protocol [ 20 ]. Polymer-based microfluidic chip is highly automated (multistep continuous reactions can be realized via sophisticated microstructures) and integrated, which has been widely used for infectious disease detections [ 60 ].…”

“…Moreover, because of the relatively independent reaction space on the microfluidic platforms, the sensitivity and specificity of each assay can be ensured in multiplex detection. Therefore, multiplex detection technology based on microfluidic platforms shows great potential for the diagnosis and mass screening of infectious diseases [ 19 , 20 ]. Although there are several reviews that comprehensively discuss microfluidic detection technologies for infectious disease diagnosis [ 21 , 22 , 23 ], few of them focus on microfluidic platforms for multiplex detection [ 21 ].…”

CRISPR/Cas-Powered Amplification-Free Detection of Nucleic Acids: Current State of the Art, Challenges, and Futuristic Perspectives

“…Single-molecule sensitivity was achieved by harnessing an ultralocalized/confined reactor or microvolume, such as through the use of droplet microfluidics, microchamber-array technologies, etc. , This not only improves the reaction efficiency via the confinement effect but also reduces the requirement for manual operation and shortens turnaround times through miniaturization and integration. Besides, this makes multiplexed detection possible …”

“…Besides, this makes multiplexed detection possible. 33 Zhou and co-workers proposed an ultralocalized Cas13a assay on the basis of the natural confinement effect, which limited the RNA-triggered Cas13a catalytic reaction system to a cell-sized reactor via droplet microfluidics for the enhancement of the target and reporter concentrations simultaneously (Figure 3A). 34 The microfluidic was a T-junction and explored the generation of water-in-oil droplets in picoliter sizes.…”