Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms

Abstract

In the fight against the worldwide pandemic coronavirus disease 2019 (COVID-19), simple, rapid, and sensitive tools for nucleic acid detection are in urgent need. PCR has been a classic method for nucleic acid detection with high sensitivity and specificity. However, this method still has essential limitations due to the dependence on thermal cycling, which requires costly equipment, professional technicians, and long turnover times. Currently, clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensors have been developed as powerful tools for nucleic acid detection. Moreover, the CRISPR method can be performed at physiological temperature, meaning that it is easy to assemble into point-of-care devices. Microfluidic chips hold promises to integrate sample processing and analysis on a chip, reducing the consumption of sample and reagent and increasing the detection throughput. This review provides an overview of recent advances in the development of CRISPR-based biosensing techniques and their perfect combination with microfluidic platforms. New opportunities and challenges for the improvement of specificity and efficiency signal amplification are outlined. Furthermore, their various applications in healthcare, animal husbandry, agriculture, and forestry are discussed.

Recent Advances, Opportunities, and Challenges in Developing Nucleic Acid Integrated Wearable Biosensors for Expanding the Capabilities of Wearable Technologies in Health Monitoring

“…One of the important aspects of the sensing layer is the consideration for the biorecognition element immobilization. These layers need to be compatible with the functionalization chemistry without affecting its transduction performance [ 49 ] many different sensing materials have been utilized, such as gold thin films [ 29 , 41 , 50 , 51 ] and graphene monolayers. Materials such as carbon nanomaterials, although cost-effective and easy to fabricate, pose limitations such as electrical performance deterioration and high electrical resistance.…”

“…Cas enzymes, upon binding to these crRNAs, form a complex which can search for and cleave target sequences complementary to the crRNAs ( Figure 3 c) [ 96 ]. The advantages of such a system include its inherent high sensitivity (detection limits as low as ~50 fM, as reported in the literature) [ 97 ], due to Cas enzymes’ specific sequence requirements for cleavage, leading to resolution down to a single base pair, low development cost, rapid reaction time (0.5–2 h), and programmability to target any nucleic acid sequence or protein sequences if coupled with aptamer/DNAzyme-based assays [ 25 , 51 , 98 ]. However, CRISPR-based biosensors have been integrated with mainly fluorescent transduction and lateral flow strips, which sometimes rely on heavy instruments, and have low sensitivity [ 76 ].…”

“…However, CRISPR-based biosensors have been integrated with mainly fluorescent transduction and lateral flow strips, which sometimes rely on heavy instruments, and have low sensitivity [ 76 ]. Additionally, for low target concentrations, CRISPR-based biosensors are usually coupled with a nucleic acid amplification method to augment signal strength, making portability a challenge [ 51 ]. As discussed earlier, since PCR requires specific equipment and professional operators, isothermal nucleic acid amplification methods may be used with CRISPR-based sensing devices.…”

“…As discussed earlier, since PCR requires specific equipment and professional operators, isothermal nucleic acid amplification methods may be used with CRISPR-based sensing devices. Successful attempts have been made to develop amplification-free CRISPR-based assays, by using gold nanostructures [ 51 ]. In the context of wearable biosensors, Nguyen et al recently used a CRISPR system as an unlocker, which activates a subsequent recognition element in their sensing device.…”

Point‐of‐care CRISPR/Cas biosensing technology: A promising tool for preventing the possible COVID‐19 resurgence caused by contaminated cold‐chain food and packaging

“…Compared with some standard test methods, the micro amount can significantly improve the reaction rate, efficiently increase the detection throughput, and greatly shorten the test time (Bing Chen, Li, Xu, & Yang, 2022 ; Fattahi & Hasanzadeh, 2022 ; Yahui Wang, Ma, et al., 2021 ). Due to its superior performances (e.g., small size, low reagent/sample consumption, easy integration, and rapid analysis speed), microfluidic chips have been successfully integrated with CRISPR/Cas system‐based biosensors for the rapid and POC diagnosis of SARS‐CoV‐2 (B. Chen et al., 2022 ; F.‐E. Chen et al., 2021 ; Fattahi & Hasanzadeh, 2022 ; Ramachandran et al., 2020 ).…”