Nucleic acids are essential biomarkers in molecular diagnostics. The CRISPR/Cas system has been widely used for nucleic acid detection. Moreover, canonical CRISPR/Cas12a based biosensors can ...specifically recognize and cleave target DNA, as well as single-strand DNA serving as reporter probe, which have become a super star in recent years in the field of nucleic acid detection due to its high specificity, universal programmability and simple operation. However, canonical CRISPR/Cas12a based biosensors are hard to meet the requirements of higher sensitivity, higher specificity, higher efficiency, larger target scope, easier operation, multiplexing, low cost and diversified signal reading. Then, advanced non-canonical CRISPR/Cas12a based biosensors emerge. In this review, applications of non-canonical CRISPR/Cas12a-based biosensors in nucleic acid detection are summarized. And the principles, peculiarities, performances and perspectives of these non-canonical CRISPR/Cas12a based biosensors are also discussed.
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•Brief overview of canonical/non-canonical CRISPR/Cas12a biosensors.•Characterization and mechanism of non-canonical CRISPR/Cas12a biosensors.•Advance and perspective of CRISPR/Cas12a biosensors for nucleic acid detection.
MicroRNA (miRNA) has emerged as a promising biomarker for disease diagnosis and a potential therapeutic targets for drug development. The detection of miRNA can serve as a noninvasive tool in ...diseases diagnosis and predicting diseases prognosis. CRISPR/Cas12a system has great potential in nucleic acid detection due to its high sensitivity and specificity, which has been developed to be a versatile tool for nucleic acid-based detection of targets in various fields. However, conversion from RNA to DNA with or without amplification operation is necessary for miRNA detection based on CRISPR/Cas12a system, because dsDNA containing PAM sequence or ssDNA is traditionally considered as the activator of Cas12a. Until recently, direct detection of miRNA by CRISPR/Cas12a system has been reported. In this review, we provide an overview of the evolution of biosensors based on CRISPR/Cas12a for miRNA detection from indirect to direct, which would be beneficial to the development of CRISPR/Cas12a-based sensors with better performance for direct detection of miRNA.
Foodborne pathogens can cause illnesses. Existing tools for detecting foodborne pathogens are typically time-consuming or require complex protocols. Here, we report an assay to directly analyze ...pathogenic genes based on CRISPR-Cas12. This new test, termed proximal DNA probe-based CRISPR-Cas12 (PPCas12), facilitates the detection of foodborne pathogens without amplification steps. The elimination of the nucleic acid amplification process dramatically reduced the processing time, complexity, and costs in the analysis of foodborne pathogens. The substitution of the frequently used dually labeled DNA reporter with a proximal DNA probe in the PPCas12 assay led to a 4-fold sensitivity enhancement. PPCas12 offered a limit of detection of 619 colony-forming units in the detection of Salmonella enterica (S. enterica) without the nucleic acid amplification process. The specific recognition of genes via PPCas12 allowed distinguishing S. enterica from other foodborne pathogens. The PPCas12 assay was applied in the screening of S. enterica contamination on fresh eggs with high precision. Hence, the new PPCas12 assay will be a valuable tool for on-site monitoring of foodborne pathogens.
Real-time quantitative PCR (qPCR) is a sought-after method for quantifying nucleic acids. A series of commercial instruments based on qPCR has been developed and applied to detect several diseases. ...However, commercial instruments have some limitations in terms of cost, bulk, and efficiency. Herein, we proposed a novel qPCR device using water-cooling technology to shorten the period of the PCR reaction. The device was made of an amplification module with a novelty water-cooling-based PCR chip, a custom-made fluorescence detection module, and a control module based on economical off-the-shelf electronics. All these modules were assembled in a 3D-printed frame with a size of 11 cm × 9.7 cm × 8.9 cm and a weight of 0.5 kg. With the help of water-cooling technology, the total time required to complete a PCR reaction was shortened, with a maximum cooling rate of 12 ℃/s, which is much faster than the rate of conventional cooling methods. Compared with commercial qPCR instruments, our device's cost, efficiency, and portability have been significantly optimized. To test the proposed device, a qPCR experiment was successfully performed with cDNA of Vesicular stomatitis virus at concentrations ranging from 0.005 μg/mL to 50 μg/mL.
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•A water-cooling-based qPCR device for rapid analysis of nucleic acid.•The qPCR device is portable and low-cost (11 cm × 9.7 cm × 8.9 cm, 0.5 kg, $197.91).•Simple and low-cost fabrication process for PCR chip based on water-cooling technology.•The maximum cooling rate of the PCR reaction is about 12 ℃/s.
Recent advances in Argonaute (Ago)-mediated biotechnology have provided new insights into the development of programmable and highly sensitive nucleic acid detection platforms. This study provides an ...overview of recent research on Ago-based nucleic acid detection. The potential applications of these emerging nucleic acid biosensors and the challenges associated with their use have been discussed.
Circulating tumor cells (CTCs) are an important biomarker for various types of cancers. As CTCs are extremely rare in the bloodstream, the accurate analysis and efficient separation of CTCs is a ...challenging task. In order to develop new diagnostic approaches for cancer research, understanding the functional characteristics of biomolecules at the single-molecule level in individual cancer cells is required. Meanwhile, microfluidic platforms have emerged as a promising technology for analyzing single molecules and single cells. In this review, we discuss some label-free techniques for biomolecule detection and highlight some reports that have employed combined label-free techniques with microfluidics for individual molecule or cancer cell analysis. Finally, we offer our perspective of the integration of label-free techniques with microfluidic technology on future applications.
•Label-free techniques were utilized for biomolecules detection.•Microfluidic were utilized for analyzing single molecules and single cells.•Analysis of biomolecule and CTCs based on label-free techniques with microfluidic.
•Recent advances towards infectious pathogens diagnostics are introduced.•Integrated microfluidic devices for infectious disease diagnostics are summarized.•Current challenges and future perspectives ...for POC diagnostics are discussed.
Rapid and timely diagnosis of infectious diseases is a critical determinant of clinical outcomes and general public health. For the detection of various pathogens, microfluidics-based platforms offer many advantages, including speed, cost, portability, high throughput, and automation. This review provides an overview of the recent advances in microfluidic technologies for point-of-care (POC) diagnostics for infectious diseases. The key aspects of such technologies for the development of a fully integrated POC platform are introduced, including sample preparation, on-chip nucleic acid analysis and immunoassay, and system integration/automation. The current challenges to practical implementation of this technology are discussed together with future perspectives.
Evanescent-wave optical biosensors have become an attractive alternative for the screening of nucleic acids in the clinical context. They possess highly sensitive transducers able to perform ...detection of a wide range of nucleic acid-based biomarkers without the need of any label or marker. These optical biosensor platforms are very versatile, allowing the incorporation of an almost limitless range of biorecognition probes precisely and robustly adhered to the sensor surface by covalent surface chemistry approaches. In addition, their application can be further enhanced by their combination with different processes, thanks to their integration with complex and automated microfluidic systems, facilitating the development of multiplexed and user-friendly platforms. The objective of this work is to provide a comprehensive synopsis of cutting-edge analytical strategies based on these label-free optical biosensors able to deal with the drawbacks related to DNA and RNA detection, from single point mutations assays and epigenetic alterations, to bacterial infections. Several plasmonic and silicon photonic-based biosensors are described together with their most recent applications in this area. We also identify and analyse the main challenges faced when attempting to harness this technology and how several innovative approaches introduced in the last years manage those issues, including the use of new biorecognition probes, surface functionalization approaches, signal amplification and enhancement strategies, as well as, sophisticated microfluidic solutions.
Herein, a DNA junction scaffold structure was exploited as the only immobilization probe to construct a simple and multifunctional electrochemical sensing platform. The programmable operation of a ...catalytic DNA circuit reaction on it afforded the amplified and well-controllable signal responses for the individual and simultaneous analysis of two nucleic acid targets. Also, it could be flexibly harnessed for each target assay based on multilevel signal response behaviours including single-signal, dual-signal and ratiometric signal responses. Then, its application for the operation of two logic devices of “AND” and “OR” gates toward different target inputs was demonstrated. Interestingly, the “OR” gate could be in-situ evolved from the “AND” status by simply controlling signal output. The low detection limit could reach 10 fM whether for the simultaneous or individual analysis of two targets in buffer or serum. Current sensing platform by only a DNA junction scaffold probe features in facile biosensor fabrication and operation, sensitive and selective analysis of multiplexed targets, well-controllable and multilevel signal response behaviours, and expandable logic operation. It thus may open a promising avenue toward the development of advanced biosensors or logic devices applied for molecular diagnosis or information processing.
•An unique electrochemical DNA junction scaffold-based sensing platform was developed.•A catalytic DNA circuit reaction was programed for the amplified and well-controllable signal response.•It could achieve the simultaneous and individual analysis of two nucleic acids with a high sensitivity and selectivity.•It could be flexibly harnessed for target monitoring by a multilevel signal response mechanism.•Its application for the construction and in-situ transformation from “AND” to “OR” gate was demonstrated.
Nucleic acid analysis using ultrasensitive and simple methods is critically important for the early‐stage diagnosis and treatment of diseases. The CRISPR/Cas proteins, guided by a single‐stranded RNA ...have shown incredible capability for sequence‐specific targeting and detection. Herein, in order to improve and expand the application of CRISPR/Cas technology to the electrochemical interface‐based nucleic acids analysis, the authors develop a CRISPR/Cas12a powered DNA framework‐supported electrochemical biosensing platform via the cis and trans cleavage of Cas12a on the heterogeneous carbon interface (the existing publications which commonly adopted trans‐cleavage). Their solid‐liquid interface is first immobilized by 3D tetrahedral framework nucleic acids (FNAs) with specific DNA recognition probe. Based on the recognition of the complementary target through protospacer adjacent motif (PAM) confirmation and CRISPR‐derived RNA (crRNA) matching, the easily formed Cas12a/crRNA duplex can get access to the interface, and the cis and trans cleavage of Cas12a can be easily activated. In combination with the enzyme catalyzed reaction, they achieved an ultralow limit of detection (LOD) of 100 fm in HPV‐16 detection without pre‐amplification. Furthermore, the platform is compatible with a spike‐in human serum sample and has superior stability. Thus, their reported platform offers a practical, versatile, and amplification‐free toolbox for ultrasensitive nucleic acid analysis.
CRISPR/Cas12a powered DNA framework supported electrochemical biosensing platform via the cis and trans cleavage of Cas12a provide high accessibility of the Cas12a/crRNA and target to the report probe on the heterogeneous interface, meanwhile repelling adsorption of non‐specific report DNA, thus leading to an ultrahigh sensitivity without further signal amplification.
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