Detection of small-sized DNA fragments in a glassy nanopore by utilization of CRISPR-Cas12a as a converter system

Zhang, Shumin; Liu, Minyi; Cui, Haofa; Ziaee, Muhammad Asad; Sun, Rongwei; Chen, Liting; Chen, Daqi; Garoli, Denis; Wang, Jiahai

doi:10.1039/d1an02313f

Cited by 19 publications

(22 citation statements)

References 66 publications

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“…61 Very recently, Zhang et al also proposed a Gnanopore sensing platform for detection of small DNA fragments by the combined use of a DNA tetrahedron as a signal sensor and CRISPR-Cas12a as a converter system, with the l1 gene encoding HPV18 (a type of human papillomavirus, belonging to the high-risk group) as the target DNA molecule (concentration range: 0.5−50 nM) for the detection of cervical cancer cases (Figure 5e). 80 The G-nanopore at the tip of the glass capillaries is the only connection between the internal and external electrolyte solution. Under a bias potential, ionic current blockage changes caused by single molecule translocation can be detected in the sensing region.…”

Section: Molecule Detection Of Tumorsmentioning

confidence: 99%

Glass Capillary-Based Nanopores for Single Molecule/Single Cell Detection

Guan

Chen

et al. 2023

ACS Sens.

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A glass capillary-based nanopore (G-nanopore), due to its tapered tip, easy tunability in orifice size, and especially its flexible surface modifications that can be tailored to effectively capture and enhance the ionic current signal of single entities (single molecules, single cells, and single particles), offers a powerful and nanoconfined sensing platform for diverse biological measurements of single cells and single molecules. Compared with other artificial two-dimensional solid-state nanopores, its conical tip and high spatial and temporal resolution characteristics facilitate noninvasive single molecule and selected area (subcellular) single cell detections (e.g., DNA mutations, highly expressed proteins, and small molecule markers that reflect the change characteristics of the tumor), as a small G-nanopore (≤100 nm) does negligible damage to cell functions and cell membrane integrity when inserted through the cell membrane. In this brief review, we summarize the preparation of G-nanopores and discuss the advantages of them as solid-state sensing platforms for single molecule and single cell detection applications as well as for cancer diagnosis and treatment applications. We also describe the current bottlenecks that limit the widespread use of G-nanopores in clinical applications and provide an outlook on future developments. The brief review will provide the reader with a quick survey of this field and facilitate the rapid development of a Gnanopore sensing platform for future tumor diagnosis and personalized medicine based on single-molecule/single-cell bioassay.

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Section: Molecule Detection Of Tumorsmentioning

confidence: 99%

Glass Capillary-Based Nanopores for Single Molecule/Single Cell Detection

Guan

Chen

et al. 2023

ACS Sens.

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“…Previous studies have shown that if BSA was present in the reaction buffer, the noise level of a glass nanopore would severely deteriorate due to the strong adsorption of BSA on the surface of the glass nanopore. 16,17 Unfortunately, for many kinds of enzyme-involved commercial reaction buffers, such as the commercial Cas12a reaction buffer and PCR reaction buffer applied in this experiment, BSA is an essential component that can help to improve the stability of the enzyme during the reaction. Previous works chose to seek an alternative reaction buffer, carry out extra purification steps or apply BSA-free reactions to design sensing strategies.…”

Section: Optimization Of the Signal Transducermentioning

confidence: 99%

“…15 (2) The widespread presence of protein interferents such as bovine serum albumin (BSA) and recombinant albumin in the commercial reaction buffer of enzyme-involved biochemical reactions that are often used to establish sensing or signal amplification strategies will greatly deteriorate the noise level of glass nanopores, drowning the translocation signals that should be generated by the analyte, leading to an experiment failure. 16,17 Valuable works have been done to overcome these two challenges. For the first challenge, researchers have tried to use signal transducers or amplification methods to increase the size of the analyte, enhancing the interaction between target molecules and the glass nanopore to produce better translocation signals.…”

Section: Introductionmentioning

confidence: 99%

“…However, the signal transducer or amplification method they selected was still not good enough owing to the unevenness in size and the relatively low SNR translocation signal, which increased the difficulty of subsequent analyses. For the second challenge, although several attempts have been tried, including optimizing the buffer component to replace BSA, 16,17,19,20 introducing purification steps to remove BSA, 21 or designing sensing strategies with BSA-free methods, such as hybridization chain reaction (HCR) 22,23 or loop-mediated isothermal amplification (LAMP), 24,25 they are still not good enough to overcome the second challenge because these methods consume additional time to make changes to commercial reaction buffers only especially for nanopore applications, require additional experimental steps and cost, and can only be compatible with limited biochemical reactions. These defects hamper the practical use of glass nanopores, which greatly limits their application.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A signal on–off strategy based on the digestion of DNA cubes assisted by the CRISPR–Cas12a system for ultrasensitive HBV detection in solid-state nanopores

Wang

Luo

et al. 2022

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Nucleic acid nanostructure for delivery of CRISPR/Cas9‐based gene editing system

Tang

Liu

Ding

2022

Interdisciplinary Medicine

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CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9)-based gene editing system has aroused great interest in many research fields. However, the efficient and safe delivery of this gene editing system into the target tissues and cells remain a major challenge.During the past decades, nucleic acid nanostructures have been widely developed for drug delivery. In this perspective, we will introduce and discuss the recent progress in the design of multifunctional nucleic acid nanostructures, including RCA-derived DNA, branched DNA, and hybrid DNA, for delivery of the CRISPR/Cas9-based gene editing system. Furthermore, we prospect the challenges and future opportunities of nucleic acid nanotechnology in the delivery of gene editing systems. K E Y W O R D SCRISPR/Cas9, drug delivery, gene editing, gene therapy, nucleic acid nanostructure 1 | INTRODUCTION CRISPR/Cas9 system is an adaptive immune system of bacteria and archaea, which plays a significant role in preventing foreign nucleic acids. 1,2 When foreign viral or plasmid DNA enters the cell, the specialized Cas proteins snip the foreign DNA into small pieces and paste them into contiguous stretches of DNA, called the CRISPR array. Then, CRISPR RNAs (crRNAs) encoded by the CRISPR array are obtained and maturated with the assistance of trans-activating crRNA (tracrRNA), ribonuclease III, and Cas9. The tracrRNA is hybridized with crRNA to form a two-RNA structure as guide RNA (gRNA), which directs the Cas9 to foreign DNA based on sequence specificity. Finally, the gRNA/Cas9 complex is responsible for the cleavage of target DNA to achieve DNA double-strand breaks (DSBs) to interfere with foreign invaders. 3,4 Inspired by the CRISPR/Cas9 system in bacteria,This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Detection of small-sized DNA fragments in a glassy nanopore by utilization of CRISPR-Cas12a as a converter system

Abstract: The fabrication of nanopore with matched pore size, and the existence of multi interferents make the reproducible detection of small-sized molecules by means of solid-state nanopore still challenging. A useful...

Cited by 19 publications

References 66 publications

Glass Capillary-Based Nanopores for Single Molecule/Single Cell Detection

Glass Capillary-Based Nanopores for Single Molecule/Single Cell Detection

A signal on–off strategy based on the digestion of DNA cubes assisted by the CRISPR–Cas12a system for ultrasensitive HBV detection in solid-state nanopores

Nucleic acid nanostructure for delivery of CRISPR/Cas9‐based gene editing system

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