Dual-Engine Powered Paper Photoelectrochemical Platform Based on 3D DNA Nanomachine-Mediated CRISPR/Cas12a for Detection of Multiple miRNAs

Abstract

This work proposed a novel double-engine powered paper photoelectrochemical (PEC) biosensor based on an anode–cathode cooperative amplification strategy and various signal enhancement mechanisms, which realized the monitoring of multiple miRNAs (such as miRNA-141 and miRNA-21). Specifically, C3N4 quantum dots (QDs) sensitized ZnO nanostars and BiOI nanospheres simultaneously to construct a composite photoelectric layer that amplified the original photocurrent of the photoanode and photocathode, respectively. Through the independent design and partition of a flexible paper chip to functionalize injection holes and electrode areas, the bipolar combination completed the secondary upgrade of signals, which also provided biological reaction sites for multitarget detection. With the synergistic participation of a three-dimensional (3D) DNA nanomachine and programmable CRISPR/Cas12a shearing tool, C3N4 QDs lost their attachment away from the electrode surface to quench the signal. Moreover, electrode zoning significantly reduced the spatial cross talk of related substances for multitarget detection, while the universal trans-cleavage capability of CRISPR/Cas12a simplified the operation. The designed PEC biosensor revealed excellent linear ranges for detection of miRNA-141 and miRNA-21, for which the detection limits were 5.5 and 3.4 fM, respectively. With prominent selectivity and sensitivity, the platform established an effective approach for trace multitarget monitoring in clinical applications, and its numerous pioneering attempts owned favorable reference values.

Ultrasensitive Electrochemiluminescence Biosensor Based on 2D Co3O4 Nanosheets as a Coreaction Accelerator and Highly Ordered Rolling DNA Nanomachine as a Signal Amplifier for the Detection of MicroRNA

“…Inversely, an obvious increase of ECL signal was obtained with MCF-7 cell numbers increasing from 10 1 to 10 5 , indicating miRNA-21 exhibited high expression in MCF-7 cells. The above results confirmed the expression level of miRNA-21 was higher in MCF-7 cells than in HeLa cells, which was consistent with reported works. , The real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was also supplemented to confirm the above finding (Figure S9). In addition, comparative experiments were performed to demonstrate that the cell lysis buffer had no effect on the biosensor (Figure S10).…”

Chemiluminescence Sensor for miRNA-21 Detection Based on CRISPR-Cas12a and Cation Exchange Reaction

“…MiRNA-21, highly expressed in most malignant tumors, including lung cancer, breast cancer, and cervical carcinomas, − is one of the earliest miRNAs to be identified and widely studied . A variety of methods are currently available for the determination of miRNA-21, such as fluorescence, electrochemistry, photoelectrochemical (PEC), electrochemiluminescence (ECL), colorimetry, chemiluminescence (CL), Raman detection, and single molecule detection. − Among them, chemiluminescence has been widely used in quantitative analysis due to its characteristics of no light source and no filter, high sensitivity, wide dynamic linear range and ease of operation . Luminol-H 2 O 2 , as a classical CL system, is used extensively in the field of the biological community.…”

Ultrasensitive CRISPR/Cas13a-Mediated Photoelectrochemical Biosensors for Specific and Direct Assay of miRNA-21

“…In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems containing CRISPR/Cas9, Cas12, Cas13, and Cas14 have been widely applied to biomedical research and clinical diagnostics. − Among these systems, Cas13a (previously known as C2c2) or Cas12a, as an RNA-guided ribonuclease, can be programmed with CRISPR RNAs (crRNAs) to nonspecifically cleave (termed trans -cleavage) nearby RNAs or DNAs, respectively, in the presence of its target RNA or DNA. − On average, this trans -cleavage activity activated by a single target nucleic acid can cleave numerous nonspecific nucleic acid strands at physiological temperature, resulting in signal amplification. − More importantly, compared to the previously reported signal amplification strategies including HCR, catalytic hairpin assembly, and DNA nanomachine, the CRISPR/Cas system can greatly simplify the operation difficulty and significantly improve the analytical performance of the biosensing platform without the need for complex nucleic acid sequence design and excessive experimental manipulations. Given these advantages, several CRISPR/Cas12a-assisted PEC biosensors have been developed for the sensitive detection of miRNAs. − Moreover, to further achieve a desired detection sensitivity, the CRISPR/Cas12a system was commonly integrated with nucleic acid pre-amplification for miRNA detection, such as polymerase chain reaction, rolling circle amplification, loop-mediated isothermal amplification, recombinase polymerase amplification, and so forth. − However, the integration strategies of the pre-amplification step and CRISPR assay are carried out in steps and usually involve the transfer of amplified products. It not only increases the detection time and operational complexity but also easily causes aerosol contamination.…”