Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Ноя. 18, 2023

Abstract Nucleic acid detection powered by CRISPR technology provides a rapid, sensitive, and deployable approach to molecular diagnostics. While exciting, there remain challenges limiting its practical applications, such as the need for pre-amplification lack of quantitative ability. Here, we develop an asymmetric assay cascade signal amplification nucleic acids leveraging trans -cleavage behavior competitive crRNA. We discover that reaction between full-sized crRNA split CRISPR-Cas12a can induce amplification, significantly improving target signal. In addition, find recognize fragmented RNA/DNA targets, enabling direct RNA Cas12a. Based on these findings, apply our quantitatively detect microRNA without pre-amplification, achieving sensitivity 856 aM. Moreover, using this method, analyze quantify miR-19a biomarker in plasma samples from bladder cancer patients. This has potential be widely applied simple sensitive various diagnostic settings.

Язык: Английский

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CRISPR/Cas13a‐Based MicroRNA Detection in Tumor‐Derived Extracellular Vesicles

Advanced Science, Год журнала: 2023, Номер 10(24)

Опубликована: Июнь 20, 2023

MicroRNAs (miRNAs) in extracellular vesicles (EVs) play essential roles cancer initiation and progression. Quantitative measurements of EV miRNAs are critical for diagnosis longitudinal monitoring. Traditional PCR-based methods, however, require multi-step procedures remain as bulk analysis. Here, the authors introduce an amplification-free extraction-free miRNA detection method using a CRISPR/Cas13a sensing system. components encapsulated liposomes delivered them into EVs through liposome-EV fusion. This allows accurately quantify specific miRNA-positive counts 1 × 108 EVs. The show that miR-21-5p-positive range 2%-10% ovarian EVs, which is significantly higher than positive from benign cells (<0.65%). result excellent correlation between analysis with gold-standard method, RT-qPCR. also demonstrate multiplexed protein-miRNA tumor-derived by capturing EpCAM-positive quantifying ones subpopulation, plasma patients healthy controls. developed system provides intact without RNA extraction opens up possibility single protein markers.

Язык: Английский

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Split crRNA with CRISPR-Cas12a enabling highly sensitive and multiplexed detection of RNA and DNA

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Сен. 27, 2024

Язык: Английский

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Programmable Entropy-Driven Circuit-Cascaded Self-Feedback DNAzyme Network for Ultra-Sensitive Fluorescence and Photoelectrochemical Dual-Mode Biosensing

Analytical Chemistry, Год журнала: 2024, Номер 96(18), С. 7274 - 7280

Опубликована: Апрель 24, 2024

Inspired by natural DNA networks, programmable artificial networks have become an attractive tool for developing high-performance biosensors. However, there is still a lot of room expansion in terms sensitivity, atom economy, and result self-validation current microRNA sensors. In this protocol, miRNA-122 as target model, ultrasensitive fluorescence (FL) photoelectrochemical (PEC) dual-mode biosensing platform developed using entropy-driven circuit (EDC) cascaded self-feedback DNAzyme network. The well-designed EDC realizes full utilization the strands improves atomic economy signal amplification system. unique rational design double-CdSe quantum-dot-released substrate network significantly avoids high background signals enhances sensitivity specificity. Also, enzyme-free, effectively risk leakage accuracy sensor. Moreover, introduction superparamagnetic Fe

Язык: Английский

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Universal crRNA Acylation Strategy for Robust Photo‐Initiated One‐Pot CRISPR–Cas12a Nucleic Acid Diagnostics

Angewandte Chemie International Edition, Год журнала: 2024, Номер 63(23)

Опубликована: Апрель 2, 2024

Abstract Spatiotemporal regulation of clustered regularly interspaced short palindromic repeats (CRISPR) system is attractive for precise gene editing and accurate molecular diagnosis. Although many efforts have been made, versatile efficient strategies to control CRISPR are still desirable. Here, we proposed a universal accessible acylation strategy regulate the CRISPR–Cas12a by 2′‐hydroxyls (2′‐OH) on crRNA strand with photolabile agents (PLGs). The introduction PLGs confers suppression function rapid restoration reaction upon light exposure regardless sequences. Based this strategy, constructed PhotO‐Initiated Robust One‐pot Testing (POIROT) platform integrated recombinase polymerase amplification (RPA), which showed two orders magnitude more sensitive than conventional one‐step assay comparable two‐step assay. For clinical sample testing, POIROT achieved high‐efficiency detection performance gold‐standard quantitative PCR (qPCR) in sensitivity specificity, but faster qPCR method. Overall, believe will promote development other photo‐controlled technologies one‐pot assay, even expand applications fields controllable CRISPR‐based genomic editing, disease therapy, cell imaging.

Язык: Английский

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Catapult-like DNA actuator for highly stable and ultrasensitive detection of urinary miRNA

Chemical Engineering Journal, Год журнала: 2025, Номер unknown, С. 159806 - 159806

Опубликована: Янв. 1, 2025

Язык: Английский

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Double CRISPR/Cas12a-drived hyperbranched rolling circle amplification with triple signal amplification enables low background miRNA detection

Sensors and Actuators B Chemical, Год журнала: 2024, Номер 408, С. 135490 - 135490

Опубликована: Фев. 13, 2024

Язык: Английский

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Weakly ionized gold nanoparticles amplify immunoassays for ultrasensitive point-of-care sensors

Science Advances, Год журнала: 2024, Номер 10(28)

Опубликована: Июль 10, 2024

Gold nanoparticle-based lateral flow immunoassays (AuNP LFIAs) are widely used point-of-care (POC) sensors for in vitro diagnostics. However, the sensitivity limitation of conventional AuNP LFIAs impedes detection trace biomarkers. Several studies have explored size and shape factors AuNPs derivative nanohybrids, showing limited improvements or enhanced at cost convenience affordability. Here, we investigated surface chemistry on LFIAs. By modifying ligands, a strategy involving weakly ionized enables ultrasensitive naked-eye (~100-fold sensitivity). We demonstrated how this chemistry-amplified immunoassay approach modulates nanointerfacial bindings to promote antibody adsorption higher activity adsorbed antibodies. This design eliminates complex nanosynthesis, auxiliary devices, additional reagents while efficiently improving with advantages: simplified fabrication process, excellent reproducibility reliability, ultrasensitivity toward various The using represents versatile sensitizing POC sensors.

Язык: Английский

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Biporous silica nanostructure-induced nanovortex in microfluidics for nucleic acid enrichment, isolation, and PCR-free detection

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Фев. 14, 2024

Abstract Efficient pathogen enrichment and nucleic acid isolation are critical for accurate sensitive diagnosis of infectious diseases, especially those with low levels. Our study introduces a biporous silica nanofilms-embedded sample preparation chip enrichment/isolation. This features unique nanostructures comprising large small pore layers. Computational simulations confirm that these enhance the surface area promote formation nanovortex, resulting in improved capture efficiency. Notably, demonstrates 100-fold lower limit detection compared to conventional methods used detection. Clinical validations using patient samples corroborate superior sensitivity when combined luminescence resonance energy transfer assay. The enhanced efficiency chip, along facile straightforward synthesis nanostructures, offers promising solution polymer chain reaction-free acids.

Язык: Английский

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Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA

Analytica Chimica Acta, Год журнала: 2024, Номер 1303, С. 342477 - 342477

Опубликована: Март 22, 2024

Язык: Английский

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