FARPA-based tube-array coupled with quick DNA extraction enables ultra-fast bedside-detection of antibiotic-resistant pathogens DOI
Jinling Huang,

Huijie Yue,

Wei Wei

et al.

The Analyst, Journal Year: 2024, Volume and Issue: 149(13), P. 3607 - 3614

Published: Jan. 1, 2024

Rapid and accurate detection of pathogens antimicrobial-resistant (AMR) genes the are crucial for clinical diagnosis effective treatment infectious diseases. However, time-consuming steps conventional culture-based methods inhibit precise early application anti-infection therapy. For prompt pathogen-infected patients, we have proposed a novel tube array strategy based on our previously reported FARPA (FEN1-aided recombinase polymerase amplification) principle ultra-fast antibiotic-resistant site. The entire process from "sample to result" can be completed in 25 min by combining quick DNA extraction urine sample with avoid usually complicated step. Furthermore, 36-tube made commercial 384-well titre plates was efficiently introduced perform portable analyser, achieving an increase loading throughput (from several tens), which is quite suitable point-of-care testing (POCT) multiple samples. Finally, tested 92 samples verify performance method. sensitivities

Language: Английский

PAM‐Engineered Toehold Switches as Input‐Responsive Activators of CRISPR‐Cas12a for Sensing Applications DOI
Neda Bagheri, Alejandro Chamorro-García, Andrea Idili

et al.

Angewandte Chemie International Edition, Journal Year: 2024, Volume and Issue: 63(17)

Published: Jan. 29, 2024

Abstract The RNA‐programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio‐engineering strategies are required to achieve control over activity. Here, we show that Toehold Switch DNA hairpins, presenting rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used response inputs. Reconfiguring from hairpin duplex conformation through strand displacement reaction provides an effective means modulate accessibility of PAM, thereby controlling binding cleavage activities Cas12a. Through this approach, showcase potential trigger downstream activity by leveraging proximity‐based reactions target binding. By utilizing trans ‐cleavage signal transduction method, demonstrate versatility our approach sensing applications. Our system enables rapid, one‐pot detection IgG antibodies small molecules with high sensitivity specificity even within complex matrices. Besides bioanalytical applications, switchable PAM‐engineered Switches serve programmable tools capable regulating Cas12a‐based targeting processing inputs hold promise wide array biotechnological

Language: Английский

Citations

12
Combination of nucleic acid amplification and CRISPR/Cas technology in pathogen detection DOI Creative Commons
Dandan Zeng, Jinlong Jiao,

Tianlu Mo

et al.

Frontiers in Microbiology, Journal Year: 2024, Volume and Issue: 15

Published: Feb. 6, 2024

Major health events caused by pathogenic microorganisms are increasing, seriously jeopardizing human lives. Currently PCR and ITA widely used for rapid testing in food, medicine, industry agriculture. However, due to the non-specificity of amplification process, researchers have proposed combination nucleic acid technology with novel CRISPR detection, which improves specificity credibility results. This paper summarizes research progress conjunction CRISPR/Cas detection pathogens, provides a reference theoretical basis subsequent application field pathogen detection.

Language: Английский

Citations

9
Facile and Ultrafast Microfluidic Photothermal PCR for Autonomous and Quantitative Point‐of‐Care Pathogen Detection DOI Open Access
Yuan Zou,

Mingxu Liu,

Zixi Gao

et al.

Small, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 13, 2025

Abstract Point‐of‐care (POC) pathogen detection is highly desirable in diverse fields such as infectious disease diagnosis, food safety testing, and environmental monitoring. Herein, the study seeks to address this critical need by developing an automated microfluidic photothermal quantitative polymerase chain reaction (AMP‐qPCR) system a greatly simplified format. A key element of AMP‐qPCR architecture that combines design clockwork‐like, magnetically‐driven multi‐chamber cartridge with use cheap black tape beneath PCR chamber fast photothermal‐responsive engine. This not only enables unprocessed sample be lysed, purified, subjected real‐time fluorescence ultracompact autonomous manner but also eliminates for sophisticated photonic material/device fabrication frequently required performing ultrafast PCR. It shown can accomplish high‐efficient bacterial DNA extraction within 18.5 min, enabling accurate quantification bacteria concentration from 10 8 2 CFU·mL −1 . Furthermore, its practical applicability demonstrated detecting Neisseria gonorrhoeae sexually transmitted infection‐suspected patients using clinical urine cervical swab specimens, exhibiting matched performance benchtop machine. The presented platform enhances availability POC molecular diagnostics on‐site in‐home testing.

Language: Английский

Citations

1
PAM‐Engineered Toehold Switches as Input‐Responsive Activators of CRISPR‐Cas12a for Sensing Applications DOI
Neda Bagheri, Alejandro Chamorro-García, Andrea Idili

et al.

Angewandte Chemie, Journal Year: 2024, Volume and Issue: 136(17)

Published: Jan. 29, 2024

Abstract The RNA‐programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio‐engineering strategies are required to achieve control over activity. Here, we show that Toehold Switch DNA hairpins, presenting rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used response inputs. Reconfiguring from hairpin duplex conformation through strand displacement reaction provides an effective means modulate accessibility of PAM, thereby controlling binding cleavage activities Cas12a. Through this approach, showcase potential trigger downstream activity by leveraging proximity‐based reactions target binding. By utilizing trans ‐cleavage signal transduction method, demonstrate versatility our approach sensing applications. Our system enables rapid, one‐pot detection IgG antibodies small molecules with high sensitivity specificity even within complex matrices. Besides bioanalytical applications, switchable PAM‐engineered Switches serve programmable tools capable regulating Cas12a‐based targeting processing inputs hold promise wide array biotechnological

Language: Английский

Citations

8
Temperature-programmed microfluidic CRISPR diagnostics enable rapid and automatous point-of-care testing for syphilis DOI
Bowen Shu,

Jianjiang Yang,

Wentao Chen

et al.

Chemical Engineering Journal, Journal Year: 2024, Volume and Issue: 496, P. 154174 - 154174

Published: July 21, 2024

Language: Английский

Citations

8
Next-generation CRISPR-based diagnostic tools for human diseases DOI
Ting Wang, Ziwei Wang,

Linlin Bai

et al.

TrAC Trends in Analytical Chemistry, Journal Year: 2023, Volume and Issue: 168, P. 117328 - 117328

Published: Sept. 25, 2023

Language: Английский

Citations

16
A Semisynthetic Bioluminescence Sensor for Ratiometric Imaging of Metal Ions In Vivo Using DNAzymes Conjugated to An Engineered Nano‐Luciferase DOI
Mengyi Xiong, Yuting Wu,

Gezhi Kong

et al.

Angewandte Chemie International Edition, Journal Year: 2023, Volume and Issue: 62(37)

Published: Aug. 7, 2023

DNA-based probes have gained significant attention as versatile tools for biochemical analysis, benefiting from their programmability and biocompatibility. However, most existing rely on fluorescence the signal output, which can be problematic due to issues like autofluorescence scattering when applied in complex biological materials such living cells or tissues. Herein, we report development of bioluminescent nucleic acid (bioLUNA) sensors that offer laser excitation-independent ratiometric imaging target vivo. The system is based computational modelling mutagenesis investigations a genetic fusion between circular permutated Nano-luciferase (NLuc) HaloTag, enabling conjugation protein with DNAzyme. In presence Zn

Language: Английский

Citations

14
CRISPR-Cas based diagnostic tools: Bringing diagnosis out of labs DOI
Abu Sufiyan Chhipa,

Ekta Radadiya,

Snehal S. Patel

et al.

Diagnostic Microbiology and Infectious Disease, Journal Year: 2024, Volume and Issue: 109(2), P. 116252 - 116252

Published: March 8, 2024

Language: Английский

Citations

4
Development of Luminescent Biosensors for Calprotectin DOI
Tong Lan, Tomasz Ślężak, Jinyue Pu

et al.

ACS Chemical Biology, Journal Year: 2024, Volume and Issue: 19(6), P. 1250 - 1259

Published: June 6, 2024

Calprotectin, a metal ion-binding protein complex, plays crucial role in the innate immune system and has gained prominence as biomarker for various intestinal systemic inflammatory infectious diseases, including bowel disease (IBD) tuberculosis (TB). Current clinical testing methods rely on enzyme-linked immunosorbent assays (ELISAs), limiting accessibility convenience. In this study, we introduce Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), novel quantitative method calprotectin measurement. FESCA utilizes two new fragment antigen binding proteins (Fabs), CP16 CP17, that bind to different epitopes of complex. These Fabs are fused with split NanoLuc luciferase fragments, enabling reconstitution active upon either solution or varied immobilized assay formats. FESCA's output luminescence can be measured standard laboratory equipment well consumer-grade cell phone cameras. detect physiologically relevant levels across sample types, serum, plasma, whole blood. Notably, abnormally elevated native from TB patients. summary, presents convenient, low-cost, assessing biological samples, potential improve diagnosis monitoring especially at-home point-of-care settings.

Language: Английский

Citations

4
Engineered CRISPR/Cas Ribonucleoproteins for Enhanced Biosensing and Bioimaging DOI

Linxin Cao,

Zeyuan Wang, Chunyang Lei

et al.

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 11, 2025

CRISPR-Cas systems represent a highly programmable and precise nucleic acid-targeting platform, which has been strategically engineered as versatile toolkit for biosensing bioimaging applications. Nevertheless, their analytical performance is constrained by inherent functional activity limitations of natural CRISPR/Cas systems, underscoring the critical role molecular engineering in enhancing capabilities. This review comprehensively examines recent advancements ribonucleoproteins (RNPs) to enhance capabilities advanced detection cellular imaging. We explore innovative strategies developing enhanced RNPs, including Cas protein through mutagenesis fusion techniques, guide RNA via chemical structural modifications. Furthermore, we evaluate these RNPs' applications sensitive biomarker live-cell genomic DNA monitoring, while analyzing current challenges prospective developments RNP bioimaging.

Language: Английский

Citations

0