Enhanced CRISPR/Cas-Based Immunoassay through Magnetic Proximity Extension and Detection DOI
Fangchi Shao, Jiumei Hu, Pengfei Zhang

и другие.

medRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Сен. 10, 2024

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-associated systems have recently emerged as a focal point for developing next-generation molecular diagnosis, particularly nucleic acid detection. However, the detection of proteins is equally critical across diverse applications in biology, medicine, and food industry, especially diagnosing prognosing diseases like cancer, Alzheimer's cardiovascular conditions. Despite recent efforts to adapt CRISPR/Cas protein with immunoassays, these methods typically achieved sensitivity only femtomolar picomolar range, underscoring need enhanced capabilities. To address this, we developed CRISPR-AMPED, an innovative CRISPR/Cas-based immunoassay by magnetic proximity extension This approach combines assay (PEA) beads that converts into DNA barcodes quantification effective washing steps minimize non-specific binding hybridization, therefore reducing background noise increasing sensitivity. The resulting are then detected through isothermal amplification testing (NAAT) using recombinase polymerase (RPA) coupled CRISPR/Cas12a system, replacing traditional PCR. integration eliminates thermocycling bulky equipment, reduces time, provides simultaneous target signal amplification, thereby significantly boosting CRISPR-AMPED achieves attomolar level sensitivity, surpassing ELISA over three orders magnitude outperforming existing systems. Additionally, our smartphone-based device demonstrates potential point-of-care applications, digital format extends dynamic range enhances quantitation precision. We believe represents significant advancement field

Язык: Английский

Tuning the Dynamic Reaction Balance of CRISPR/Cas12a and RPA in One Pot: A Key to Switch Nucleic Acid Quantification DOI
Zhihao Yao, Kaiyu He, Hongmei Wang

и другие.

ACS Sensors, Год журнала: 2024, Номер 9(7), С. 3511 - 3519

Опубликована: Апрель 23, 2024

Excavating nucleic acid quantitative capabilities by combining clustered regularly interspaced short palindromic repeats (CRISPR) and isothermal amplification in one pot is of common interest. However, the mutual interference between CRISPR cleavage primary obstacle to detection. Though several works have demonstrated enhanced detection sensitivity reducing inhibition on pot, few paid attention process even dynamic reaction processes two. Herein, we find that DNA quantification can be realized regulating either recombinase polymerase (RPA) efficiency or CRISPR/Cas12a cleaving (namely, tuning balance) pot. The sensitive utilizing dual PAM-free crRNAs for recognition. varied RPA primer concentration with stabilized systems significantly affects performances. Alternatively, also achieved stabilizing while concentration. capability proved detecting targets from Lactobacillus acetotolerans SARS-CoV-2. performance toward real samples comparable real-time PCR L. spiked fermented food SARS-CoV-2 clinical samples. We expect presented method will a powerful tool quantifying other targets.

Язык: Английский

Процитировано

15
STAMP-Based Digital CRISPR-Cas13a for Amplification-Free Quantification of HIV-1 Plasma Viral Loads DOI
Reza Nouri, Yuqian Jiang, Anthony J. Politza

и другие.

ACS Nano, Год журнала: 2023, Номер 17(11), С. 10701 - 10712

Опубликована: Май 30, 2023

Quantification of HIV RNA in plasma is critical for identifying the disease progression and monitoring effectiveness antiretroviral therapy. While RT-qPCR has been gold standard viral load quantification, digital assays could provide an alternative calibration-free absolute quantification method. Here, we reported a Self-digitization Through Automated Membrane-based Partitioning (STAMP) method to digitalize CRISPR-Cas13 assay (dCRISPR) amplification-free HIV-1 RNAs. The Cas13 was designed, validated, optimized. We evaluated analytical performances with synthetic With membrane that partitions ∼100 nL reaction mixture (effectively containing 10 input sample), showed samples spanning 4 orders dynamic range between 1 fM (∼6 RNAs) pM (∼60k be quantified as fast 30 min. also examined end-to-end performance from extraction STAMP-dCRISPR using 140 μL both spiked clinical samples. demonstrated device detection limit approximately 2000 copies/mL can resolve change 3571 (equivalent 3 RNAs single membrane) 90% confidence. Finally, 20 patient (10 positives negatives) benchmarked RT-PCR. results agree very well RT-PCR all negative high positive Ct < 32. However, limited detecting low > 32 due subsampling errors. Our platform offer accessible By further addressing issue approaches such preconcentration, this exploited quantitatively determining array infectious diseases.

Язык: Английский

Процитировано

22
MicroRNA Sensors Based on CRISPR/Cas12a Technologies: Evolution From Indirect to Direct Detection DOI
Songcheng Yu,

Xueying Lei,

Chenling Qu

и другие.

Critical Reviews in Analytical Chemistry, Год журнала: 2024, Номер unknown, С. 1 - 17

Опубликована: Март 15, 2024

MicroRNA (miRNA) has emerged as a promising biomarker for disease diagnosis and potential therapeutic targets drug development. The detection of miRNA can serve noninvasive tool in diseases predicting prognosis. CRISPR/Cas12a system great nucleic acid due to its high sensitivity specificity, which been developed be versatile acid-based various fields. However, conversion from RNA DNA with or without amplification operation is necessary based on system, because dsDNA containing PAM sequence ssDNA traditionally considered the activator Cas12a. Until recently, direct by reported. In this review, we provide an overview evolution biosensors indirect direct, would beneficial development CRISPR/Cas12a-based sensors better performance miRNA.

Язык: Английский

Процитировано

8
Detection and absolute quantification biosensing tools for food authentication: CRISPR/Cas, digital CRISPR and beyond DOI
Xiaolin Wu,

Xuanming Lou,

Hanzhang Zhou

и другие.

Trends in Food Science & Technology, Год журнала: 2024, Номер 145, С. 104349 - 104349

Опубликована: Янв. 29, 2024

Язык: Английский

Процитировано

7
DECODE: Contamination-Free Digital CRISPR Platform for Point-of-Care Detection of Viral DNA/RNA DOI
Sheng Li, Haofan Yin, Jiale Zheng

и другие.

ACS Sensors, Год журнала: 2024, Номер 9(8), С. 4256 - 4264

Опубликована: Июль 20, 2024

Rapid and precise nucleic acid testing at the point-of-care (POC) is essential for effective screening management of infectious diseases. Current polymerase-based molecular diagnostics often suffer from potential cross-contamination issues, particularly in POC settings. Here, we introduce DECODE, a contamination-free detection platform integrating digital microfluidics (DMF) extraction CRISPR amplification-free assay pathogen detection. The demonstrates sensitivity, detecting target DNA RNA reaction mixture concentrations 10 5 copies/μL, respectively. Leveraging DMF-extracted samples enhances performance assay. DECODE offers sample-to-result workflow 75 min using compact devices. Validation studies clinical confirm DECODE's robust performance, achieving 100% sensitivity specificity HPV18 cervical epithelial cells influenza A nasal swabs. represents versatile, poised to enhance integrated public health surveillance efforts.

Язык: Английский

Процитировано

7
Next-generation CRISPR-based diagnostic tools for human diseases DOI
Ting Wang, Ziwei Wang,

Linlin Bai

и другие.

TrAC Trends in Analytical Chemistry, Год журнала: 2023, Номер 168, С. 117328 - 117328

Опубликована: Сен. 25, 2023

Язык: Английский

Процитировано

16
Recent advances of nanoparticles-assisted CRISPR/Cas biosensors DOI
Sitong Liu, Xu Li,

Zhaohe Huang

и другие.

Microchemical Journal, Год журнала: 2024, Номер 199, С. 109930 - 109930

Опубликована: Янв. 9, 2024

Язык: Английский

Процитировано

6
CRISPR/Cas-Based Techniques for Live-Cell Imaging and Bioanalysis DOI Open Access
Shuo Huang, Rui Dai,

Zhiqi Zhang

и другие.

International Journal of Molecular Sciences, Год журнала: 2023, Номер 24(17), С. 13447 - 13447

Опубликована: Авг. 30, 2023

CRISPR/Cas systems have found widespread applications in gene editing due to their high accuracy, programmability, ease of use, and affordability. Benefiting from the cleavage properties (trans- or cis-) Cas enzymes, scope has expanded beyond they been utilized various fields, particularly live-cell imaging bioanalysis. In this review, we summarize some fundamental working mechanisms concepts systems, describe recent advances design principles mediated techniques employed bioanalysis, highlight main biosensing a wide range molecular targets, discuss challenges prospects biosensing. By illustrating bio-sensing processes, hope review will guide best use quantifying biological clinical elements inspire new ideas for better tool

Язык: Английский

Процитировано

12
Rapid and portable quantification of HIV RNA via a smartphone-enabled digital CRISPR device and deep learning DOI Creative Commons
Hoan T. Ngo, Patarajarin Akarapipad, Pei‐Wei Lee

и другие.

Sensors and Actuators Reports, Год журнала: 2024, Номер 8, С. 100212 - 100212

Опубликована: Июнь 20, 2024

For the 29.8 million people in world living with HIV/AIDS and receiving antiretroviral therapy, it is crucial to monitor their HIV viral loads. To this end, rapid portable diagnostic tools that can quantify RNA are critically needed. We report herein a quantitative digital CRISPR-assisted detection assay has been implemented within smartphone-based device as potential solution. Specifically, we first developed fluorescence-based reverse transcription recombinase polymerase amplification (RT-RPA)-CRISPR efficiently detect at 42°C. then commercial stamp-sized chip, where molecules were quantified strongly fluorescent reaction wells. The isothermal condition strong fluorescence chip simplified design of thermal optical modules, allowing us engineer palm-size measuring 70 × 115 80 mm weighing less than 0.6 kg. also capitalized smartphone by developing custom app control device, perform assay, capture images throughout assay. Moreover, trained verified deep learning-based algorithm for analyzing identifying positive wells high accuracy. Using our smartphone-enabled CRISPR successfully detected low 75 copies just 15 min, showing its toward monitoring loads aiding global effort combat epidemic.

Язык: Английский

Процитировано

5
CRISPR-Based Strategies for Sample-to-Answer Monkeypox Detection: Current Status and Emerging Opportunities DOI Creative Commons
Md. Ahasan Ahamed, Anthony J. Politza, Tianyi Liu

и другие.

Nanotechnology, Год журнала: 2024, Номер 36(4), С. 042001 - 042001

Опубликована: Окт. 21, 2024

Abstract The global health threat posed by the Monkeypox virus (Mpox) requires swift, simple, and accurate detection methods for effective management, emphasizing growing necessity decentralized point-of-care (POC) diagnostic solutions. clustered regularly interspaced short palindromic repeats (CRISPR), initially known its nucleic acid abilities, presents itself as an attractive strategy. CRISPR offers exceptional sensitivity, single-base specificity, programmability. Here, we reviewed latest developments in CRISPR-based POC devices testing strategies Mpox detection. We explored crucial role of genetic sequencing designing crRNA reaction understanding transmission mutations. Additionally, showed integration CRISPR-Cas12 strategy with pre-amplification amplification-free methods. Our study also focused on significant Cas12 proteins effectiveness coupled recombinase polymerase amplification (RPA) envision future prospects challenges, positioning CRISPR-Cas12-based a frontrunner next generation molecular biosensing technologies.

Язык: Английский

Процитировано

5