iScience
·
2024
·
IF 4.6
Xuan Wu,Rong Xiang,Danting Yang,Xiaoyao He,Lu Zhu,Fangfang Sun,Hanqing Li,Ningning Pi,Yi Li
Global pandemic has emphasized the needs for advanced pathogen diagnosis in dealing with newly emerged infectious threats, including the Langya henipavirus (LayV). LayV, as an emerging zoonotic pathogen, has potential to cause pandemic, but lacks of rapid diagnostic tools, particularly at point-of-care level. Here, we leveraged the merits of CRISPR-Cas12a biosensing and established a highly sensitive LayV detection method. By integrating CRISPR-Cas12a with RPA, 10 copies/μL ultra-sensitive LayV RNA detection has been achieved at room temperature within 30 min. More importantly, this study developed a special horseradish peroxidase (HRP)-single-stranded DNA (ssDNA) reporter enabling CRISPR-Cas12a to detect LayV RNA without pre-amplification, achieving a sensitivity of 1,200 copies/μL detectable by the naked eye. These explorations can serve as accelerator for CRISPR-Cas biosensing toward rapid response for newly emerged biological threats, and also provide a method to realize simple, precise, and amplicon-free point-of-care pathogen screening for resource limited or underdevelopment regions.
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高影响因子
ANALYST
·
2024
·
IF 3.6
Chengchen Tan,Xiaolong Yan,Xingchang Lu,Jianxiu Wang,Xinyao Yi
Breast cancer, as a malignant tumor with the highest incidence in the world, poses a great threat to human life and health. Early diagnosis of breast cancer may help enhancing the survival rate. In this work, a colorimetric and fluorescent dual-mode biosensor based on CRISPR-Cas12a system was constructed for the detection of breast cancer biomarker BRCA1. The intact G4 DNA could form DNAzyme in the assistance of K+ and hemin, catalyzed the oxidation of o-phenylenediamine (OPD) under the assistance of hydrogen peroxide (H2O2), and generate the oxidation product 2,3-diaminophenazine (DAP) with obvious absorption and fluorescence peaks. The presence of the target BRCA1 can activate the trans-cleavage activity of CRISPR-Cas12a, cleaving the G4 DNA and inhibiting the catalytic oxidation of OPD. The target BRCA1 was quantitatively determined by detecting both the absorbance and fluorescence intensity of DAP. Compared with single signal analysis, dual-signal output mode provided more reliable detection results. The detection limits were calculated to be 0.615 nM for colorimetric method and 0.289 nM for fluorescence method. The dual-mode biosensor showed good selectivity for BRCA1 and can resist the interference of complex substrates, which has great potential in biomedical detection.
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TALANTA
·
2024
·
IF 5.6
Hongmin Gao,Hehua Zhang,Xue Qi,Meng Miao,Longbin Que,Xin Gu,Dong Chang,Hongzhi Pan
Accurate and reliable detection of Staphylococcus aureus ( S. aureus ) is essential for preventing infections, particularly in healthcare and food safety contexts. This work presents a novel dual-mode biosensor that integrates the CRISPR/Cas12a system with an enzyme-free isothermal amplification method for detecting S. aureus . Hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) amplify the aptamer-triggered response, significantly enhancing sensitivity. CRISPR/Cas12a′s nuclease activity is utilized in two modes: cis cleavage generates a fluorescence signal, while trans cleavage produces an electrochemical signal, enabling dual-mode detection. The biosensor demonstrates outstanding performance, with a limit of detection (LOD) as low as 5.7 CFU mL −1 in electrochemical mode and 133.7 CFU mL −1 in fluorescence mode, showcasing excellent accuracy, stability, and sensitivity. It has been successfully applied to detecting actual samples, confirming its practical applicability. This innovative approach offers a powerful tool for the swift and precise identification of S. aureus and paves the way for developing next-generation dual-mode biosensors for various analytes. Future research will aim to simplify the detection process further, making it more accessible for use in resource-limited settings.
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Songkuan Zhuang,Xiaojun Huang,Muyan Diao,Ruiling Dong,Yueyu Dong,Jun Liu,Yizhen Liu
Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.
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ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Hong Zhang,Xuekun Bai,Chenxi Wang,Wei Pang,Jiaxin Zhang,Junhuai Song,Huanying Zhou,Zhixian Gao,Bin Zheng
Background Biorhythmic played an important role in physical health. Biorhythmic disorders led to various health issue, including cardiovascular diseases and metabolic disorders. Early diagnosis of biorhythmic disorders was crucial in slowing the progression of disease. Therefore, it was important to develop technologies for detecting biorhythmic and their disorder markers. This study selected HMGB1(High mobility group box 1), BMAL1 (Brain and Muscle Aryl hydrocarbon receptor nuclear translocator-like 1), and MICU (mitochondrial calcium uptake 1) mRNA for detection. Results CRISPR/Cas12a/SERS (surface-enhanced Raman spectroscopy) ultrasensitive biosensing platform was developed for the detection of biorhythmic markers. The biosensor was partly an ultrabright Raman signal reporter (on) of a novel satellite structure formed by gold/silver bimetallic nanoparticles. This novel satellite structure utilizes the potential difference between gold and silver nanoparticles to promote the transfer of electrons from the core structure to the AuNPs and MBA (4-mercaptobenzoic acid) molecules, while enhancing the electromagnetic field distribution. Compared with the Raman signal of the satellite structure composed of Au/Au bimetallic, the Raman signal of the satellite structure in this study is increased by about 10 times. CRISPR/Cas12a recognized the target molecule and activated trans -cleavage activity, cleaving the DNA/RNA hairpin structure and releasing RNA 2∗ . RNA 2∗ caused the satellite structure to disperse, significantly attenuating the ultra-strong Raman signal (off), thereby enabling quantitative detection of the target nucleic acid. The LOD for three targets were of less than 1 fM. Significance In summary, the CRISPR/Cas12a/SERS biosensing platform enables mRNA detection without preamplification. This biosensing platform can detect target molecules at the fM level, achieving ultra-sensitive detection. In addition, the satellite structure exhibits long-term stability, providing a theoretical basis for the practical application of this biosensing platform.
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BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Yining Zhao,Ziye Liu,Ziting Liu,Juan Zhang,Jinghui Peng,Genxi Li
Prosthetic joint infection (PJI), which is still difficult to be diagnosing, is caused by bacterial infection. Rapid detection for trace amounts of bacteria should be a solution; however, the existing assays cannot serve for PJI diagnosis due to the inherent limitations. Herein, a strategy based on supramolecular chemistry mediated Cas12a catalytic aggregation (CCA) is explored in this work for ultrasensitive detection of universal bacteria. In the design, supramolecular chemistry is introduced to impede the cis-cleavage of activated Cas12a, resulting in its aggregation in the surrounding of bacteria with the linkage of CCA probe. Since Cas12a aggregation owns excellently enhanced catalytic efficiency, the CCA-based strategy can be used for bacterial analysis with a limit of detection as low as 1 CFU/mL. With high sensitivity, reproducibility, generalizability, and stability, the CCA-based strategy can be applied for the detection of clinical effusion samples. The remarkable performance of CCA-based strategy has also demonstrated its robust potential to be a simple, rapid, and highly sensitive bacteria diagnostic tool, paving the way for prompt diagnosis and management of PJI.
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Biosensors-Basel
·
2025
·Vol. 15
·期 8
·479
·
IF 5.6
Xiaotian Guan,Peizheng Wang,Yi Wang,Shuqing Sun
The CRISPR/Cas system has attracted increasing attention in accurate nucleic acid detection. Herein, we reported a CRISPR/Cas12a-chemiluminescence cascaded bioassay (CCCB) for the amplification-free and sensitive detection of human papillomavirus type 16 (HPV-16) and parvovirus B19 (PB-19). A magnetic bead (MB)-linking single-stranded DNA (LssDNA)-alkaline phosphatase (ALP) complex was constructed as the core component of the bioassay. During the detection process, the single-stranded target DNA was captured and enriched by LssDNA and then activated the trans-cleavage activity of Cas12a. Due to the Cas12a-mediated cleavage of LssDNA, ALP was released from the MB, subsequently catalyzing the substrate to generate a chemiluminescence (CL) signal. Given the cascade combination of CRISPR/Cas12a with the CL technique, the limits of detection for HPV-16 and PB-19 DNA were determined as 0.14 pM and 0.37 pM, respectively, and the whole detection could be completed within 60 min. The practicality and reliability of the platform were validated through target-spiked clinical specimens, and the recovery rate was 93.4–103.5%. This dual-amplification strategy—operating without target pre-amplification—featured high specificity, low contamination risk, facile preparation, and robust stability. It provides a novel approach for sensitive nucleic acid detection, with the potential for rapid extension to the diagnosis of various infectious diseases.
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Tianyi Zhang,Yingying Zhao,Cong Zhu,Xi Zhu,Xiaohua Zhu,Ye Qiu,Zhou Nie,Chunyang Lei
Viral proteases are critical molecular targets in viral pathogenesis, representing pivotal biomarkers for understanding viral infection mechanisms and developing antiviral therapeutics. This study introduces a label-free electrochemical biosensor that enables sensitive viral protease detection by integrating protease-responsive CRISPR/Cas protein switches (CasPSs) with a hemin aptamer-functionalized electrochemical interface. The biosensor’s mechanism relies on viral protease-mediated proteolysis, which leads to the release of active Cas12a proteins from CasPSs and generates amplified electrochemical responses through continuous cleavage of immobilized redox-active hemin/aptamer complexes. This biosensor achieved specific hepatitis C virus NS3/4A protease sensing with femtomolar sensitivity and could be readily expanded to other viral proteases by replacing the CasPS module. The feasibility of this biosensor was demonstrated by monitoring enterovirus 71 3C protease activities in virus-infected cell samples with different viral loads and postinfection times. This study provides a promising strategy for integrating CRISPR biosensing with electrochemical platforms, offering a helpful analytical tool for viral infection monitoring and antiviral drug screening.
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Food Quality and Safety
·
2025
·
IF 4.4
Hu Jinyan,Liu Xiaogang,Liu Tiantian,Zhao Ting,Li Mingliang,Yang Qilin,Jiang Xiaofeng,Xiong Yanfei,Shen Caihong,Liu Shuangping,Mao Jian,Tu Rongkun
ObjectivesYeast plays a crucial role in fermented foods, yet the application of the CRISPR/Cas system for yeast detection has been rarely reported. The objective of this study is to develop a rapid and quantitative detection method for brewing yeast based on the CRISPR-Cas12a system, aiming to achieve higher sensitivity than the quantitative real-time polymerase chain reaction (qPCR) assay and fill the gap in the application of CRISPR for yeast detection in fermented foods.Materials and Methods: This work established one-step RPA-CRISPR/Cas12a (ORCC) detection for quantifying yeasts in fermented foods by integrating target amplification and the CRISPR system into a one-pot reaction.ResultsA numerical standardized definition was established for the quantifiable state in ORCC detection of yeasts. Using ORCC detection, which occurs at 37 °C, absolute quantification of Saccharomyces cerevisiae, Wickerhamomyces anomalus, and Zygosaccharomyces bailii can be completed in less than 45 min. Under optimal conditions, the limits of quantification and the limits of detection for three yeasts were 100 copies/μL and 1 copy/μL, with no cross-reaction observed among non-target yeasts. In pure yeast samples, ORCC detection of the target yeasts achieved a detection rate of 91.60% - 116.57%. In simulated fermentation samples, the recovery rate for spiked target yeasts using ORCC detection ranged from 92.78% to 106.90%. In original naturally fermented samples, target yeasts can be quantified by ORCC detection but not by qPCR assay. In spiked naturally fermented samples, the recovery rates of the qPCR assay were generally lower than those of ORCC detection.ConclusionORCC detection is simple, rapid, accurate, and highly sensitive for detecting three yeasts in fermented foods. It has significant potential for precisely monitoring yeast growth and fermentation process in various fermentation systems.
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ANALYTICAL CHEMISTRY
·
2021
·
IF 6.986
Chunyan Wang,Cuiyan Han,Xiaoxue Du,Weiwei Guo
In addition to their roles as revolutionary genome engineering tools, CRISPR-Cas systems are also highly promising candidates in the construction of biosensing systems and diagnostic devices, which have attracted significant attention recently. However, the CRISPR-Cas system cannot be directly applied in the sensing of non-nucleic acid targets, and the needs of synthesizing and storing different vulnerable guide RNA for different targets also increase the application and storage costs of relevant biosensing systems, and therefore restrict their widespread applications. To tackle these barriers, in this work, a versatile CRISPR-Cas12a-based biosensing platform was developed through the introduction of an enzyme-free and robust DNA reaction network, the entropy-driven dynamic DNA network. By programming the sequences of the system, the entropy-driven catalysis-based dynamic DNA network can respond to different types of targets, such as nucleic acids or proteins, and then activate the CRISPR-Cas12a to generate amplified signals. As a proof of concept, both nucleic acid targets (a DNA target with random sequence, T, and an RNA target, microRNA-21 (miR-21)) and a non-nucleic acid target (a protein target, thrombin) were chosen as model analytes to address the feasibility of the designed sensing platform, with detection limits at the pM level for the nucleic acid analytes (7.4 pM for the DNA target T and 25.5 pM for miR-21) and 0.4 nM for thrombin. In addition, the detection of miR-21 or thrombin in human serum samples further demonstrated the applicability of the proposed biosensing platform in real sample analysis.
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2025
·
IF 3.7
Weiyu Xie,Zhenkun Cai,Zehua Bao
Saccharomyces cerevisiae is an important organism for basic research and applied biotechnology. Genome editing techniques, particularly CRISPR/Cas9 from Streptococcus pyogenes, have greatly facilitated saturation genome editing in yeast to understand mutant functions on a large scale. However, Cas9 is restricted by its targeting preference for G-rich protospacer-adjacent motif (PAM) sequences. To broaden the targeting scope, we established an efficient homology-integrated CRISPR/Cas12a system to install genetic variants through homologous recombination by targeting T-rich PAMs. We benchmarked the PAM compatibility of PAM-relaxed Cas12a variants and identified the improved LbCas12a (impLbCas12a) as the most efficient and PAM-relaxed variant in S. cerevisiae, showing high editing purity and an editing window centering the double-strand break. We show that our system can be used to perform targeted saturation mutagenesis to reveal functional variants not captured previously. By using a homology-integrated CRISPR RNA array, we utilized the multiplexing capability of CRISPR/Cas12a to realize multiplex genetic variant installation. Our system enriches the yeast genetic variant engineering toolbox, complementing the commonly used CRISPR/Cas9 system.
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BIOSENSORS & BIOELECTRONICS
·
2021
·
IF 10.618
Bin Qiao,Jiakun Xu,Wenhao Yin,Wanmeng Xin,Lixin Ma,Jie Qiao,Yi Liu
Herein, we report a method that combined “aptamer-locker” DNA with CRISPR/Cas12a-based biosensing for sensitive and rapid melamine analysis. Three strategies were harnessed for designing the DNA sensors that were well characterized by circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC) in the absence and presence of melamine. The detection parameters were optimized to achieve good analytic performance. As a result, a limit of detection (LOD) as low as 38 nM was achieved, which is below the threshold (1.0 mg/kg) of allowable melamine in infant milk products. In addition, the sensors show high selectivity for melamine against other analogues such as cyanuric acid , ammeline and ammelide . Moreover, our method was effective for rapid melamine analysis in whole milk samples, with or without sample pretreatment , in less than 20 min. Adopting a commercially available portable fluorimeter, on-site analysis of melamine in milk was accomplished. The strategies demonstrated here can expand to detect other non-nucleic-acid targets by simply replacing the aptamers.
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