ANALYST
·
2025
·
IF 3.3
Yang Li,Yikan Zhao,Zhihao Yi,Shitong Han
Timely and accurate diagnosis of RNA viruses, represented by the SARS-CoV-2, is the foundation for protecting people from health threats. Currently, direct detection of viral RNA genes remains the most accurate method. Herein, a rapid, ultrasensitive, and no heating equipment required CRISPR/Cas13a based evanescent wave fluorescence biosensing platform for quantitative detection of the SARS-CoV-2 gene is reported. The collateral effect of CRISPR/Cas13a for RNA is combined with a self-driven enzyme-free hybridization chain reaction (HCR) as a signal amplification step. When the initiator RNA strand is cleaved by Cas13a, the downstream signal amplification induced by HCR is blocked, and a multiple crRNA strategy is used to enhance the cleavage efficiency. The newly designed HCR assemblies are captured by the cDNA-modified optical fiber and generate a higher-intensity fluorescence signal induced by the evanescent field. The CRISPR/Cas13a-HCR evanescent wave fluorescence biosensing platform is capable of detection of SARS-CoV-2 with a LOD of 0.47 copies per μL and the detection time is within 35 min. The spike recovery tests in saliva and high specificity have demonstrated the potential of this method for point-of-care diagnosis. This method is also suitable for the detection of other RNA viruses, without the need to alter any design of the HCR component, and only the corresponding crRNA needs to be replaced.
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高影响因子
ACS Sensors
·
2026
·
IF 10.9
Zhenzhen Liu,Jianrong Qi,Lili Sun,Junhua Su,Wenjun Li,Lei Wu,Yufeng Liang,Chuhong Wei,Fangfang He,Yao Han,Yansong Sun,Huacheng Yan,Hao Li,Rui Xiao
The outbreak of Chikungunya virus (CHIKV) has caused widespread acute morbidity with severe polyarthralgia or chronic arthritis, placing a great challenge to public health and socioeconomic development. Establishing a rapid and highly sensitive detection technology is crucial for achieving precise control and prevention. Here, we established a CRISPR/Cas13a-mediated SERS lateral flow immunoassay platform for rapid and highly sensitive detection of CHIKV. One-dimensional nanowires loading Au nanoparticles were used to prepare SERS tags, presenting excellent SERS-enhanced performances and superior applicability to directional flow on the test strip. For the best performance, the structure of WOX nanowires was regulated by adjusting the addition concentration of ascorbic acid during the synthesis process, resulting in a stronger LSPR effect derived from more Au NPs in situ grown on highly reducing WOX. Furthermore, the lyophilized CRISPR/Cas system greatly simplified the workflow. In the optimal conditions, the limit of detection reached 0.56 and 1.03 copies/μL for the CHIKV plasmid and inactivated viruses by this method, respectively. Furthermore, 34 clinical serum samples were accurately diagnosed by our proposed method, 100% consistent with qPCR. This platform with the advantages of simple operation and rapid response provides a reliable technical tool for the early precise identification and efficient monitoring of CHIKV.
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高影响因子
MICROCHIMICA ACTA
·
2024
·Vol. 191
·期 8
·1-12
·
IF 5.3
He Qian,Chen Qun,Lian Lijin,Qu Jiuxin,Yuan Xi,Wang Chuhui,Xu Lidan,Wei Jiazhang,Zeng Shaoling,Yu Dongmei,Dong Yuhan,Zhang Yongbing,Deng Lin,Du Ke,Zhang Canyang,Pandey Vijay,Gul Ijaz,Qin Peiwu
The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13’s reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg 2+ concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform. Graphical Abstract
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高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Zhongheng Wei,Huini Luo,Danlu Huang,Peixin Wei,Jiayi Zhang,Jihua Wei,Qianli Tang,Longjian Huang,Kai Zhang,Xianjiu Liao
Flap endonuclease 1 (FEN1) is crucial for DNA replication, repair, and telomere maintenance. Its dysregulation is linked to various cancers and diseases. Accurate detection of FEN1 is essential for early diagnosis and therapeutic monitoring. Thus, a novel electrochemiluminescent (ECL) biosensor has been developed for the structure-specific and detection of FEN1. The strategy integrates dumbbell DNA probe-mediated transcription and CRISPR/Cas13a-induced trans-cleavage of RNA G-quadruplexes. In the presence of FEN1, the 5′-flap structure of the probe was selectively cleaved and subsequently ligated by T4 DNA ligase to form a closed circular template. This enabled T7 RNA polymerase to transcribe crRNA-encoded RNA strands, which activated Cas13a to cleave surface-tethered G-quadruplexes/hemin complexes on a Ru(II)/Ti 3 C 2 /AuNPs-modified electrode, thereby restoring the quenched ECL signal. The biosensor exhibited an ultralow detection limit of 4.82 × 10 -9 U μL -1 and a wide dynamic range (1 × 10 -8 to 1 × 10 -5 U μL -1 ), along with excellent specificity and stability. Successful application in human serum validated its reliability for complex biological samples. This work presents a powerful platform for sensitive FEN1 monitoring, holding potential for clinical diagnostics and enzymatic analysis.
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高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2023
·
IF 8.4
Qing Han,Xinlan Bian,Yue Chen,Bingzhi Li
Flap endonuclease I (FEN1) is a structure-specific nuclease that participates in DNA repair, and its overexpression is related to an increased risk of cancer. Currently, few FEN1 assays can meet the requirements for diagnostic purposes due to their high complexity, low specificity, and limited sensitivity. Herein, we developed a biosensor adopting synthetic biology strategy, called CLAIM (cleavage-ligation-activation-involved monitoring) for FEN1 quantification. CLAIMintegrates ligation-based T7 promoter repair, RNA generation, and CRISPR/Cas13a. FEN1 cleavages the DNA probe with a 5′-flap to generate a nick site that can be ligated to form a T7 promoter. Subsequent transcription produces abundant RNA that can activate Cas13a, generating a fluorescence signal due to the trans -cleavage of Cas13a to a fluorogenic reporter. CLAIM was able to detect FEN1 with low femtomolar sensitivity, and its accuracy was validated using samples of cancer and non-tumorigenic cell lines. Interestingly, we found that the utilization of thermostable T4 DNA ligase is critical to make the cascade of FEN1 cleavage, ligation, and transcription take place in one-pot. CLAIM was also repurposed for validating FEN1 inhibitors, indicating its potential in drug screening. This work provides a diagnostic tool for FEN1 analysis and opens up opportunities for FEN1 biosensing and related drug discovery.
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BIOELECTROCHEMISTRY
·
2025
·
IF 4.5
Zhaofan Zhou,Ye Li,Dongya Wen,Xuyu Zhang,Ni Li,Jiangbing Shuai,Xiaofeng Zhang,Xiaoye Zheng,Zhaojun Ban,Farouk Amr,Xiaoping Yu,Jun Huang
Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen responsible for porcine epidemic diarrhea, which causes severe diarrhea and high mortality rates in neonatal piglets, leading to substantial economic losses in the swine industry. Therefore, there is an urgent need for rapid, sensitive, and accurate detection methods for PEDV. In this study, we develop a rapid, ultrasensitive electrochemical CRISPR-based biosensor for detecting PEDV with high sensitivity and specificity. The sensor integrates reduced graphene oxide-polypyrrole‑gold nanoparticle (rGO-PPy-AuNP) nanocomposites to enhance sensitivity and CRISPR-Cas13a for target-specific recognition. This dual-signal amplification strategy achieves an excellent analytical detection limit of 1.01 fg/mL and high linearity (R 2 = 0.9979) across a broad dynamic range (0.005–100,000 pg/mL) within 45 min, eliminating the need for nucleic acid amplification. By synergizing Cas13a-assisted signal enhancement with nanoparticle-mediated electron transfer, the biosensor outperforms conventional methods in sensitivity while maintaining excellent stability, and specificity, demonstrating strong potential for PEDV detection in clinical diagnostics.
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高影响因子
ANALYTICA CHIMICA ACTA
·
2026
·
IF 6.1
Xinying Wang,Yujie Chang,Yiyan Zhang,Xuan Yu,Yixuan Zou,Zheqi Liu,Fuchuan Qi,Jingjing Ming
Accurate and rapid determination of aflatoxin M1 (AFM1) in food is crucial for ensuring the safety of the human diet. In view of this, we proposed a proximity-inducible Cas13a activity and hydrogel-assisted paper-based distance sensor for the detection of aflatoxin M1. The single-stranded DNA (ssDNA) and AFM1 aptamer were split into two parts and linked to the terminus of two separate DNA activator strands, respectively. By introducing AFM1, it stimulated these two DNA activator strands to assemble with CRISPR RNA (crRNA) through spatial proximity mechanism between the target and these two DNA activators to activate the Cas13a trans-cleavage towards the RNA-containing site within hairpin probe to release a DNA initiator. The released DNA initiator triggered the catalytic hairpin assembly (CHA) between magnetic beads (MBs)-DNA capture probe and trypsin-DNA detection probe. After hydrolysis of gelatin hydrogel by trypsin, water released from the hydrogel flowed along the pH indicator strip and exhibited a distinct mark. Depending on this portable method, the limit of detection (LOD) was achieved at 23.6 ng L -1 . Furthermore, our developed strategy can be applied to measure AFM1 in ultra-high temperature (UHT) milk with satisfactory effects, and its excellent specificity and reproducibility also demonstrate that our platform has great potential for application in environmental monitoring.
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BIOSENSORS & BIOELECTRONICS
·
2022
·
IF 12.545
Yang Sun,Cheng Yang,Xiaolin Jiang,Pengbo Zhang,Shuo Chen,Fengxia Su,Hui Wang,Weiliang Liu,Xiaofei He,Lei Chen,Baoyuan Man,Zhengping Li
False detection of SARS-CoV-2 is detrimental to epidemic prevention and control. The scalar nature of the detected signal and the imperfect target recognition property of developed methods are the root causes of generating false signals. Here, we reported a collaborative system of CRISPR-Cas13a coupling with the stabilized graphene field-effect transistor, providing high-intensity vector signals for detecting SARS-CoV-2. In this collaborative system, SARS-CoV-2 RNA generates a “big subtraction” signal with a right-shifted feature, whereas any untargets cause the left-shifted characteristic signal. Thus, the false detection of SARS-CoV-2 is eliminated. High sensitivity with 0.15 copies/μL was obtained. In addition, the wide concerned instability of the graphene field-effect transistor for biosensing in solution environment was solved by the hydrophobic treatment to its substrate, which should be a milestone in advancing it's engineering application. This collaborative system characterized by the high-intensity vector signal and amazing stability significantly advances the accurate SARS-CoV-2 detection from the aspect of signal nature.
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高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Jingzhi Zhou,Xichen Shi,Chaoqin He,Xiuwen Zheng,Ruo Yuan,Xia Yang
Background Entropy-Driven Circuits (EDC), distinguished by their spontaneous operation and absence of enzymatic reactions, represent a superior strategy for integration with CRISPR/Cas systems, as they obviate the potential for interference among various enzymes during the process of DNA amplification and CRISPR/Cas system integration. Due to the wide band gap of TiO 2 , its response to visible light is limited, and owing to its high crystallinity and exceptionally stable crystal lattice, the charge transfer (CT) process in TiO 2 is suboptimal. Results In this study, lychee-like Fe–TiO 2 was firstly prepared to serve as Raman enhanced substrate, facilitating exciton capture and separation to exhibit an excellent Surface-enhanced Raman spectroscopy (SERS) performance. It is proven that the incorporation of Fe results in a significantly narrower band gap for TiO 2 , facilitating exciton resonance. The amount of Fe in TiO 2 was optimized to fabricate a SERS biosensor for detection of miRNA-21 based on the Cas13a assisted entropy-driven system. The detection limit of miRNA was 43.88 fmol/L. Significance This work proposes a sensing strategy that integrates the CRISPR/Cas system with EDC, leveraging a semiconductor substrate exhibiting superior SERS performance to provide a stable Raman signal, thereby enabling highly sensitive detection of miRNA-21, which has a potential application in disease early warning and treatment.
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ACS Sensors
·
2026
·
IF 10.9
Tianchi Zhuang,Yan Long,Yongtong Xu,Qiumei Li,Zhiming Liu,Xueshan Zhong,Dawei Wu,Yu Ma,Wenzhe Su,Xiaokang Li,Zunfu Ke,Cheng Guo
Early and accurate diagnosis of Chikungunya virus (CHIKV) infection is critical for controlling its outbreaks. CRISPR/Cas-based detection offers promise for pathogen identification, yet one-step CRISPR/Cas systems are limited by suboptimal sensitivity, field-deployability, and adaptability to complex clinical samples, hindering their use in rapid outbreak response. Here, we developed a CRISPR/Cas13a-based One-Step System for Rapid Detection of Emerging Viruses (CRISPR-CORE) and applied it during the CHIKV outbreak in Guangdong Province, China. Multidimensional optimizations enabled the CRISPR-CORE system to achieve a limit of detection of 5 copies/μL within 40 min. An extraction-free RNA release protocol for CHIKV in blood samples and a premixed reagent approach were implemented. Furthermore, a portable fluorescence detector was used to enhance user-friendliness in point-of-care (POC) settings. The clinical CHIKV genomic information was identified through hybrid capture sequencing, informing the design of highly specific CRISPR RNA (crRNA). Clinical validation across three regions yielded 92.6% sensitivity and 100% specificity, underscoring the applicability and reliability of the CRISPR-CORE system. Our system demonstrates its suitability for CHIKV outbreak detection. It facilitates rapid and POC testing for emerging viruses in resource-limited settings. Furthermore, it provides a universal strategy for the prevention and control of infectious diseases.
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高影响因子
Sensors and Actuators Reports
·
2025
·
IF 7.6
Yanli Sun,Hongying Li,Tao Wang,Kai Zhang,Xueliang Wang
MicroRNA-17 (miR-17) is a critical biomarker linked to various cancers and diseases, underscoring the need for sensitive and selective detection methods. Herein, an ultra-sensitive electrochemiluminescent (ECL) sensor for miR-17 detection utilizing MOF-derived porous CuFe 2 O 4 nanospheres and CRISPR/Cas13a trans-cleavage-triggered hybridization chain reaction (HCR) amplification was presented. The porous CuFe 2 O 4 nanospheres possessed a high surface area and excellent catalytic activity, significantly enhancing ECL emission. To the best of our knowledge, this is the first report demonstrating that MOF-derived CuFe 2 O 4 can effectively enhance the electrochemiluminescence of Ru(bpy) 3 2 ⁺ for miR-17 detection. The CRISPR/Cas13a system provided miR-17 recognition and trans-cleavage activity, triggering HCR for signal amplification. Under optimized conditions, the biosensor exhibited a wide linear detection range from 20 aM to 5000 aM, with a detection limit of 9.23 aM. The system discriminated miR-17 from closely related miRNAs with negligible cross-reactivity, which demonstrated its outstanding selectivity. Practical applicability was confirmed by successful quantification of miR-17 in diluted human serum which achieved recovery rates between 95.2% and 104.8%. The results validated the biosensor’s potential for early disease diagnosis and clinical monitoring of miR-17-associated disorders.
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Analytical Methods
·
2025
·
IF 2.6
Zixun Wang,Dong Zhang,Yuxuan Wu,Xu Ye,Yayun Qian,Fengsong Chen
The low early diagnosis rate of hepatocellular carcinoma (HCC) severely impacts patient prognosis, making the development of highly sensitive and specific early diagnostic technologies crucial. MicroRNA-221 (miR-221), an aberrantly overexpressed biomarker in HCC, holds significant diagnostic potential. This paper constructed a capillary surface-enhanced Raman scattering (SERS) sensing platform utilizing CRISPR/Cas13a trans-cleavage and double-shell gold-silver nanorods (DS Au-AgNRs) to detect serum miR-221 in HCC patients. DS Au-AgNRs were synthesized and assembled onto aminated capillaries, followed by conjugation of Cy5-labeled single-stranded DNA (ssDNA) to the DS Au-AgNR surface via Au–S bonds. In the presence of miR-221, activated CRISPR/Cas13a trans-cleavage cleaves the ssDNA, releasing Cy5 from the sensor surface and diminishing the SERS signal, enabling miR-221 quantification. The synthesized DS Au-AgNRs exhibit uniform morphology and size, are uniformly distributed on the capillary, and form numerous “hotspots”, thereby significantly enhancing the SERS signal. According to the characteristic peak of Cy5 at 1074 cm−1, a linear relationship is established between the log concentration of miR-221 and the measured SERS intensity (y = −3527.97 × −35369.60, R2 = 0.97767), with a LOD as low as 4.17 × 10−17 M. The sensor demonstrated high specificity and high sensitivity, and its capacity to detect miR-221 expression aligned with qRT-PCR results when analyzing serum samples, confirming that hepatocellular carcinoma patients exhibited significantly higher miR-221 levels compared to healthy individuals. The capillary SERS sensor thus provides an accurate and convenient approach for early HCC diagnosis.
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