SENSORS AND ACTUATORS B-CHEMICAL
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2022
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IF 9.221
Yu Tao,Ke Yi,Haixia Wang,Kai Li,Mingqiang Li
Since hepatitis B virus (HBV) infection can cause chronic viral hepatitis, increase the risk of liver fibrosis and cirrhosis, and ultimately result in hepatoma, it is of great significance to explore convenient, rapid, and sensitive diagnostic strategies for HBV DNA detection. In this study, we developed a novel and sensitive fluorescent biosensor for HBV detection based on the sensing system consisting of CRISPR-Cas12a enzymes and metal nanoclusters as the luminescent nanoprobes, involving gold nanoclusters (AuNCs), silver nanoclusters (AgNCs), and copper nanoclusters (CuNCs). The DNA probes could effectively be applied as the template for the preparation of fluorescent metal nanoclusters. In the presence of the HBV target, the trans-cleavage capability of Cas12a was able to be initiated to degrade DNA probes, which brought about the inhibition of metal nanoclusters formation, thereby giving minimal fluorescence signals. In comparison with AuNCs and AgNCs, the CuNCs-based nanosensor exhibited much higher sensitivity. In addition, the fluorescence response of CuNCs to HBV detection was significantly faster, which could be completed within 25 min without the requirements of sophisticated equipment or harsh reaction conditions. Moreover, the practical application of this analysis method was also demonstrated to detect HBV DNA target in human serum samples with satisfactory recovery, which was promising for simple, rapid, sensitive, and label-free HBV DNA bioanalysis .
研究领域:
分子生物学
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医学诊断
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纳米技术
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DOI
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2023
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IF 15.1
Yuqing Zhu,Xiaohe Zheng,Rui Zhu,Hongrui Zhao,Hongli Zhai,Feiyang Qian,Tenghua Zhang,Zihui Xie,Silan Liu,Bowen Jiang,Yan Sheng,Jiaming Hu
Detection of multiple biomarkers for accurate diagnosis of disease is highly desirable but remains challenging. Here, we developed a versatile diagnostic biosensor (SMARTLOCK) through the unique combination of DNA tetrahedron, nonlinear HCR and CRISPR-Cas12a system for simultaneous detection of multiple nucleic acids, small molecules, proteins and different types of combinations. Two DNA tetrahedrons are assembled into a functional dumbbell lock, which was unlocked by the target molecules, thereby triggering a two-stage signal amplification of nonlinear HCR and CRISPR-Cas12a to generate powerful fluorescence signals. The capability of multi-target simultaneous detection of SMARTLOCK is demonstrated in the detection of miR-141, miR-375, miR-21, ATP, cortisol, insulin and their combinations. SMARTLOCK could selectively (specificity = 93.3%) and sensitively (sensitivity = 90%) distinguish early-stage prostate cancer (PCa) patients (n = 10) from healthy donors (n = 30). Measurement of two PCa-related RNAs (miR-141 and miR-375) shows the potential of SMARTLOCK to be a highly accurate and low-cost tool for the screening, diagnosis and prognosis of various diseases.
研究领域:
生物技术
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医学诊断
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分子生物学
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DOI
高影响因子
Nature Communications
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2025
·Vol. 16
·期 1
·1-14
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IF 15.7
Hu Hao,Guo Songcheng,Li Yiyuan,Dong Kejun,Lu Yan,Ye Keyi,Li Longjie,Zhou Xiaoyu,Cheng Liming,Xiao Xianjin
Detecting small molecules is pivotal across fields like clinical diagnostics, environmental monitoring, and food safety. The CRISPR-Cas12a system, known for its simplicity and sensitivity, offers a promising basis for small molecule detection. However, current CRISPR-based detection methods face challenges, including complex design requirements, high background noise, and limited adaptability to different targets. In our study, we introduce the SBS-Cas system, leveraging a split crRNA mode to induce spatial hindrance on the scaffold strand through molecular binding. This approach prevents the assembly with Cas12a, effectively masking its trans-cleavage activity. By introducing small molecules that competitively bind to the macromolecule, we eliminate this spatial hindrance, activating Cas12a. Our results demonstrate high sensitivity, versatility, and adaptability in small molecule detection across multiple reactions, with successful intracellular imaging and responsive fluctuations in complex environments underscoring the system’s robustness. This innovative CRISPR-Cas12a-based approach establishes a low-background, highly sensitive platform for small molecule detection. SBS-Cas promises not only to enhance tools for clinical, environmental, and food safety applications but also to advance CRISPR research, providing insights and expanding possibilities in molecular detection science.
研究领域:
分子生物学
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临床诊断
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环境科学
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食品安全
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2023
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IF 7.4
Yin Zhao,Dongjuan Chen,Zhichen Xu,Tao Li,Jiang Zhu,Rui Hu,Guoyong Xu,Ying Li,Yunhuang Yang,Maili Liu
Fast, simplified, and multiplexed detection of human papillomaviruses (HPVs) is of great importance for both clinical management and population screening. However, current HPV detection methods often require sophisticated instruments and laborious procedures to detect multiple targets. In this work, we developed a simple microfluidic dual-droplet device (M-D3) for the simultaneous detection of HPV16 and HPV18 by combining the CRISPR-Cas12a system and multiplexed recombinase polymerase amplification (RPA) assay. A new approach of combining pressure/vacuum was proposed for efficient droplet generation with minimal sample consumption. Two groups of droplets that separately encapsulate the relevant Cas12a/crRNA and the fluorescent green or red reporters are parallelly generated, followed by automatic imaging to discriminate the HPV subtypes based on the specific fluorescence of the droplets. The M-D3 platform performs with high sensitivity (∼0.02 nM for unamplified plasmids) and specificity in detecting HPV16 and HPV18 DNA. By combining the RPA and Cas12a assay, M-D3 allows on-chip detection of HPV16 and HPV18 DNA simultaneously within 30 min, reaching a detection limit of 10–18 M (∼1 copy/reaction). Moreover, the outstanding performance of M-D3 was validated in testing 20 clinical patient samples with HPV infection risk, showing a sensitivity of 92.3% and a specificity of 100%. By integrating the dual-droplet generator, CRISPR-Cas12a, and multiplexed RPA, the M-D3 platform provides an efficient way to discriminate the two most harmful HPV subtypes and holds great potential in the applications of multiplexed nucleic acid testing.
研究领域:
分子生物学
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医学检测
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生物技术
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2021
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IF 6.986
Wei S. Zhang,Jianbin Pan,Feng Li,Min Zhu,Mengting Xu,Hongyan Zhu,Yanyan Yu,Gaoxing Su
The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV–vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.
研究领域:
分子生物学
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病毒学
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诊断学
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2021
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IF 7.46
Lijuan Yin,Ninghui Duan,Si Chen,Yuan Yao,Jifeng Liu,Long Ma
Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenient. In this study, we developed a G-quadruplex-based CRISPR-Cas12a bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella specific invA gene activated the trans -cleavage activity of Cas12a and triggered CRISPR-Cas12a based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K + . This DNAzyme could catalyze the TMB-H 2 O 2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R 2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity and on-site capability.
研究领域:
食品科学
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分子生物学
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生物技术
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DOI
高影响因子
MICROCHEMICAL JOURNAL
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2025
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IF 5.1
Ping Qiu,Longjian Huang,Jiayi Zhang,Wenbo Li,Xudong Xin,Luchang Chen,Qianli Tang,Kai Zhang,Xianjiu Liao,Jihua Wei
We report the development of a novel split aptamer-driven electrochemical biosensor integrating entropy-driven Signal amplification (EDSA) and CRISPR-Cas12a technology for the highly sensitive and specific detection of adenosine deaminase (ADA). ADA is an important biomarker involved in immune regulation and purine metabolism, and its accurate quantification is critical for diagnosing various diseases. In our system, ADA catalyzes the deamination of adenosine to inosine, resulting in the release of Aptamer1 from a split aptamer structure. The liberated Aptamer1 initiates a strand displacement reaction within a Blocker/Bandage/Scaffold complex, triggering a cascade of entropy-driven amplification cycles that generate a DNA duplex capable of activating CRISPR-Cas12a. Activated Cas12a exhibits trans -cleavage activity, specifically cleaving a ferrocene-labeled single-stranded DNA (ssDNA) probe immobilized on a gold electrode, leading to a measurable decrease in electrochemical signal. The biosensor operates under isothermal and enzyme-free conditions, providing simplicity, rapid detection, and low cost. It achieves a low detection limit of 0.688 U·L −1 with a wide linear range from 1 to 20,000 U·L −1 . The system demonstrates excellent specificity against ADA analogues and common interfering proteins and performs reliably in diluted human serum samples. This strategy represents a versatile and modular platform for point-of-care diagnostics. By altering the aptamer sequence, the biosensor can be adapted for the detection of other clinically relevant proteins and enzymes, contributing to advances in personalized medicine.
研究领域:
生物传感技术
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分子诊断
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医学检测
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DOI
高影响因子
ANALYTICA CHIMICA ACTA
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2023
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IF 6.2
Shiqi Xu,Songtao Wang,Ling Guo,Yuqin Tong,Lina Wu,Xingxu Huang
CRISPR-based detection was often based on the target preamplification to realize the high sensitivity. Here, we prepared a CRISPR-Cas12a system for the colorimetric detection of lead ion (Pb 2+ ) based on the assistance of DNAzyme and nanozyme instead of preamplification. The recognition between GR-5 DNAzyme and Pb 2+ could trigger the CRISPR-Cas12a system. MnO 2 nanozymes connected with magnetic beads through single stranded DNA were prepared as the colorimetric signal probes and catalyst of CRISPR-Cas12a system for the strong oxidase-like activity inducing the color change of 3,3′,5,5′-tetramethylbenzidine. The nanozyme-catalysed CRISPR-Cas12a system could be used to detect Pb 2+ through the color change with high specificity and sensitivity. The linear range of this approach was 0.8 nM–2500 nM, with a limit of detection of 0.54 nM. This method was applied for the detection of the Pb 2+ in food samples indicating good accuracy and anti-interference ability.
研究领域:
生物传感器
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分析化学
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环境科学
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食品安全
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产品关联
DOI
PubMed
高影响因子
Biosensors-Basel
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2025
·Vol. 15
·期 12
·821
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IF 5.6
Jie Chen,Shutao Liu,Sunyi Chen,Jingwen Mai,Maiwula Abudukadi,Yao Chen,Jie Lu,Guanglei Li,Chenchen Ge
Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor’s response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.
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DOI
PubMed
高影响因子
TALANTA
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2025
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IF 6.1
Mengqiu Xiong,Yao Wang,Shuhan Lu,Nasifu Lubanga,Tao Li,Zhihao Li,Bangshun He,Ying Li
Multiple infections of respiratory viruses are common in patients with clinical respiratory diseases, but current detection methods still have problems such as complex equipment and long detection time. Rapid, low-cost, and on-site detection of human respiratory viruses is crucial for both clinical diagnosis and population screening. In this research, we created a space-coded microfluidic chip (SC-Chip) for the recognition of nine respiratory viruses: influenza A virus, influenza B virus, severe acute respiratory syndrome coronavirus 2, human coronavirus OC43, human coronavirus NL63, human coronavirus HKU1, human respiratory syncytial virus, human parainfluenza virus, and human metapneumovirus. For the first time, a comprehensive sequence comparison among these viruses was performed to design the recombinase polymerase amplification (RPA) primers and Cas12a-crRNAs. The SC-Chip partitions samples amplified by RPA into spatially coded wells preloaded with CRISPR-Cas12a detection reagents, enabling the identification of all nine viral targets in a single test using a single fluorescence probe. The chip-based assay displays 9 respiratory viruses in less than 40 min with a minimum detection limit at a concentration of 10 −18 M (∼1 copy/reaction). Additionally, the efficacy of the method was assessed through its application to 35 clinical patient samples identified as being at risk for respiratory virus infection, yielding a sensitivity of 90 % and a specificity of 100 %. In summary, this space-coded microfluidic CRISPR system offers several advantages, including ease of operation, cost-effectiveness, and rapid data acquisition, thereby holding great potential for multiplexed detection of nucleic acid targets in a clinical setting.
研究领域:
分子生物学
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医学诊断
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微流控技术
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DOI
PubMed
高影响因子
Synthetic and Systems Biotechnology
·
2025
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IF 4.4
Po Li,Xueying Lei,Xiaoying Niu,Wen Tian,Zhehuang Li,Songcheng Yu,Peng Zhang
MicroRNA (miRNA) is promising candidate for non-invasive diagnostic biomarker. Conventional CRISPR/Cas12a-based miRNA detection systems are constrained by reliance on reverse transcription, nucleic acid pre-amplification and costly fluorescently labeled reporters which introduce chemical modification complexity and background noise. To address these limitations, we herein developed a multifunctional switch that integrated target recognition, CRISPR/Cas12a system activation, intrinsic fluorescence signaling, and autonomous signal amplification within a single molecular architecture. As a proof of concept, this switch enabled a label-free CRISPR/Cas12a biosensing for miR-21 detection with a detection limit of 4.8 nM and robust performance in accuracy, precision, and selectivity. This proposed label-free CRISPR/Cas12a platform could be applied for real sample and is a promising candidate for point-of-care miRNA detection.
研究领域:
生物传感技术
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分子诊断
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CRISPR 基因检测
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核酸扩增
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即时检测
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产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2024
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IF 8.5
Sujun Liu,Kai Shi
The residue of clenbuterol (CLB) in food poses a potential harm to human health. Herein, we presented an electrochemical aptasensor ( E -A-CRISPR) based on employing an aptamer as a specific recognition element and asymmetric CRISPR-Cas12a as signal amplifiers for sensitive, and selective detection of CLB. In this E -A-CRISPR system, the target CLB bound to the aptamer and initiated cascade signal amplification through the DNase activity of CRISPR-Cas12a with two competitive gRNAs. Upon amplification, the active Cas12a cleaved the methylene blue-labeled hairpin probe on the electrode, reducing the peak current. Under optimal conditions, the E -A-CRISPR system showed a wide linear range (1 pM-100 nM) and a low detection limit (500 fM). This system could detect CLB in potable water, pig liver, and pork samples, showing significant potential for food safety monitoring. To our knowledge, this study is the first to use a CRISPR-Cas12a powered system for electrochemical sensing of CLB.
研究领域:
食品安全
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生物传感
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分子生物学
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）