SENSORS AND ACTUATORS B-CHEMICAL
·
2024
·
IF 8
Xin Gao,Yun Yin,Junyu Xie,Shaohua Gong,Xuemei Li
CRISPR/Cas13a system has been combined with different isothermal amplification strategies for RNA analysis. Nevertheless, expensive enzymes are usually indispensable in these isothermal amplification methods, which increases the detection cost. In this study, a CRISPR/Cas13a-mediated triple signal amplification strategy has been designed for selective, sensitive and inexpensive detection of viral RNA. SARS-CoV-2 S RNA activated CRISPR/Cas13a to cut hairpin DNA, triggering catalytic hairpin assembly (CHA) and subsequent hybridization chain reaction (HCR) to form a long-stranded Y-type DNA. G-quadruplexes were distributed on the branch of the long-stranded DNA and bound with N-methyl mesoporphyrin IX (NMM) to enhance the fluorescence signal. SARS-CoV-2 S RNA was specifically detected with a limit of detection (LOD) of 285 fM. Notably, no additional expensive enzymes were required in the entire detection process. The CRISPR/Cas13a-mediated triple signal amplification strategy holds great potential for specific and sensitive viral RNA detection with low cost.
研究领域:
生物技术
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分子生物学
·
病毒学
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产品关联
DOI
高影响因子
TALANTA
·
2022
·
IF 6.556
Bin Yang,Xiaowei Zeng,Jin Zhang,Jilie Kong,Xueen Fang
Delta (B.1.617.2), a highly infectious variant of SARS-CoV-2, has been sweeping the world, and threatening the safety of human life seriously. It is urgent to develop a highly selective and sensitive assay to accurately identify the SARS-CoV-2 variant Delta. In this work, we constructed a graphene/CRISPR-dCas9 electrochemical biosensor to accurately identify SARS-CoV-2 variant Delta, where the signal was further amplified by embedded electrochemical probe [Ru(phen) 2 dppz]BF 4 . This detection assay could be finished within 47 min totally, with the detection limit of 1.2 pM and good reproducibility with a C·V.% of 2.48% (n = 5). And the biosensor could selectively identify Delta among SARS-CoV-2 and other variants, including Alpha, Beta, Gamma. This assay was further validated by 26 real clinical samples, showing 100% clinical sensitivity and 100% clinical specificity, which provides a new direction for identifying other SARS-CoV-2 variants in the future.
研究领域:
生物技术
·
电化学
·
分子生物学
·
病毒学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Yuwan Du,Shuaifeng Ji,Qingyang Dong,Jiang Wang,Dianpeng Han,Zhixian Gao
Nucleic acid markers have been widely used in the detection of various virus-related diseases, including hepatitis B virus (HBV), which is spreading worldwide. The trans -activated CRISPR-Cas system has shown excellent sensitivity and specificity in nucleic acid detection. However, nucleic acid testing usually requires amplification of the target nucleic acid for more accurate and specific detection; furthermore, current nucleic acid assays are time-consuming, costly, and are limited by non-specific cross-reactivity. We developed an amplification-free viral DNA biosensor-based diagnostic method that uses a clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas)-based approach with surface enhanced Raman spectroscopy . This method can specifically identify the target site by changing the crRNA sequence. In addition, the incubation period and development of the disease can be determined by quantitative detection of viral DNA. This system could achieve rapid and highly sensitive detection of HBV DNA within 50 min and vast detection range from 0.1 pM to 1 nM. Therefore, a combined CRISPR/Cas12a-SERS-based assay would improve the sensitivity of detection in assays using multiple biomarkers. In conclusion, our CRISPR/Cas12a-based biosensor would enable rapid, simple, and sensitive detection of HBV nucleic acids.
研究领域:
病毒学
·
分子诊断
·
生物医学工程
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2023
·
IF 12.6
Hua Wang,Yang Sun,Yuan Zhou,Yujie Liu,Shuo Chen,Wenbo Sun,Zidong Zhang,Junqing Guo,Cheng Yang,Zhengping Li,Lei Chen
At present, the 100% case fatality and the cross-infection of virus strains make the ASFV 's harm to society continue to expand. The absence of an effective commercial vaccine poses early detection remains the most effective means of curbing ASFV infection. Here, we report a cascaded detection platform based on the CRISPR-Cas12a system combined with graphene field-effect transistor sensors. The cascade platform could detect ASFV as low as 0.5 aM within 30 min and achieve typing of wild and vaccine strains of ASFV in a single detection system. The evaluation of 16 clinical samples proved that, compared with the gold standard Real-time PCR method, this platform has outstanding advantages in sensitivity, specificity and typing. Combining CRISPR-Cas12a's high specificity with the bipolar electric field effect of graphene field-effect transistor, the cascade platform is expected to achieve clinical application in the field of DNA disease detection, and provides a new direction for multi-strain disease typing.
研究领域:
分子生物学
·
病毒学
·
生物传感器
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2020
·
IF 5.977
Lei Peng,Jin Zhou,Lijuan Yin,Shuli Man,Long Ma
For the first time, three 2-input elementary AND, OR, INHIBIT logic gates have been constructed by using CRISPR-Cas12a system. These logic gates utilised the intrinsic advantages of programmability, sequence specificity and high base resolution of CRISPR-Cas12a system. Among them, the AND gate owned the potentials as a built-in biosensor that responded rapidly to external pathogenic bacteria such as Staphylococcus aureus with high sensitivity and specificity. We applied the CRISPR-Cas12a based bacterial detection after a target-amplification using PCR. The total sample-to-answer time was appropriately 2.0 h, the limit of detection (LOD) was 10 3 CFU/mL, and the dynamic range was 10 3 –10 7 CFU/mL. Also, the sequence addressability enabled this AND logic gate to accurately trace back and distinguish input genes. These above-mentioned features were highly ideal to incur a rapid response to pathogenic bacteria for decision making. Our results not only validated the possibility of using CRISPR-Cas systems for constructing bio-computing devices but also provided a prototype of biosensor for rapid and intelligent pathogenic bacteria detection.
研究领域:
生物技术
·
分子生物学
·
微生物学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2024
·
IF 5.7
Haolin Sun,Xiaoyu Zhang,Hainan Ma,Lina Zhang,Yang Zhang,Ruimeng Sun,Haoran Zheng,Han Wang,Jiayu Guo,Yanqi Liu,Yurou wang,Yanfei Qi
Rapid and sensitive detection of pathogenic bacteria is crucial for disease prevention and control. The CRISPR/Cas12a system with the DNA cleavage capability holds promise in pathogenic bacteria diagnosis. However, the sensitivity of CRISPR-based assays remains a challenge. Herein, we report a versatile and sensitive pathogen sensing platform (HTCas12a) based on the CRISPR/Cas12a system, hybridization chain reaction (HCR) and Poly T-copper fluorescence nanoprobe . The sensitivity is improved by HCR and the Poly-T-Cu reporter probe reduces the overall experiment cost to less than one dollar per sample. Our results demonstrate the specific recognition of target nucleic acid fragments from other pathogens. Furthermore, a good linear correlation between fluorescence intensity and target quantities were achieved with detection limits of 23.36 fM for Target DNA and 4.17 CFU/mL for S.aureus , respectively. The HTCas12a system offers a universal platform for pathogen detection in various fields, including environmental monitoring, clinical diagnosis, and food safety.
研究领域:
生物技术
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医学诊断
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食品安全
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Zhenzhen Jia,Yujie Zhang,Chuanyu Zhang,Xueyong Wei,Mingzhen Zhang
With an increasing incidence worldwide, inflammatory bowel disease (IBD) is a chronic inflammatory disease affecting the gastrointestinal tract, which impairs the life quality of patients. Therefore, it is of great significance to construct a sensitive, simple, and convenient biosensor to analyze IBD-associated biomarkers for an auxiliary diagnosis of IBD. Intestinal alkaline phosphatase (IAP), expressed by the intestinal epithelium, is an endogenous protein that is thought to play a vital role in maintaining intestinal homeostasis and is considered a potential biomarker for IBD. Here, an IAP detection method was developed using pregnancy test strips by dephosphorylation. Initially, a double-stranded DNA (dsDNA) was designed to respond to IAP and acted as an activator of Cas12a. In the presence of IAP, the designed dsDNA was not digested by lambda exonuclease (λ exo), which hybridized to the Cas12a-crRNA duplex and resulted in the activation of the trans-cleavage of Cas12a. Further, the activated Cas12a cleaved the single-strand DNA (ssDNA) linker in the MBs-ssDNA-hCG probe, triggering the release of hCG. With magnetic separation, the released hCG could be quantitatively detected by pregnancy test strips. IAP levels were analyzed in feces from colitis and healthy mice by pregnancy test strips. The results showed that the IAP level of colitis mice (3.89 ± 1.92 U/L) was much lower than that of healthy mice (39.64 ± 24.93 U/L), indicating the correlation between IAP and intestinal inflammation. Taken together, a sensitive, user-friendly detection assay based on pregnancy test strips was constructed to monitor IAP and used as an auxiliary diagnostic approach for IBD in a clinical scene.
研究领域:
医学诊断
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分子生物学
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生物传感技术
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产品关联
DOI
PubMed
高影响因子
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.
研究领域:
生物传感器
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分子诊断学
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纳米技术
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CRISPR技术
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RNA检测
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生物医学工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Xiaobo Zhang,Xiuwen Wang,Longjiao Zhu,Jinyan Zhu,Qiuyue Zheng,Jingli Yuan,Wentao Xu,Jijuan Cao
Salmonella typhimurium ( S. typhimurium ), as common and highly pathogenic foodborne pathogen, poses a significant risk to public safety worldwide. The development of highly sensitive, rapid and on-site method for S. typhimurium analysis is urgently needed to prevent bacterial infections. Herein, we introduced a CRISPR/Cas12a-driven electrochemiluminescence (ECL) sensor based on luminol/O 2 binary systems for S. typhimurium detection, employing ultrafine Pt nanoclusters-anchored 2D delaminated-MXenes (Pt NCs/D-MXenes) as the co-reactant accelerator. The ultrathin D-MXenes support regulates the size and dispersibility of Pt NCs and facilitates the full exposure of active sites, and synergistic interactions between D-MXenes and Pt NCs improves electrocatalytic properties toward the reduction of O 2 , which promotes the generation of ROS for boosting ECL emission. Using target responsive-regulated CRISPR/Cas12a system, the ECL sensor for S. typhimurium showed a broad concentration range from 10 1 to 10 6 CFU/mL and limit of detection of 6 CFU/mL, with satisfactory recoveries in spiked-actual samples.
研究领域:
生物技术
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食品安全
·
分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
TALANTA
·
2022
·
IF 6.556
Siwenjie Qian,Yanju Chen,Cheng Peng,Xiaofu Wang,Hui Wu,Yang Che,Huanying Wang,Junfeng Xu,Jian Wu
African swine fever virus (ASFV) can cause highly contagious and fatal disease among domestic pigs, resulting in considerable economic losses for swine breeders. There is a strong demand for accurate, rapid, and simple detection methods especially for on-site application. Nucleic acid testing is the most commonly used method for ASFVdetection. However, traditional nucleic acid purification step is time- and labor-consuming. The nucleic acid purification, amplification and amplicons detection rely on laboratory settings which limits the on-site detection. Here, we proposed a simple and cost-effective detection method that utilized filter paper to purify nucleic acids from swine blood and employed CRISPR/Cas12a-mediated loop-mediated isothermal amplification (LAMP) reaction to detect ASFV. The filter paper which was made into dipsticks could effectively purify nucleic acids from whole blood in 2 min. This simple and low-cost purification method avoided multiple pipetting steps and potential amplification inhibitors (e.g., ethanol) that were generally used in traditional nucleic acids extraction processes. After nucleic acid purification, the lyophilized LAMP reagent dissolved by elution solution was employed to perform isothermal amplification reaction on a portable heating block. The CRISPR/Cas12a system was designed to specifically detect amplicons. Assisted by a portable homemade device, the fluorescent signals produced by positive samples could be observed by the naked eye, while negative samples remained colorless. The whole detection procedure could be finished within 50 min with a detection limit of one copies/μL. This established method provided a novel strategy for rapid visualized detection and showed great potential for on-site application.
研究领域:
兽医学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF NANOBIOTECHNOLOGY
·
2024
·Vol. 22
·期 1
·1-13
·
IF 10.6
Zhang Lifeng,Luo Shihua,Li Wenbin,Su Wanting,Chen Siting,Liu Chunchen,Pan Weilun,Situ Bo,Zheng Lei,Li Ling,Yan Xiaohui,Zhang Ye
Rapid and sensitive nucleic acid detection is vital in disease diagnosis and therapeutic assessment. Herein, we propose a co-freezing localized CRISPR-Cas12a (CL-Cas12a) strategy for sensitive nucleic acid detection. The CL-Cas12a was obtained through a 15-minute co-freezing process, allowing the Cas12a/crRNA complex and hairpin reporter confined on the AuNPs surface with high load efficiency, for rapid sensing of nucleic acid with superior performance to other localized Cas12a strategies. This CL-Cas12a based platform could quantitatively detect targets down to 98 aM in 30 min with excellent specificity. Furthermore, the CL-Cas12a successful applied to detect human papillomavirus infection and human lung cancer-associated single-nucleotide mutations. We also achieved powerful signal amplification for imaging Survivin mRNA in living cells. These findings highlight the potential of CL-Cas12a as an effective tool for nucleic acid diagnostics and disease monitoring.Graphical abstract
研究领域:
生物技术
·
分子生物学
·
诊断学
·
纳米技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2024
·
IF 8
Dayong Li,Yanheng Yao,Wenting Cheng,Feifan Yin,Miao He,Yang Xiang
In this work, we constructed a manganese ion enhanced CRISPR fluorescence analysis platform based on magnetic separation technology and CRISPR/Cas12a system for the amplification-free detection of SARS-CoV-2 RNA. Firstly, nucleic acid functionalized magnetic beads and dual-function signal probes were used to identify conserved sequences of target RNA at the same time, and then the target was captured and separated by magnetic enrichment. The dual-function signal probe is used to bridge the recognition of the target and the output of the fluorescence signal. And the CRISPR activation sequence on the signal probe is used to initiate the downstream CRISPR/Cas12a fluorescence system to generate fluorescent signals. In this method, we used manganese ions to replace the traditional magnesium ions to assist the CRISPR/Cas12a system, which significantly enhanced the analysis effect. Moreover, we designed a variety of signal probes for different regions of the target RNA to further improve the signal intensity. This method can accurately detect the target RNA within 45 minutes, and can effectively identify the positive cases of COVID-19 infection in clinical throat swab samples, which has great potential for clinical application.
研究领域:
分子生物学
·
医学诊断
·
生物化学
✓ 已验证
产品关联
DOI
高影响因子
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