MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Xuan Wu,Ning-Ning Pi,Fei Deng,Shi-Yang Tang,Cheng-Chen Zhang,Lu Zhu,Fang-Fang Sun,Xiao-Yao He,Han-Qing Li,Shi-Long Zhao,Rong Xiang,Yi Li
Periodontitis not only leads to tooth loss but is also associated with various systemic symptoms. Porphyromonas gingivalis ( Pg ) is the microorganism most closely linked to periodontitis. A rapid and accurate Pg detection method holds significant potential health benefits for patients. While several nucleic acid amplification-based diagnostic methods have been developed, there is a current lack of rapid diagnostic tools for Pg , particularly at chair-side level. Here, two detection methods for the Pg 16S gene based on the CRISPR/Cas12a system have been developed. To achieve ultra-high sensitivity in the developed CRISPR/Cas12a assays without pre-amplification, we designed a circular DNA molecule (termed as Cir-amplifier) which amplifies the signal output of the Cas12a reactions in a cascade response manner. The Cir-amplifier enhanced Fluorescence-Cas12a reaction achieved a minimum detectable limit of 0.39 CFU of Pg within 80-minute under room-temperature. In addition, the Cir-amplifier enhanced Lateral Flow Detection (LFD)-Cas12a reaction has demonstrated the ability to detect a minimum of 3.9 CFU of Pg in 90 min, with results observable by the naked eye. The potential of the two methods for Pg detection in clinical samples has been demonstrated, showing 100 % sensitivity compared to the traditional real-time PCR method. In summary, we have established two efficient detection methods for Pg that eliminate the need for pre-amplification, hence no amplicon contaminations, making them promising candidates for chair-side Pg diagnosis. Furthermore, the Cir-amplifier has been demonstrated to be adaptable to CRISPR/Cas systems producing various signal formats. The Cir-amplifier is expected to become a universal CRISPR/Cas enhancer.
研究领域:
分子生物学
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微生物学
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诊断学
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生物技术
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DOI
高影响因子
Device
·
2024
·
IF 0
Siyi Zou,Jingnan Li,Sheng Lu,Danyu Li,Yuxuan Liu,Weiguo Zhang,Xiaolin Cui,J. Justin Gooding,Kang Tian,Guozhen Liu
Inflammation quantification through the detection of cytokines and inflammatory factors in a point-of-care scenario is crucial for managing and preventing diseases. Achieving early-stage inflammation quantification in osteoarthritis (OA), before the radiographic findings become apparent, may provide an opportunity for accurate diagnosis and effective therapy, potentially preventing joint degeneration. Here, we developed a glass-fiber-based universal system with CRISPR-Cas12a-based immunosensors (GUSCI) for simultaneous detection of multiple inflammation biomarkers using a portable fluorescence detection device. GUSCI enables the detection of five inflammation biomarkers (interleukin-4 [IL-4], IL-6, tumor necrosis factor alpha [TNF-α], interferon γ [IFN-γ], and matrix metalloproteinase 3 [MMP-3]) with picomolar sensitivity in a 10-μL sample while having less than 75 min of assay time and a smartphone signal readout. A cohort study of 35 synovial samples from OA patients revealed differences in inflammation levels. This “sample in, answer out” platform not only aids in understanding OA progression but also offers a versatile strategy for point-of-care diagnosis of a wide range of diseases in resource-limited settings.
研究领域:
生物传感器
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分子诊断
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炎症疾病
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骨关节炎
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即时检测
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DOI
SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2024
·
IF 4.3
Bowen Xiong,Xiang Li,Yuting Zhang,Changling Yan,Gongke Wang
The overexpression of vascular endothelial growth factor 165 (VEGF 165 ) in cancer cells plays a pivotal role in promoting tumor metastasis by facilitating their excessively rapid proliferation and division. Hence, the development of analytical methods possessing high sensitivity and resistance to interference is imperative for the detection of VEGF 165 . Various types of aptasensors have been devised for VEGF 165 detection; however, the performance of these biosensors can be influenced by non-target signals caused by conformational changes in unbound aptamers. The paper shows the creation of a precise and sensitive fluorescence biosensor designed to detect VEGF 165 by using a VEGF 165 -specific split aptamer. Additionally, this biosensor employs nicking enzyme-assisted DNA walker coupling with CRISPR-Cas12a to achieve dual-signal amplification. The VEGF 165 calibration curve shows a detection limit of 268 fM and has a broad linear range from 5 to 4000 nM. The fluorometric biosensor was utilized to detect VEGF 165 in human serum and cellular homogenate samples, yielding good outcomes. The innovative design serves as proof of concept and demonstrates significant potential in detecting various targets.
研究领域:
生物技术
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分子生物学
·
医学诊断
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DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2023
·Vol. 190
·期 11
·1-9
·
IF 5.7
Wang Jiaying,Wang Xianliang,Li Bin,Zhang Kai,Mao Jingyuan
An electrochemical biosensor is reported for controlling CRISPR/Cas12a activity through the utilization of entropy-driven reactions, alongside the construction of a highly sensitive biosensor for B-type natriuretic peptide (BNP) detection. In the biosensor, entropy-driven reactions are employed to regulate the activity of CRISPR/Cas12a - a gene editing tool - capable of nonspecific cleavage of single-stranded DNA (ssDNA). The biosensor architecture encompasses an electrode that is modified with ssDNA probes designed to hybridize with target BNP aptamers. These aptamers, furnished with labeled ssDNA triggers, facilitate the activation of CRISPR/Cas12a through interaction with its guide RNA. Upon the presence of BNP, it associates with the aptamers, subsequently liberating the triggers that instigate the entropy-driven reactions. As a consequence of these reactions, more stable duplexes emerge between the triggers and guide RNA, thereby activating CRISPR/Cas12a. The activated CRISPR/Cas12a subsequently executes cleavage of ssDNA probes residing on the electrode surface, culminating in the generation of an electrochemical signal directly (the calibration plots of differential pulse voltammetric detection were acquired at a working potential of 0.2 V (vs. ref. electrode)) proportional to the BNP concentration. Validation of the biosensor’s performance is undertaken, wherein BNP detection is demonstrated in both buffer and human serum samples. Evident in the findings is the biosensor’s discernible sensitivity and specificity for BNP detection, exemplified by a detection limit of 13.53 fM and a lack of interference originating from other cardiac biomarkers, respectively. Furthermore, the biosensor’s potential to discriminate between healthy individuals and those afflicted by heart failure, predicated on distinctive BNP levels, is illustrated. Graphical Abstract
研究领域:
分子生物学
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生物医学工程
·
临床检验
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DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
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2020
·Vol. 412
·期 3
·601-609
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IF 3.637
Zhao Xianxian,Zhang Wenqing,Qiu Xiaopei,Mei Qiang,Luo Yang,Fu Weiling
Numerous studies have shown that exosomes are closely related to the pathogenesis of various diseases, especially cancers. Therefore, a rapid and sensitive method for exosome detection will be of great importance for the diagnosis and prognosis of diseases. We report here a method for exosome detection based on the CD63 aptamer and clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system. This method consists mainly of exosomal membrane protein recognition based on the CD63 aptamer and signal amplification based on CRISPR/Cas12a. The CD63 aptamer, as an easily adaptable nucleic acid strand, is responsible for the conversion of the amounts of exosomes into nucleic acid detection, whereas CRISPR/Cas12a is responsible for highly specific nucleic acid signal amplification. The detection range of the method was determined as 3 × 10 3 –6 × 10 7 particles per microliter. Additionally, we successfully applied this method to detect exosomes in clinical samples from both healthy individuals and patients with lung cancer, and the results were highly consistent with those obtained by nanoparticle tracking analysis. In general, this method provides a highly sensitive and specific method for the detection of exosomes and offers an avenue toward future exosome-based diagnosis of diseases.
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产品关联
DOI
PubMed
高影响因子
ACS Sensors
·
2026
·
IF 10.9
Lin Hu,Honghua Hu,Fang Liu,Xingrong Li,Qinghua Wu,Xuhuai Fu,Diangeng Li,Xuan Wu,Rong Xiang,Rongzhong Huang,Xiaosong Li,Yi Li
DNA methyltransferases (MTases) are important epigenetic enzymes involved in DNA methylation regulation and have attracted considerable interest as analytical targets in disease-related epigenetic studies. However, traditional detection methods are constrained by laboratory-bound, sophisticated instrumentation and time-consuming protocols, while emerging approaches either depend on complex enzymatic amplifications or suffer from uncontrolled background signals, limiting their bioanalysis utility for precision-medicine-related applications. Inspired by the structure-controlled activation mechanism of CRISPR/Cas12a, we designed a branched split structure to regulate Cas12a for stepwise enzymatic activation and established the Cas12a-induced DNA MTase Sensor (CDMS), which enables highly sensitive MTase activity detection without the need for nucleic-acid amplification step. The core strategy lies in designing split-and-reconstituted Cas12a-activating DNA fragments with steric hindrance effects. This design significantly inhibits nonspecific Cas12a activation while efficiently restoring its activity upon removal of steric hindrance in response to DNA MTase activity. This method achieves ultra-low detection limit of 1.06 × 10-6 U/mL for M.SssI MTase and 7.41 × 10-8 U/mL for Dam MTase, exhibiting excellent specificity and robust applicability for MTase inhibitor screening. We also developed a palm-sized portable optical device, named iCas-Analyzer, to further explore the CDMS's potential for point-of-care testing (POCT), which can be easily operated by nonexpert end users. With device integration, the CDMS extends CRISPR-based diagnostics into the field of epigenetics, holding its potential for future decentralized MTase analysis and POCT-oriented assay development.
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高影响因子
New Biotechnology
·
2021
·
IF 5.079
Ke Liu,Yang Gao,Zhen-Hai Li,Min Liu,Feng-Qing Wang,Dong-Zhi Wei
Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum , including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.
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PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2026
·Vol. 54
·期 5
·
IF 15
Wang Zhongzhong,Wu Yi,Wang Zixuan,Zhang Simeng,Liu Hao,Nie You,Chen Kexin,Huang Yanjun,Zhou Yu,Cao Yuanyuan,Sun Lin,Hao Rongzhang
CRISPR–Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.
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DOI
PubMed
高影响因子
TALANTA
·
2023
·
IF 6.1
Wenping Xing,Qian Li,Cong Han,Dongdong Sun,Zheng Zhang,Xiaona Fang,Yu Guo,Feng Ge,Wei Ding,Zhaofeng Luo,Liyun Zhang
The CRISPR/Cas systems have provided wide biosensing applications. Particularly, the aptamer-involved CRISPR/Cas sensor system powerfully expanded to non-nucleic-acid targets. However, tailoring the sequence of the aptamer to explore the relationship between affinity and the activation of CRISPR/Cas12a trans -cleavage activity has not been reported yet. Herein, we developed a series of new aptamers toward the spike protein 1(S1) of SARS-CoV-2. Surface plasmon resonance measurements showed that the affinity of these aptamers to S1 was at the nM level. Subsequently, a “SET” effect (Sequence Essential Trans -cleavage activity) is discovered for the activation of CRISPR/Cas12a trans -cleavage activity. That is, an aptamer, as the activator, sequence needs to be tailored to activate CRISPR/Cas12a efficiently. A balance should be reached between affinity and activation ability. On the one hand, high affinity ensures target recognition performance, and on the other hand, activation can achieve adequate amplification and output of recognition signals. The optimized sequence (with 27 nucleotides, for short 27-nt) not only recognizes the target with a high affinity and specificity but also can trigger the CRISPR/Cas12a trans -cleavage activity efficiently, showing an excellent detection performance in electrochemical biosensors. The detection limit for SARS-CoV-2 S1 can be low at 1.5 pg mL −1 . The new CRISPR/Cas12a-derived aptasensor also displays a remarkable ability to detect Beta, Delta, and Omicron variants but is selective toward other kinds of proteins. Above all, it is robust for point-of-care testing (POCT) in complex biological fluids, such as saliva, urine, and serum, and provides a universal and scalable detecting platform. Our results provide new insights into aptamer development and a different strategy for COVID-19 antigen detection and biosensor development.
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DOI
PubMed
高影响因子
ACS Sensors
·
2021
·
IF 7.711
Yongzhong Jiang,Menglu Hu,An-An Liu,Yi Lin,Linlin Liu,Bo Yu,Xiaoming Zhou,Dai-Wen Pang
The outbreak of COVID-19 caused a worldwide public health crisis. Large-scale population screening is an effective means to control the spread of COVID-19. Reverse transcription–polymerase chain reaction (RT-qPCR) and serology assays are the most available techniques for SARS-CoV-2 detection; however, they suffer from either less sensitivity and accuracy or low instrument accessibility for screening. To balance the sensitivity, specificity, and test availability, here, we developed enhanced colorimetry, which is termed as a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (M-CDC), for SARS-CoV-2 detection. By this method, SARS-CoV-2 RNA from synthetic sequences and cultured viruses can be detected by the naked eye based on gold nanoparticle (AuNP) probes, with a detection limit of 50 RNA copies per reaction. With CRISPR/Cas12a-assisted detection, SARS-CoV-2 can be specifically distinguished from other closely related viruses. M-CDC was further used to analyze 41 clinical samples, whose performance was 95.12%, consistent with that of an approved Clinical RT-qPCR Diagnosis kit. The developed M-CDC method is not dependent on sophisticated instruments, which makes it potentially valuable to be applied for SARS-CoV-2 screening under poor conditions.
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DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2021
·
IF 10.618
Peng-Fei Liu,Kai-Ren Zhao,Zhi-Jun Liu,Li Wang,Shu-Ying Ye,Guo-Xi Liang
CRISPR/Cas system have drawn increasing attention in accurate and sensitive nucleic acids detection. Herein, we reported a novel Cas12a-based electrochemiluminescence biosensor for target amplification-free human papilloma virus subtype (HPV-16) DNA detection. During this detection process, Cas12a employed its two-part recognition mechanism to improve the specificity and trans-cleavage capability to achieve signal amplification , while L-Methionine stabilized gold nanoclusters (Met-AuNCs) were served as high-efficiency ECL emitters to achieve ECL signal transition. Given the unique combination of Cas12a with ECL technique, the detection limit was determined as 0.48 pM and the whole detection could be completed within 70 min. We also validated the practical application of the proposed biosensor by using undiluted human blood samples, which gives impetus to the design of new generations of CRISPR/Cas detection system beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies.
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产品关联
DOI
PubMed
高影响因子
Analytical Methods
·
2023
·Vol. 15
·期 17
·2105-2113
·
IF 3.1
Furong Zhao,Pei Wang,Haoxuan Wang,Sirui Liu,Muhammad Sohail,Xing Zhang,Bingzhi Li,He Huang
The spread of the monkeypox virus (MPXV) from Central and West Africa to previously non-endemic regions has caused a global panic. In this context, the rapid, specific, and ultrasensitive detection of MPXV is crucial to contain its spread, though such technology has seldom been reported. Herein, we proposed an MPXV assay combining recombinase-aided amplification (RAA) and CRISPR/Cas12a. This assay targeted the highly conserved MPXV F3L gene and demonstrates a low detection limit (LOD) of 101 copies per μL. By leveraging the high specificity nature of RAA and CRISPR/Cas12a, we rationally optimized probes and conditions to achieve high selectivity that differentiates MPXV from other orthopox viruses and current high-profile viruses. To facilitate on-site screening of potential MPXV carriers, a kit integrating lateral flow strips was developed, enabling naked-eye MPXV detection with a LOD of 104 copies per μL. Our RAA-Cas12a-MPXV assay was able to detect MPXV without the need for sophisticated operation and expensive equipment. We believe that this assay can be rapidly deployed in emerging viral outbreaks for on-site surveillance of MPXV.
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PubMed
高影响因子
危险品化学品经营许可证（带存储）