Biomolecules
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2025
·Vol. 15
·期 3
·315
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IF 4.8
Dongxia Pan,Mukaddas Mijit,Hui Wang,Chaoqun Sun,Bantan Pingcuo,Zhixue Yu,Benhai Xiong,Xiangfang Tang
The A-to-G mutation (FecB) in theBMPR1Bgene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of theFecBgene is therefore critical for advancing sheep breeding programs. This study aimed to develop a fast and accurate method for detecting theFecBmutation and genotyping the gene to enhance sheep reproduction and productivity. To achieve this, we integrated the CRISPR-Cas12a system with an optimized amplification refractory mutation system (ARMS). A similar DNA origami technique-based fluorescence reporter nanotree structure was synthesized using gold nanomagnetic beads as carriers to amplify the fluorescence signal further. The resulting biosensing platform, termed CRISPR-ARMS, demonstrated excellent sensitivity for detectingFecBmutations, with a detection limit as low as 0.02 pmol. Therefore, this innovative approach shows great promise for single-base mutation detection and represents a pioneering tool for high-yield genetic screening.
研究领域:
分子生物学
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遗传学
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动物科学
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PubMed
高影响因子
TALANTA
·
2023
·
IF 6.1
Dan Zhao,Jiutang Tang,Qin Tan,Xiaohong Xie,Xin Zhao,Dingpei Xing
Constructing an ultrasensitive CRISPR/Cas-based biosensing strategy is highly significant for the detection of trace targets. Here we presented a dual-amplified biosensing method based on CRISPR/Cas13a-triggered Cas12a, namely, Cas13a-12a amplification. As proof-of-principle, the developed strategy was used for miRNA-155 detection. The target bound to the Cas13a-crRNA complex and activated the cleavage activity of Cas13a for cleaving uracil ribonucleotides (rU) in the bulge structure of blocker strand (BS), resulting in the release of primer strand (PS) from the BS modified on magnetic beads. Then, the released PS activated the cleavage activity of Cas12a to cleave single-strand DNA reporter probes, producing a significantly increased fluorescent signal. The detection limit of the Cas13a-12a amplification using synthetic miRNA-155 was as low as 0.35 fM, which was much lower than that of the only Cas13a-based assay. The applied performance of this amplification strategy was verified by accurately quantifying miRNA-155 expression levels in different cancer patients. Therefore, the developed strategy offers a supersensitive and highly specific miRNAs sensing platform for clinical application.
研究领域:
分子生物学
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医学诊断
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生物技术
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DOI
PubMed
高影响因子
CHINESE JOURNAL OF CHEMISTRY
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2023
·
IF 5.4
Xiuyuan Wang,Shusheng Yu,Yuqiu He,Fuan Wang,Xiaoqing Liu
Comprehensive Summary A label-free deoxyribozyme (DNAzyme) biosensor for m 6 A demethylase activity detection is developed. When demethylase FTO (Fat mass and obesity-associated protein), an important m 6 A demethylase of ALKBH demethylase family is present, the m 6 A group is specifically demethylated, and the cleavage activity of DNAzyme is restored. This allows the cleavage product that contains G-quadruplex sequence to bind to thioflavin T (ThT) and generate fluorescence signals. The biosensor shows high specificity and sensitivity, and fast reaction speed. Our study demonstrates a new design of allosteric DNAzyme for sensing. This method represents the first label-free nucleic acid biosensor for FTO assay, providing a feasible route towards inhibitors screening.
研究领域:
生物化学
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分子生物学
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高影响因子
Analytical Methods
·
2015
·Vol. 7
·期 16
·6712-6717
·
IF 1.821
Weiwen Hu,Yong Ning,Lianzhi Li,Jinming Kong,Xueji Zhang
In this work, a highly effective biosensor supported on morpholino-functionalized magnetic microspheres was investigated for the sequence-specific detection of DNA. Briefly, morpholino was first modified on the surface of magnetic microspheres through amido bonds, and then hybridized with target DNA in the ensuing step. Biotin-labeled signal probe DNA was designed to hybridize with the DNA nearby the 5′ end of sequence, and the biotin was used to bind with streptavidin-alkaline phosphatase (SA-ALP). L-Ascorbic acid-2-phosphate (AAP) was hydrolyzed by ALP to generate L-ascorbic acid, which could reduce resazurin to resorufin, resulting in a turn-on fluorescence signal. UV-vis absorbance spectroscopy was applied to prove the conjugation of ALP with signal probe DNA. Under optimal conditions, this biosensor displayed a good linear relationship between the fluorescence intensity and logarithm of single-stranded DNA concentrations in the range of 0.1 pM to 0.1 nM with a low detection limit of 9.32 fM. Moreover, fully complementary versus single-base mismatched, three-base mismatched and non-complementary DNA could be effectively distinguished. In addition, this novel approach rendered satisfactory analytical results for the determination of DNA in serum, thus exhibiting practical significance. These results demonstrated that this assay method displays excellent sensitivity and specificity for DNA detection and has great potential for practical applications.
研究领域:
生物化学
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分子生物学
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DOI
高影响因子
MICROCHEMICAL JOURNAL
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2023
·
IF 4.8
Xianfeng Wang,Chengxiang Chu,Yuanyi Deng,Yi Ma,Mei Yang,Huibo Luo,Danqun Huo,Changjun Hou
Accurate identification of Staphylococcus aureus ( S. aureus ) is crucial for food safety and clinical diagnosis. Particularly, identifying drug-resistant strains guides effective treatment and prevention of infectious diseases. Herein, we presented a highly efficient programmable CRISPR/Cas12a system for identification of methicillin-resistant S. aureus (MRSA). Genus-specific gene (spec gene) and methicillin-resistant gene (mecA gene) of S. aureus were amplified by PCR. According to the sequences of spec gene and mecA gene, corresponding programmable crRNAs were designed and prepared to construct CRISPR/Cas12a system, which recognized and bound to the PCR products through complementary interactions to activate the trans -cleavage activity of Cas12a. This led to the indiscriminate digestion of surrounding FQ-reporter, releasing a distinct fluorescence signal for MRSA identification. In addition, we devised a pair of AuNPs-DNA probes for colorimetric recognition of S. aureus . Target-triggered trans -cleavage for linker probe led to an non-aggregation of AuNPs-DNA probes pair. This led to a noticeable appearance of red color, enabling naked-eye detection. Thus, MRSA identification was realized with remarkable sensitivity and exceptional selectivity. Successful identification of MRSA in clinical samples validated the practical applications of the proposed CRISPR/Cas12a assay, establishing it as a universal platform for diagnosing pathogenic microorganisms with versatility and compatibility.
研究领域:
分子生物学
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医学诊断
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食品安全
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DOI
高影响因子
TALANTA
·
2025
·
IF 6.1
Tao Meng,Qi Kang,Jiashuo Xu,Shuang Zhao,Tianqi Liu,Dianming Zhou,Xiaoqun Gong,Jianyu Zhang
The CRISPR/Cas12a system has become a powerful tool in biosensing because of its specific target recognition ability and highly efficient trans -cleavage activity. However, a problem faced by the CRISPR/Cas12a system when directly used for trace detection is the linear amplification efficiency of single-cycle digestion. Here, we present a novel hairpin reporter-driven CRISPR/Cas12a (HR-CRISPR) amplification system that establishes a positive feedback loop within the CRISPR/Cas12a platform to finish an exponential and sensitive signal amplification in a one-step reaction. As proof of concept, we applied this strategy to the terminal deoxynucleotidyl transferase (TdT) activity assay without pre-amplification procedure. The polyT strand extended by TdT hybridizes with crRNA, activating Cas12a, which then cleaves the FQ-hairpin reporter. The cleavage products are further elongated by reverse transcriptase using crRNA as a template, reactivating Cas12a and producing exponentially amplified fluorescence signals. This assay offers a simple yet highly sensitive approach for quantifying TdT activity, achieving a low detection limit of 4.55 × 10 −6 U. Moreover, it is applicable for inhibitor screening and monitoring TdT activity in human serum samples.
研究领域:
分子生物学
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生物技术
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医学检测
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2024
·
IF 5.7
Bohan Wang,Lanhua Liu,Haopeng Zhang,Zhiqiang Wang,Kang Chen,Bo Wu,Limin Hu,Xiaohong Zhou,Lanlan Liu
Background Quinolones (QNs) widely exist in the environment due to their wide range of applications and poor metabolic properties, resulting in the generation and spread of resistance genes, posing a potential threat to human health. Traditional analytical methods cannot detect all broad ranges of QNs simultaneously. The development of facile, efficient and reliable method for quantification and assessment of the total QNs is a long-lasting challenge. Results We hereby provide a simple, sensitive and instantaneous group-targeting biosensor for the detection of total QNs in environmental water samples. The biosensor is based on a group-specific antibodies with high affinity against QNs. Fluorescent labeled antibodies bound to the coated antigen modified on the surface of the transducer, and excited by the evanescent waves. The detected fluorescent signal is inversely proportional to the QNs concentration. This biosensor exhibited excellent performance with detection limits lower than 0.15 μg L −1 for all five QNs variants, and even lower than 0.075 μg L −1 for ciprofloxacin (CIP) and ofloxacin (OFL). Environmental water samples can be detected after simple pretreatment, and all detection steps can be completed in 10 min. The transducer has a high regenerative capacity and shows no significant signal degradation after two hundred detection cycles. The recoveries of QNs in a variety of wastewater range from 105 to 119%, confirming its application potential in the measurement of total QNs in reality. Significance The biosensor can realize rapid and sensitive detection of total QNs in water samples by simple pretreatment, which overcomes the disadvantage of the traditional methods that require complex pretreatment and time-consuming, and pave the groundwork for expansive development centered around this technology.
研究领域:
环境科学
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分析化学
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生物技术
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DOI
PubMed
高影响因子
COLLOIDS AND SURFACES B-BIOINTERFACES
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2019
·
IF 3.973
Weipan Peng,Yiwen Huang,Qian Zhao,Guowei Lian,Minghui Chen,Jiafang Piao,Xiaoqun Gong,Jin Chang
Considered as the next-generation biomarkers, microRNAs play an important role in the early diagnosis of cancers. Here, we designed a fluorescent signal “removal” sensor for one-step, sensitive and specific detection of multiple microRNAs by flow cytometry (FCM). In this work, single-stranded DNA (ssDNA), working as the interlinkage, immobilized the fluorescent nanosphere (FS) onto the SiO 2 microspheres surface to form the SiO 2 -ssDNA-FS probes. When target miRNAs integrated with SiO 2 -ssDNA-FS probes, the duplex-specific nuclease (DSN) could cleave the ssDNA selectively and release FS with numerous cycles to enhance the fluorescent signal attenuation of SiO 2 -ssDNA-FS, so as to remarkably improve the analysis sensitivity. It achieved a simple, accurate and quantitative microRNA-21 detection for clinical blood samples. Parallel multi-target detection of microRNA-21 and Let-7d was also realized by different color labeled FS. Moreover, our designed sensor was suitable for other targets’ detection with the corresponding probes.
研究领域:
分子生物学
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医学诊断
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生物技术
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DOI
PubMed
高影响因子
SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2022
·
IF 4.831
Ning Wu,Hao Guo,Mingyue Wang,Liping Peng,Yuan Chen,Bingqing Liu,Zhilan Pan,Yinsheng Liu,Wu Yang
Ratio fluorescence has attracted much attention because of its self-calibration properties. However, it is difficult to obtain suitable fluorescent materials with well-resolved signals simultaneously under one excitation. In this work, we report a different strategy, using MIL-68(In)-NH 2 as both the fluorescence element and the scattered light unit, and coupling the fluorescence and the scattered light to construct the fluorescence and scattered light ratio system. Based on the optical properties and the second-order scattering (SOS) of the material nanoparticles , the synthesized MIL-68(In)-NH 2 can be used to realize the ratio detection of Hg 2+ . Because the scattering intensity of small particle MIL-68(In)-NH 2 is weak, SOS is not obvious. When Hg 2+ is introduced the coordination reaction between the amino nitrogen atoms of MIL-68(In)-NH 2 and Hg 2+ make the particles larger, resulting in the decrease of fluorescence and the enhancement of SOS. As a result, a novel Hg 2+ ratiometric detection method is developed by using the dual signal responses of the fluorescence and scattering. Under the optimal conditions (pH = 6, reaction time 5 min, room temperature , and the maximum excitation wavelength 365 nm), the linear range of the method is 0–100 μM, and the detection limit is 5.8 nM (Ksv = 9.89 × 10 9 M −1 ). In addition, the probe is successfully used to evaluate Hg 2+ in actual water samples. Compared with the traditional method of recording only the fluorescence signal, the proposed fluorescence-scattering method provides a new strategy for the design of ratiometric sensors.
研究领域:
材料科学
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分析化学
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环境科学
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DOI
PubMed
高影响因子
SCIENCE OF THE TOTAL ENVIRONMENT
·
2023
·
IF 9.8
Fang Deng,Jiafeng Pan,Manjia Chen,Zhi Liu,Junhua Chen,Chengshuai Liu
Polychlorinated biphenyls (PCBs) are ubiquitous persistent organic pollutants that cause harmful effects on environmental safety and human health. There is an urgent need to develop an intelligent method for PCBs sensing. In this work, we proposed a logic gate biosensing platform for simultaneous detection of multiple PCBs. 2,3′,5,5′-tetrachlorobiphenyl (PCB72) and 3,3′,4,4′-tetrachlorobiphenyl (PCB77) were used as the two inputs to construct biocomputing logic gates. We used 0 and 1 to encode the inputs and outputs. The aptamer was used to recognize the inputs and release the trigger DNA. A catalytic hairpin assembly (CHA) module is designed to convert and amplify each trigger DNA into multiple programmable DNA duplexes, which initiate the trans -cleavage activity of CRISPR/Cas12a for the signal output. The activated Cas12 cleaves the BHQ-Cy5 modified single-stranded DNA (ssDNA) to yield the fluorescence reporting signals. In the YES logic gate, PCB72 was used as the only input to carry out the logic operation. In the OR, AND, and INHIBIT logic gates, PCB72 and PCB77 were used as the two inputs. The output signals can be visualized by the naked eye under UV light transilluminators or quantified by a microplate reader. Our constructed biosensing platform possesses the merits of multiple combinations of inputs, intuitive digital output, and high flexibility and scalability, which holds great promise for the intelligent detection of different PCBs.
研究领域:
环境科学
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生物传感
·
分子生物学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Shuyu Zhu,Shiying Zhou,Liyuan Deng,Jiangbo Dong,Tao Gu,Xinyu He,Liang Jin,Changjun Hou,Danqun Huo
Background Alkaline phosphatase (ALP) can catalyze the hydrolysis of phosphate esters under alkaline conditions, mediate the dephosphorylation of substances such as proteins and nucleic acids. ALP can participate in physiological activities such as bone mineralization, cell division, and immune regulation. Abnormal ALP activity is associated with various diseases, making it an important clinical biomarker. In recent years, highly sensitive detection technologies based on isothermal amplification have developed rapidly. The Rolling Circle Amplification (RCA) technology uses T4 DNA ligase to connect the 5'-phosphate group and 3'-hydroxyl terminus of the padlock probe into a ring, achieving efficient amplification through continuous rolling circle replication. Results Leveraging this characteristic, we developed a highly sensitive sensing method (RCA-LG) based on RCA-generated G43 long nanowires for ALP detection. T4 DNA ligase circularizes the RCT into a template. Subsequently, Phi29 DNA polymerase amplifies the primer along the circular template, producing G43 long nanowires that bind with Thioflavin T (ThT) to generate fluorescent signals. When ALP is present, it dephosphorylates the 5' end of RCT, preventing circular template formation and thereby inhibiting the amplification reaction. The RCA-LG method can detect ALP concentrations as low as 1.0×10 -6 U/mL within 2.5 h. We propose a novel one-step RCA approach that integrates ligation and amplification reactions into a single step, simplifying operations while enhancing amplification efficiency. Furthermore, we physically separated the ALP dephosphorylation and RCA processes to avoid potential contamination from repeated sample handling. Significance and Novelty This strategy takes advantage of the characteristics of RCA without the need for redundant phosphorylation modifications. Moreover, we have proposed an improved one-step RCA method combined with physical partitioning, enabling highly sensitive and specific detection of ALP and allowing the detection of this target in complex samples. Furthermore, the RCA-LG strategy can also play an important role in the screening of enzyme inhibitors.
研究领域:
生物化学
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分子诊断
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医学检测
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2021
·
IF 7.46
Daliang Yu,Yingying Zha,Zheng Zhong,Yiming Ruan,Ziwei Li,Lili Sun,Sen Hou
Dichlorodihydrofluorescein diacetate (DCFH-DA) has been widely used in detection of reactive oxygen species (ROS). Unfortunately, the detection process is easily affected by light irradiation during measurement, which cannot be overlooked especially for samples with a small amount of ROS. In the study, we revealed several factors that influence the ROS detection process and developed a new ROS detection method by DCFH-DA with higher stability and sensitivity. After light irradiation, the fluorescence signal of DCFH-DA solution increased significantly with pH value higher than 7.4. The increase of fluorescence signal was attributed to a self-oxidization process of DCFH-DA, which transferred dichlorodihydrofluorescein (DCFH) to 2′,7′-dichlorofluorescein (DCF) with the presence of DCF. At the beginning stage of light irradiation, i.e., when the amount of DCF was negligible compared to DCFH (DCFH-DA), the ratio of fluorescence density between samples keeps unchanged after light irradiation. On this basis, we developed a stable and sensitive ROS detection method via light irradiation. The method was successfully applied to detect ROS in HeLa cells which was induced by hydrogen peroxide (H 2 O 2 ) and plastic nanoparticles. A new insight into the proportional increase of fluorescence signal by light irradiation was revealed.
研究领域:
生物化学
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细胞生物学
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高影响因子
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