Aggregate
·
2024
·
IF 13.9
Yi Long,Shurui Tao,Dongni Shi,Xingyu Jiang,Tian Yu,Yingxi Long,Libing Song,Guozhen Liu
Developing the sensitive point-of-care testing (POCT) of oncogenic nucleic acids from human papillomavirus (HPV) infection is essential in preventing cervical cancer, especially in resource-limited settings. Rolling circle amplification (RCA) is attractive in achieving POCT via nucleic acid-based aggregation under isothermal conditions. However, the influence of RCA product structure on the aggregation remains unexplored resulting in limited sensitivity. Here, a minimum secondary structured RCA technique (MSS-RCA) is developed by designing a unique circular template, demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer under isothermal conditions. The amplification efficiency of MSS-RCA could be kinetically manipulated by controlling the secondary structure of the circular template. Introducing the invertase probe to MSS-RCA, HPV16 E6/E7 nucleic acid target was detected with a personal glucose meter (PGM) with a sensitivity of 5 f m (50 zmol in 10 µL). This integrated MSS-RCA-PGM detection system was successfully applied to detect HPV16 E6/E7 mRNA extracted from 54 cervical swab samples reaching a positive predictive value of 100.00% and negative predictive values of 96.00% (77.77% to 99.40%, 95% CI). MSS-RCA-PGM provides a sensitive POCT platform for the detection of nucleic acid biomarkers for screening of cervical cancer or other diseases.
研究领域:
分子生物学
·
癌症筛查
·
即时诊断
·
核酸扩增
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产品关联
DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Yuqi Yang,Shengjun Bu,Xiaoying Zhang,Qiqing Duan,Heyuan Meng,Zhuo Hao,Xiuxia He,Jiayu Wan
Infectious diseases caused by the influenza virus are the primary global concern. This study aimed to develop an electrochemical biosensor for detecting influenza A H1N1 virus based on duplex-specific nuclease (DSN) and rolling circle amplification (RCA) technologies. A single-stranded DNA (ssDNA) probe, comprising a recognition sequence for a target sequence of influenza virus H1N1-specific RNA sequence and primer sequence for initiating RCA, was designed and synthesized. A specific sequence of target H1N1 RNA triggered cyclical DSN cleavage of the probe. The released primer sequence then initiated RCA to generate detectable ssDNA-labeled multiple biotins. ssDNA was captured by streptavidin (SA) on the electrode surface and labeled with free Streptavidin-Horseradish Peroxidase (SA-HRP). The H1N1 influenza virus was quantitatively analyzed based on the changing current signal generated on the electrode surface. A linear range from 10 pM to 100 nM and a limit of detection of 0.44 pM were recorded. In addition, the biosensor demonstrated excellent selectivity, repeatability, and stability for analyzing H1N1 in real samples. This biosensor could be applied for the clinical diagnosis of influenza A H1N1 virus.
研究领域:
医学诊断
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分子生物学
·
生物传感技术
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产品关联
DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Taorong Shen,Xing Huang,Yanfei Zhang,Yanli Tong,Yakun Shi,Jianhe Guo,Xiaoyong Zou,Yuzhi Xu,Zong Dai
Fat mass and obesity-associated protein (FTO) plays a crucial role in regulating the dynamic modification of N6-methyladenosine (m6A) in eukaryotic mRNA. Sensitive detection of the FTO level and efficient evaluation of the FTO demethylase activity are of great importance to early cancer diagnosis and anticancer drug discovery, which are currently challenged by limited sensitivity/precision and low throughput. Herein, a robust strategy based on the dephosphorylation switch DNAzyme-rolling circle amplification (RCA) circuit, termed DSD-RCA, is developed for highly sensitive detection of FTO and inhibitor screening. Initially, the catalytic activity of DNAzyme is silenced by engineering with an m6A modification in its catalytic core. Only in the presence of target FTO can the methyl group on DNAzyme be eliminated, resulting in the activation of the catalytic activity of DNAzyme and thus cleaving the hairpin substrate to release numerous primers. Different from the conventional methods that use the downstream cleavage primer with the original 3′-hydroxyl end directly as the RCA primer with the problem of high background signal, which should be compensated by additional separation and wash steps in heterogeneous format, our DSD-RCA assay uses the upstream cleavage primer with a 2′,3′-cyclic phosphate terminus at the 3′-end serving as an intrinsically blocked 3′ end. Only after a dephosphorylation reaction mediated by T4 polynucleotide kinase can the upstream cleavage primers with a resultant 3′-hydroxyl end be extended by RCA. With the high signal-to-noise ratio and homogeneous property, the proposed platform can sensitively detect FTO with a limit of detection of 31.4 pM, and the relative standard deviations (RSDs %) ranging from 0.8 to 2.0% were much lower than the heterogeneous methods. The DSD-RCA method was applied for analyzing FTO in cytoplasmic lysates from different cell lines and tissues of breast cancer patients and further used for screening FTO inhibitors without the need for separation or cleaning, providing an opportunity for achieving high throughput and demonstrating the potential applications of this strategy in disease diagnostics, drug discovery, and biological applications.
研究领域:
生物化学
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分子生物学
·
癌症研究
·
药物发现
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Junzhuo Shan,Yixiao Sheng,Lun Luo,Wenhai Wang,Xinjiang Liu,Yi Ma,Jufang Wang
Apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme involved in DNA repair and cellular redox regulation, and is frequently overexpressed in tumor cells. This highlights the urgent need for a rapid and high-sensitive point-of-care testing (POCT) strategy for APE1 to facilitate early cancer diagnosis. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification method; however, its application in APE1 detection remains rare. Here, we introduce a versatile RCA-Lock (Rlock) conversion platform that enables the transformation of RCA-based nucleic acid detection technologies into APE1-responsive assays. Building upon this platform, we further developed a novel POCT method for APE1 detection─Rlock-mediated, CRISPR/Cas12a-driven RCA cycle (RCRE)─which, for the first time, integrates CRISPR/Cas12a with RCA into a one-pot APE1 detection system. The RCRE assay achieves a limit of detection of 8.86 × 10–4 U/mL within 30 min, while requiring minimal equipment, low cost, and no complex handling procedures. This Rlock-based conversion strategy represents a transformative advance in the field of APE1 diagnostics and offers conceptual inspiration for the design of programmable nucleic acid–based biosensors. The resulting RCRE assay significantly broadens the technological landscape for early cancer detection and paves the way for future clinical translation.
研究领域:
分子诊断学
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生物化学
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生物技术
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癌症研究
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遗传学
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分析化学
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产品关联
DOI
PubMed
高影响因子
Biosensors-Basel
·
2024
·Vol. 14
·期 12
·618
·
IF 4.9
Shurui Tao,Yi Long,Guozhen Liu
The Phi29 DNA polymerase is renowned for its processivity in synthesizing single-stranded DNA amplicons by rolling around a circularized DNA template. However, DNA synthesis rolling circle amplification (RCA) is significantly hindered by the secondary structure in the circular template. To overcome this limitation, an engineered circular template without secondary structure could be utilized to improve the sensitivity of RCA-based assays without increasing its complexity. We herein proposed an entropy-driven special RCA technology for the detection of HPV16 E7 gene at room temperature. The strategy is composed of a molecular beacon containing a loop region for nucleic acid target recognition and a stem region to initiate RCA. With the target analyte, the stem region of the molecular beacon will be exposed and then hybridized with a special circular template to initiate the DNA amplification. We tested different designs of the molecular beacon sequence and optimized the assay’s working conditions. The assay achieved a sensitivity of 1 pM in 40 min at room temperature. The sensitivity of this assay, at 1 pm, is about a hundred-fold greater than that of conventional linear RCA performed in solution. Our proposed sensor can be easily reprogrammed for detecting various nucleic acid markers by altering the molecular beacon’s loop. Its simplicity, rapid assay time, and low cost make it superior to RCA sensors that utilize similar strategies.
研究领域:
分子生物学
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生物技术
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纳米技术
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分析化学
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产品关联
DOI
PubMed
高影响因子
Nature Protocols
·
2021
·Vol. 16
·期 12
·5460-5483
·
IF 13.491
Yao Chi,Zhang Rui,Tang Jianpu,Yang Dayong
DNA hydrogels have unique properties, including sequence programmability, precise molecular recognition, stimuli-responsiveness, biocompatibility and biodegradability, that have enabled their use in diverse applications ranging from material science to biomedicine. Here, we describe a rolling circle amplification (RCA)-based synthesis of 3D DNA hydrogels with rationally programmed sequences and tunable physical, chemical and biological properties. RCA is a simple and highly efficient isothermal enzymatic amplification strategy to synthesize ultralong single-stranded DNA that benefits from mild reaction conditions, and stability and efficiency in complex biological environments. Other available methods for synthesis of DNA hydrogels include hybridization chain reactions, which need a large amount of hairpin strands to produce DNA chains, and PCR, which requires temperature cycling. In contrast, the RCA process is conducted at a constant temperature and requires a small amount of circular DNA template. In this protocol, the polymerase phi29 catalyzes the elongation and displacement of DNA chains to amplify DNA, which subsequently forms a 3D hydrogel network via various cross-linking strategies, including entanglement of DNA chains, multi-primed chain amplification, hybridization between DNA chains, and hybridization with functional moieties. We also describe how to use the protocol for isolation of bone marrow mesenchymal stem cells and cell delivery. The whole protocol takes ~2 d to complete, including hydrogel synthesis and applications in cell isolation and cell delivery.
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Yuan Kong,Zhaoying Liu,Guizhu Wu,Xiaoyong Liu,Song Fang,Kun Zeng,Zhen Zhang,Ming Li
Background Portable biosensing strategies are promising rapid and on-site detection methods, possessing the efficiency and accessibility for detecting target. Hydrogel present numerous advantages in rapid analysis owing to their remarkable biological loading capabilities and optical properties. However, traditional hydrogels suffer from some drawbacks, such as poor signal intensity and slow responsiveness, restricting their further utilization in practice. Results To address these challenges, an enhanced DNA cryogel biosensing platform based on dual-RCA and dual-enzyme had proposed for quantification of zearalenone (ZEN) in this study. The MB@Apt-cDNA probe and DNA cryogel were prepared in advance to provide accurate recognition and portable interface. When ZEN presented, the released cDNA aroused rolling-circle amplification (RCA) to amplify signal, and then introduced dual-enzyme system by coupling glucose oxidase and horseradish peroxidase. On the dual-RCA-based DNA cryogel, the coloration in situ displayed under self-powered H 2 O 2 system. The results identified through visualization, smartphone and color chart, exhibiting a limit of detection (LOD) at 6.95 pg/mL, with detection range between 10 pg/mL to 20 ng/mL. This biosensing showed satisfactory specificity, accuracy, efficiency and accessibility by its application in spiked and real samples. Furthermore, the highlights, including high integration, simplified operation and portable reading, had demonstrated. Significance The enhanced chromogenic cryogel platform can provide by integrating advanced strategies, which may have promising prospects for portable monitoring.
研究领域:
生物传感器
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分析化学
·
食品安全
·
分子诊断
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纳米生物技术
·
环境监测
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Applied Bio Materials
·
2019
·
IF 0
Jiantao Yu,Jing Li,Shiyao Zhai,Li Lin,Kui Wang,Bin Tang,Hong Meng,Leilei Tian
Polymerization of small interfering RNA (siRNA) has been demonstrated as a promising strategy to improve siRNA delivery, which will change the low-charge and rigid properties of single siRNA and enhance its electrostatic interactions with cationic polymers. For such polymerization strategy, a major breakthrough is still needed to fully eliminate chemical processes and further improve the nanocomplex-forming ability of polymerized siRNAs. Herein, the extremely strong interaction between the DNA product of rolling circle amplification (RCA) and linear poly(ether imide) (PEI) has been disclosed; accordingly, a stable nanocomplex is formed just at its charge neutralization point, which benefits from the high molecular weight of the RCA product (>3 000 000 Da). In addition, as the sequence of the RCA product is determined by the cyclic template, the programmable nature of DNA can simplify the optimization process and maximize the hybridization efficiency between RCA and sticky siRNAs, realizing a superior siRNA polymerization efficiency. Depending on these two effects, the RCA DNA is utilized as a cocarrier material to organize siRNA polymerization and substantially reduce the usage amount of PEI, which greatly improves RNAi efficiency of PEI/RCA-siRNA polyplex both in vitro and in vivo, providing evidence that RCA DNA is a promising material to promote the RNAi-based therapeutics.
研究领域:
分子生物学
·
纳米医学
·
治疗开发
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产品关联
DOI
PubMed
ANALYTICA CHIMICA ACTA
·
2026
·
IF 6.1
Ying Shen,Yunlong Xiao,Lu Liu,Suzhen Zhai,Renwu Mu,Jiahui Chen,Xiaoyan Yang,Xiaojun Wen,Chunlin Zhang,Hai Shi
The sensitive, point-of-care detection of acute myeloid leukemia (AML)-derived exosomes is essential for non-invasive diagnosis and therapy monitoring, but remains technically challenging. Here we report a colorimetric biosensor based on a dual -aptamer-initiated catalytic cascade, where simultaneous recognition of exosomal markers CD63 and PTK7 by engineered aptamers triggers an in situ rolling circle amplification (RCA) reaction. The resulting RCA product templates the assembly of a metal-organic framework (MOF) nanozyme, which acts as an efficient peroxidase mimic to generate a colorimetric readout. This integrated “recognition -amplification-transduction” nanoarchitecture achieves a detection limit of 0.1 ng/mL for PTK7-positive exosomes and successfully discriminates patient serum samples from healthy controls as well as AML-bearing mouse models from healthy mice, with consistent results obtained via UV-vis spectroscopy, visual colorimetry, and smartphone-based grayscale analysis. By synergizing nucleic acid nanotechnology with nanozyme catalysis, this work provides a rapid, equipment-free visual platform for exosome-based liquid biopsy in AML management.
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DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Yaqi Wang,Xinning Wang,Xin Ji,Yunxuan Weng,Xiaoying Zhao
Zearalenone (ZEN) is a mycotoxin that poses severe health risks to humans and livestock through contaminated cereals and feed. In this study, a hydrogel-based sensor was developed for sensitive visual detection of ZEN. The sensor integrates a DNA aptamer-crosslinked polyacrylamide hydrogel encapsulating gold nanoparticles (AuNPs) with a rolling circle amplification (RCA)-assisted CRISPR/Cas12a system for signal enhancement. Upon ZEN binding, the released complementary DNA triggers RCA, generating repetitive DNA strands that activate Cas12a. The activated Cas12a efficiently cleaves numerous single-stranded DNA crosslinkers within the hydrogel, leading to substantial dissociation and release of AuNPs for colorimetric readout. This strategy achieves high specificity and a significantly improved detection limit of 1.5 nM for ZEN, which is over 3000-fold lower than that of the aptamer-hydrogel system without amplification. The sensor enables rapid, simple, and cost-effective on-site quantification of ZEN, with the detection sensitivity and linear range well suited for food safety monitoring applications.
✓ 已验证
DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Qingxin Zhang,Manru Zhang,Zhiqiang Guo,Jingjing Li,Zhixue Zhu,Yu Wang,Su Liu,Jiadong Huang,Jinghua Yu
Ochratoxin A (OTA) is the most toxic class of ochratoxins and has become a major threat to the environment, humans and animals. Therefore, research on the methods for its detection is also more urgent. Herein, we propose a low-background electrochemical biosensor based on a DNA tetrahedron-besieged primer and a DNAzyme-activated programmatic rolling circle amplification (RCA) that can be ultimately utilized for OTA detection in wine samples. Low-background detection can be achieved using the besieged primer via sequenced assembly of DNA tetrahedral nanostructures so that non-specific extensions of primer can be avoided. The target OTA-mediated DNAzyme activation initiates the programmatic RCA. Additionally, the catalytic property of silver nanoclusters (AgNCs) is integrated with the electrochemical assay to achieve high sensitivity for OTA detection. Benefiting from the aforementioned processes, a low-background, and highly sensitive electrochemical biosensor has been successfully constructed. This design is capable of detecting OTA at concentrations from 1 pg/mL to 10 ng/mL, and its lowest concentration limit is 0.773 pg/mL. Simultaneously, its validation in the detection of actual samples reveals that the proposed electrochemical biosensor has a lot of potential in food safety and environmental detection.
研究领域:
生物传感
·
食品安全
·
环境监测
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Water
·
2026
·Vol. 18
·期 9
·1017
·
IF 3.5
Lanhua Liu,Shuchi Li,Jingli Wu,Yuting Mei,Jiahui Su,Bohan Wang,Qiuren Wei,Bo Shen
Mercury ions (Hg2+), a heavy metal contaminant of strong biotoxicity, pose a serious threat to ecosystems and human health in aquatic environments. Developing highly sensitive and specific detection methods is therefore of great importance. This study presents a novel label-free fluorescent biosensor for Hg2+by ingeniously coupling target-induced aptamer switching with rolling circle amplification (RCA). Upon Hg2+binding, the conformational change releases a sequestered primer to initiate RCA, generating G-quadruplex-rich DNA products that produce a strong “turn-on” signal withN-methylmesoporphyrin IX (NMM). Under optimized conditions, the assay exhibits excellent linearity from 10 to 1000 nM with a detection limit of 3.2 nM, along with high selectivity over competing metal ions. Validation using spiked environmental water samples yielded accurate and reproducible recoveries in the range of 93.8% to 106.0%. With its operational simplicity, high sensitivity, and robust performance in complex matrices, this label-free strategy offers a reliable and promising platform for detecting Hg2+in environmental waters.
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DOI
高影响因子
危险品化学品经营许可证（带存储）