ANALYTICAL CHEMISTRY
·
2024
·
IF 6.7
Jian Lv,Xue Wu,Mansha Wu,Xiaoyuan Wang,Lijuan Gong,Dawei Li,Ruocan Qian
Glass nanopipets have been demonstrated to be a powerful tool for the sensing and discrimination of biomolecules, such as DNA strands with different lengths or configurations. Despite progress made in nanopipet-based sensors, it remains challenging to develop effective strategies that separate and sense in one operation. In this study, we demonstrate an agarose gel-filled nanopipet that enables hyphenated length-dependent separation and electrochemical sensing of short DNA fragments based on the electrokinetic flow of DNA molecules in the nanoconfined channel at the tip of the nanopipet. This nanoconfined electrokinetic chromatography (NEC) method is used to distinguish the mixture of DNA strands without labels, and the ionic current signals measured in real time show that the mixed DNA strands pass through the tip hole in order according to the molecular weight. With NEC, gradient separation and electrochemical measurement of biomolecules can be achieved simultaneously at the single-molecule level, which is further applied for programmable gene delivery into single living cells. Overall, NEC provides a multipurpose platform integrating separation, sensing, single-cell delivery, and manipulation, which may bring new insights into advanced bioapplication.
研究领域:
生物分析化学
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纳米技术
·
分子生物学
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DOI
PubMed
高影响因子
JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY
·
2016
·Vol. 92
·期 4
·819-826
·
IF 2.738
Xiao Liang,Ying Ge,Zhenglong Wu,Weidong Qin
BACKGROUND Hg(II) and MeHg(I) are toxic pollutants causing wide concerns. Polyamidoamine-grafted magnetic silica nanoparticles (Fe 3 O 4 -SiO 2 -PAMAMs) were synthesized as beads, on which herring sperm DNA fragments were immobilized as reacting moieties for removal of Hg(II) and MeHg(I) from water matrices. The factors influencing the removal, such as DNA fragments, generation of the polyamidoamine spacers and pH, were studied. Further, the adsorption mechanisms were explored by means of adsorption kinetics and isotherms. RESULTS Assembling DNA fragments on the polyamidoamine-grafted beads promoted sharply the removal of both Hg(II) and MeHg(I). Adsorptions of Hg(II) and MeHg(I) followed pseudo-second-order kinetics; but their adsorption isotherms obeyed Langmuir and Freundlich models, respectively. Under the optimum conditions, the adsorption equilibrium time was approximately 5 min for Hg(II) and 5 s for MeHg(I) with maximum percentage removals of ∼95% and ∼90%, respectively. The adsorbent could be regenerated easily without significant decrease in removal efficiencies. CONCLUSION The Fe 3 O 4 -SiO 2 -PAMAM beads could bind DNA steadily, and the DNA fragments interacted strongly with mercury species. The strategy enabled the water-soluble DNAs to work as efficient removing reagents for rapid and cost-effective removal of Hg(II) and MeHg(I). © 2016 Society of Chemical Industry
研究领域:
环境科学
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生物化学
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DOI
高影响因子
Scientific Reports
·
2017
·Vol. 7
·期 1
·1-9
·
IF 4.259
Li Lanhui,Jin Mingliang,Sun Chenglong,Wang Xiaoxue,Xie Shuting,Zhou Guofu,van den Berg Albert,Eijkel Jan C. T.,Shui Lingling
DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling system. We expect that hydrodynamic forces generated during the bubbling process shear the DNA molecules, extending and breaking them at the points where shearing forces are larger than the strength of the phosphate backbone. Factors of applied pressure, bubbling time and temperature have been investigated. Genomic DNA could be fragmented down to controllable 1–10 Kbp fragment lengths with a yield of 75.30–91.60%. We demonstrate that the ends of the genomic DNAs generated from hydrodynamic shearing can be ligated by T4 ligase and the fragmented DNAs can be used as templates for polymerase chain reaction. Therefore, in the bubbling system, DNAs could be hydrodynamically sheared to achieve smaller pieces in dsDNAs available for further processes. It could potentially serve as a DNA sample pretreatment technique in the future.
研究领域:
生物技术
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分子生物学
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DOI
PubMed
高影响因子
COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS
·
2020
·
IF 3.99
Si Sun,Qian-Ru Xiao,Cui-Fang Ye,Yong Jiang
Fluorescent nanoparticles have been widely used in the field of biomedical engineering because they are usually biocompatible, cost-effective and homogeneously uniform. In this study, we describe a biomacromolecular fluorescent nanoparticle prepared with a facile disolvating process using bovine serum albumin, DNA segments and SYBR Green I. The obtained co-assemblies, termed [email protected] nanoparticles, had a narrow and relatively uniform size distribution with an average size of about 122 nm and the PDI value 0.087. Cellular uptake results demonstrated well internalization of [email protected] nanoparticles by the Michigan Cancer Foundation-7 (MCF-7) cells. Together with the fluorescent tag of SYBR Green I molecules, [email protected] nanoparticles accumulated in the cell cytoplasm after cellular internalization enabled the MCF-7 cells for fluorescent imaging. This study provided a new strategy for constructing biomacromolecular fluorescent nanoparticles for applications in biomedical engineering and biomedical imaging.
研究领域:
生物医学工程
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分子生物学
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产品关联
DOI
高影响因子
MOLECULAR AND CELLULAR BIOCHEMISTRY
·
2023
·
IF 4.3
Lu Junnan,Tong Ying,Dong Rui,Yang Yijun,Hu Wen,Zhang Minghong,Liu Quan,Zhao Siting,Adams John H.,Qin Li,Chen Xiaoping
CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, to produce “marker-free” genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed.
研究领域:
寄生虫学
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分子生物学
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基因组编辑
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DOI
PubMed
高影响因子
ANALYST
·
2022
·Vol. 147
·期 5
·905-914
·
IF 5.227
Shumin Zhang,Minyi Liu,Haofa Cui,Muhammad Asad Ziaee,Rongwei Sun,Liting Chen,Daqi Chen,Denis Garoli,Jiahai Wang
The fabrication of nanopores with a matched pore size, and the existence of multiple interferents make the reproducible detection of small-sized molecules by means of solid-state nanopores still challenging. A useful method to solve these problems is based on the detection of large DNA nanostructures related to the existence of small-sized targets. In particular, a DNA tetrahedron with a well-defined 3D nanostructure is the ideal candidate for use as a signal transducer. Here, we demonstrate the detection of an L1-encoding gene of HPV18 as a test DNA target sequence in a reaction buffer solution, where long single-stranded DNA linking DNA tetrahedra onto the surface of the magnetic beads is cleaved by a target DNA-activated CRISPR-cas12 system. The DNA tetrahedra are subsequently released and can be detected by the current pulse in a glassy nanopore. This approach has several advantages: (1) one signal transducer can be used to detect different targets; (2) a glassy nanopore with a pore size much larger than the target DNA fragment can boost the tolerance of the contaminants and interferents which often degrade the performance of a nanopore sensor.
研究领域:
生物技术
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纳米技术
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分子生物学
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产品关联
DOI
PubMed
高影响因子
STAR Protocols
·
2023
·
IF 0
Xiaoqian Zeng,Shuliu Wang,Mindong Liang,Weishan Wang,Yue Jiang,Fei Xu,Leshi Liu,Hao Yan,Yaojun Tong,Lixin Zhang,Gao-Yi Tan
Summary Large biosynthetic gene cluster (BGC) cloning is important for discovering natural product-based drugs and remains challenging in high GC content microorganisms (e.g., Actinobacteria). Here, we present an in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments. We describe steps for crRNA design and preparation, genomic DNA isolation, and CRISPR-Cas12a cleavage and capture plasmid construction and linearization. We then detail target BGC and plasmid DNA ligation and transformation and screening for positive clones. For complete details on the use and execution of this protocol, please refer to Liang et al. 1
研究领域:
分子生物学
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生物技术
·
基因工程
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DOI
PubMed
TALANTA
·
2025
·
IF 6.1
Luke Wei,Yuxin Hu,Jiamin Wu,Jing Mao,Tianqin Yin,Jieqiong Qiu
Doxorubicin (DOX), a widely utilized chemotherapeutic antibiotic, increasingly threatens environmental and public health due to its persistence in marine ecosystems, bioaccumulation in organisms, and contamination of agricultural systems. In response to the urgent demand for effective DOX detection, we have developed a label-free fluorescent aptamer biosensor (FAB) that employs SYBR Green I (SG I)-mediated signal amplification. A key innovation in this design is the strategic fragmentation of the 41-mer complementary strand (CP) of the DOX aptamer into three 11-mer fragments. It significantly mitigates nonspecific SG I binding interference caused by the longer ssDNA CP, enhancing the fluorescence signal ratio (F dsDNA /F ssDNA ) from 4.6-fold to 12.6-fold. Upon DOX introduction, competitive binding between DOX, SG I, and DNA further suppresses background noise, yielding an exceptional fluorescence quenching ratio (F 0 /F) of 52.8. The optimized FAB demonstrates ultra-sensitive DOX detection, with a detection limit of 1.41 nM and a linear response between 1.41 nM and 300 nM. The recoveries of DOX spiked into pond water and drinking water samples ranged from 95.1% to 101.8%, with relative standard deviations (RSDs) below 3.46%, demonstrating negligible matrix effects and confirming the FAB method’s robustness for environmental monitoring applications. This strategy synergistically enhances signal transduction efficiency, and strengthens detection reliability, all while maintaining operational simplicity. As a result, this FAB for DOX detection holds transformative potential, poised to revolutionize biosensor technology in the fields of food safety, environmental monitoring, and biosecurity.
研究领域:
生物传感器
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环境科学
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分析化学
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分子生物学
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生物化学
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纳米技术
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公共卫生
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海洋生态学
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DOI
PubMed
高影响因子
Metallomics
·
2021
·Vol. 13
·期 10
·
IF 4.526
Ding Haoxuan,Zhang Qian,Yu Xiaonan,Chen Lingjun,Wang Zhonghang,Feng Jie
Iron overload is an important contributor to disease. The liver, the major site of iron storage in the body, is a key organ impacted by iron overload. While several studies have reported perturbations in liver lipids in iron overload, it is not clear, on a global scale, how individual liver lipid ions are altered. Here, we used lipidomics to study the changes in hepatic lipid ions in iron-overloaded mice. Iron overload was induced by daily intraperitoneal injections of 100 mg/kg body weight iron dextran for 1 week. Iron overload was verified by serum markers of iron status, liver iron quantitation, and Perls stain. Compared with the control group, the serum of iron-overload mice exhibited low levels of urea nitrogen and high-density lipoprotein (HDL), and high concentrations of total bile acid, low-density lipoprotein (LDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), suggestive of liver injury. Moreover, iron overload disrupted liver morphology, induced reactive oxygen species (ROS) production, reduced superoxide dismutase (SOD) activity, caused lipid peroxidation, and led to DNA fragmentation. Iron overload altered the overall composition of lipid ions in the liver, with significant changes in over 100 unique lipid ions. Notably, iron overload selectively increased the overall abundance of glycerolipids and changed the composition of glycerophospholipids and sphingolipids. This study, one of the first to report iron-overload induced lipid alterations on a global lipidomics scale, provides early insight into lipid ions that may be involved in iron overload-induced pathology.
研究领域:
肝病学
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脂质组学
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病理学
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分子生物学
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DOI
PubMed
高影响因子
NEW JOURNAL OF CHEMISTRY
·
2024
·
IF 2.7
Shuwei Shen,Binbin Huang,Zhiyi Peng,Luting Yu,Lili Ren,Jia Liu,Guoguang Chen
Traditional Chinese medicine (TCM), as a significant complementary and alternative approach, has received increasing attention from medical practitioners worldwide and is now regarded as an important source for modern drug discovery owing to its special therapeutic efficiency and few side effects. In this work, a popular TCM, Ganoderma lingzhi (G. lingzhi), was used as a test drug to show how Raman spectroscopy combined with traditional chemical and biological assays could be used to monitor alternations in molecular events in G. lingzhi extract-induced cells with high sensitivity. The observations indicated that the Raman fingerprint of MDA-MB-231 cells after G. lingzhi triterpene treatment was significantly different in contrast to that of the control, and changes in the Raman bands showed that some important molecular events could be discerned, including protein denaturation, DNA fragmentation, and lipid metabolism disorder. Network pharmacology analysis further supplemented the drug's target and signaling pathways. Our work proposes an effective way for screening potential new drug products early from the complex chemical compositions of TCM, as well as for accelerating preclinical medicinal chemistry optimization cycles, playing a significant role in the development of new drugs.
研究领域:
药理学
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光谱学
·
细胞生物学
·
中药
·
药物发现
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产品关联
DOI
高影响因子
RSC Advances
·
2015
·Vol. 5
·期 57
·46301-46306
·
IF 3.84
Jing Fang Guo,Rong Mei Fang,Cheng Zhi Huang,Yuan Fang Li
In order to eliminate the scattered light induced by the signal amplifier in fluorescence anisotropy (FA) assays, a nanosized metal–organic framework MIL-101, ranging from 80–500 nm, has been synthesized through a hydrothermal method with the addition of glycerol. We chose the 100 nm MIL-101 to enhance FA for label-free detection of the respiratory syncytial virus (RSV) gene sequence and the DNA-intercalating dye SYBR Green I (SGI) as the fluorophore, based on the different affinities of MIL-101 toward ssDNA and dsDNA. The nanosized MIL-101 has a negligible scattering effect owing to its smaller particle size, so all of the experimental data of FA values were smaller than the maximum initial anisotropy of 0.4. As a specific advantage, a dual amplification result of not only an increase in the FA value of SGI/ssDNA (r1) but also a decrease in the FA value of SGI/dsDNA (r2) was presented at the same time. Consequently, a larger FA value change Δr (Δr, Δr = r1 − r2) was obtained and contributed to improve the sensitivity. In addition, the quantitative detection of the target DNA (T) was achieved according to the relationship between Δr and the concentration of T. In the presence of MIL-101, the Δr is 7-fold higher than that without MIL-101 and achieved the sensitive and selective detection of RSV DNA.
研究领域:
生物化学
·
分析化学
·
病毒学
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产品关联
DOI
高影响因子
Molecular Medicine Reports
·
2015
·Vol. 12
·期 3
·3869-3873
·
IF 1.554
Shi‑Min Yu,Dong‑Hui Hu,Jian‑Jun Zhang
Hepatocellular carcinoma (HCC) is a highly malignant tumor, associated with poor patient prognoses, and high rates of morbidity and mortality. To date, the therapeutic strategies available for the treatment of HCC remain limited. The present study aimed to elucidate the anticancer activity of umbelliferone, a naturally occurring coumarin derivative isolated from Ferula communis, against the HepG2 HCC cell line. A 3‑(4,5‑dimthylthaizol‑2‑yl)‑2,5, diphenyltetrazolium bromide assay was used to evaluate cell viability following umbelliferone treatment, and the effects of umbelliferone on cell cycle progression and apoptosis were evaluated using flow cytometry. The presence of morphological features characteristic of apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation, were evaluated in HepG2 cells following umbelliferone treatment. Cell cycle analysis conducted via propidium iodide (PI) staining indicated that umbelliferone treatment induced cell cycle arrest at S phase in HepG2 cells. Analysis with Annexin V and PI staining revealed that umbelliferone induced apoptotic events in HepG2 cells in a concentration‑dependant manner (0‑50 µM). Umbelliferone also induced dose‑dependant DNA fragmentation. In conclusion, umbelliferone was found to exhibit significant anticancer effects via the induction of apoptosis, cell cycle arrest and DNA fragmentation in HepG2 cancer cells.
研究领域:
肿瘤学
·
药理学
·
细胞生物学
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产品关联
DOI
PubMed
高影响因子
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