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  • br Experimental br Results and discussions br

    2022-05-18


    Experimental
    Results and discussions
    Conclusions In this work, a novel label-free ECL biosensing method was developed to detect TDG activity using signal amplification strategy of HCR which was triggered by DNA functionalized AuNPs. The label-free ECL biosensing platform has been constructed for TDG activity detection for the first time by utilizing of Ru(phen)32+ as an intercalated signal indicator. The high sensitivity of ECL method combined with the remarkably amplified effect of the DNA-functionalized gold nanoparticles triggered hybridization chain reaction endows this biosensing method with ultra-high sensitivity and a low detection limit. Additionally, the feasibility of this strategy for TDG detection in MCF-7 cells lysates was also confirmed. This method creates a new horizon for quantitative detection of TDG, and it shows great potential for TDG activity assay in clinical diagnostics and related research.
    Declaration of interest statement
    Acknowledgements Financial support from the National Natural Science Funds of China (Nos. 21675124, 21375102 and 201706214), the Natural Science Basic Research Plan in Shaanxi Province of China (No. 2016JM2021) and the Excellent Doctoral Dissertations Project of Northwest University (No. YYB17013) are acknowledged.
    Introduction Corynebacterium pseudotuberculosis is a Gram-positive bacterium, which together with Mycobacterium, Nocardia melanocortin 1 receptor and Rhodococcus forms the ‘CMNR group’ of potential animal and human pathogens [1]. This facultative intracellular parasite is the causative agent of caseous lymphadenitis (CLA) in sheep and goats (biovar ovis) and to a lesser extent in horses and cattle (biovar equi). Infection by C. pseudotuberculosis in sheep and goats leads to considerable economic loss because of the significant melanocortin 1 receptor in yields of wool and milk production. The infected animals suffer from weight loss which may lead to death due to the formation of abscesses in superficial and visceral lymph nodes or by caseous necrosis of the lymphatic glands [2,3]. This pathogen is capable of survival and growth in macrophages thus evading detection by the host immune system [4,5]. In macrophages, C. pseudotuberculosis is exposed to an environment rich in reactive oxygen and nitrogen species (ROS and RNS, respectively) [6], in addition to those that endogenously generated during metabolism. These molecules ultimately damage DNA molecules thereby resulting in cytotoxic or mutagenic effects on the cell [7,8]. One of the most abundant forms of genotoxic damage is the formation of 7,8‑dihydro‑8‑oxo‑Guanine (8‑oxo‑G), which arises from the oxidation of guanine by the attack of ROS [[9], [10], [11]]. The 8-oxo-G:A mispair appears primarily in DNA and failure in repairing these lesions results in deleterious G:C to T:A transversion mutations. To avoid the genotoxic effects caused by 8-oxo-G:A, cells utilize the evolutionarily conserved MutY (MUTYH in humans) adenine DNA glycosylase to selectively cleave the adenine base from 8-oxo-G:A [12,13]. Free adenine can inhibit the MutY function, the base binds to the enzyme active site pocket and prevents the interaction with adenine from the 8-oxo-G:A mispair [14,15]. The active site pockets of MutY proteins are surrounded by positively charged residues that support the DNA binding [14,15]. The interaction with the polyanion DNA is essential for MutY protein function. Polyanions are molecules with multiple negatively charges, they are common macromolecules and macromolecular complexes, with the most common been proteoglycans (heparin), nucleic acids (DNA, RNA), actin (microfilaments), tubulin (microtubules), polysialic acids, ribosomes, etc. [16]. The protein binding sites for polyanions are clusters of positively charged basic amino acids forming ion pairs with spatially defined negatively charged groups. Regarding their highly negatively charge, polyanions are potential competitors for DNA or RNA binding areas in proteins.