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  • Herein we designed a strategy

    2020-02-05

    Herein, we designed a strategy utilizing the target-protection method to produce a signal in a system involving fluorescence CuNPs stabilized by dumbbell template DNA (DT DNA) through a locked circle consisting of two poly T loops and a poly (ATTA) stem. In the presence of MTase, DT DNA was modified with methyl groups and guarded from endonuclease and exonuclease digestion, thus fluorescence could be detected after the restoration of Cu2+ in the system via the presence of DT DNA templates. In the absence MTase, the DT DNA templates were substrates of endonucleases and exonucleases, thus becoming single nucleotides. As previously reported, CuNPs have sequence specificity, and therefore, nucleotides cannot stabilize the copper restored by ascorbic acid. Thus, the fluorescence signal is associated with the detection of MTase. Additionally, signaling produces low background noise due to the sequence specificity and high specificity targeting. Through investigation of MTase, the proposed method was demonstrated to work well, with a detection limit of 1.6968 × 10−4 U/μL which is superior to some previous biosensors (Table S2). Furthermore, the results obtained using an actual sample and the application of the sensing strategy for MTase inhibitors demonstrate that this strategy is a reliable, sensitive and rapid DNA MTase detection method in medical and clinical areas.
    Experimental section
    Results and discussion
    Conclusions In summary, a method for detecting M.SssI MTase was successfully developed by creatively utilizing the features of CuNPs, which specifically bind to dsDNA or poly T DNA other than nucleotides. And the principle of target-protection enabled the system to be superior to traditional method in some aspects. This method is sensitive, with a LOD of 1.6968 × 10−4 U/μL M.SssI MTase, simple and rapid without requiring further amplification. And the selectivity assay has demonstrated this method could distinguish M.SSsI MTase from other acetaminophen paracetamol mg well. As the material to emit the fluorescence is much cheaper and more common than organic dyes, good cost performance was achieved. More importantly, the analytical performance of the proposed technique in real samples has broadened the application acetaminophen paracetamol mg of this label-free method, indicating its great potential. At the same time, this method has successfully assessed the inhibition effects of DNA MTase inhibitors using SGI-1027 and 5-Aza, thus our system can be used in screening drugs and early cancer diagnosis, which is also of great importance.
    Acknowledgments This work was financially supported through grantsfrom National Natural Science Foundation of China (No. 21402105), and Shenzhen Municipal government SZSITIC (JCYJ20160301153753269).
    Introduction Due to their unique physical, electrical, and optical properties, ultrasmall fluorescent nanoparticles have obtained extensive applications, ranging from energy conversion and storage to biomedical imaging [[1], [2], [3], [4]]. The ultrasmall fluorescent nanoparticles mainly included semiconductor nanocrystals (usually referred to as quantum dots (QDs)), fluorescent metal nanoclusters, carbon-based nanomaterials, up-conversion nanocrystals, and silicon nanoparticles. Among them, fluorescent metal nanoclusters (e.g., Au, Ag, and Cu) have attracted special research interest in the past decade due to their ease of synthesis, extreme brightness, low-toxicity, and good biocompatibility [5]. In general, fluorescent metal nanoclusters can be prepared by reduction of metal precursors or etching of large nanoparticles in the presence of strong stabilizers such as small thiol-molecules, polymers, proteins, peptides, and DNA [6]. In addition to binding to complementary nucleic acids and non-nucleic acid targets (aptamers) [7], the flexibility, nanosized structure, excellent programmable properties make the DNA molecules good templates for the synthesis of luminescent metal nanoclusters [8]. Since the first successful demonstration of DNA templated silver nanoclusters (AgNCs) by the group of Dickson in 2004 [9], DNA-AgNCs have been widely studied and successfully applied for bioimaging and biosensing [[10], [11], [12]]. In 2010, Mokhir et al. have reported that the random dsDNA could act as an efficient template for the formation of fluorescent copper nanoclusters (CuNCs) at a low concentration of CuSO4, whereas the random ssDNA or triplex template can not [13]. Subsequently, Qing et al. found that specific ssDNA (poly T) could also template CuNCs with excellent fluorescence [14]. Those DNA-CuNCs could be facilely prepared within 5 min at room temperature by reducing Cu2+ ions with ascorbic acid in the presence of nucleic acid. Due to their ease of synthesis and excellent fluorescence properties, DNA-CuNCs have been used as fluorescent indicators in the field of biochemical analysis [[15], [16], [17]]. More recently, they also investigated the effect of sequence composition on fluorescence of dsDNA templated CuNCs, and found that the dsDNA templated CuNCs were poly (AT-TA) dependent [18]. In another word, the fluorescence intensity of dsDNA-CuNCs is highly-dependent on the polymerization degree and the length of poly (AT-TA). Only the relatively long poly (AT-TA) could template fluorescent CuNCs formation, while the fluorescence induced by poly (AT-TA) of less than 12 base pairs (bp) was negligible practically, which holds an immense potential for biochemical sensing based on length-change of nucleic acid.