Fluorescence imaging of glutathione with aptasensor and monitoring deoxynivalenol-induced oxidative stress in living cells
You ZhouNuo DuanYan LvImran Mahmood KhanShuo QiXianfeng LinWenyan YuYin ZhangWei XüJianhong XuShijia WuZhouping Wang
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Nanoprobe
Aptamer
Homeostasis
The electrochemical detection of endogenous molecules from a single cell is a crucial technique for understanding fundamental cellular processes at the subcellular level and uncovering cellular heterogeneity. However, high throughput intracellular detection has posed significant challenges. This paper proposes a simple method for fabricating a nanoprobe for the electrochemical monitoring of intracellular reactive oxygen species (ROS), which are important signaling molecules that play a key role in a wide range of cellular processes, including apoptosis, proliferation, and differentiation. Combined with the automated nanoprobe platform, the study successfully achieved minimally invasive and efficient intracellular ROS detection in living cells. Moreover, the results reveal that the average ROS concentration in the cytoplasm and nucleus is uneven, which has important implications for the understanding of cellular redox signaling.
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Abstract Previous studies revealed that primarily small and relatively hydrophilic comonomers, such as TEGDMA, leach out of resin‐based restorative materials into aqueous media. Subsequently, these compounds may cause detrimental reactions with intracellular metabolic systems. The present experiments attempted to elucidate the interactions of TEGDMA with the important intracellular reducing agent glutathione (GSH). The influence of various concentrations of TEGDMA (0.5–7.5 mM) on viability and intracellular GSH concentration of primary human gingival fibroblasts was determined by means of a fluorescence assay (monobromobimane) performed in microtiter plates. Cells were treated with TEDGMA between 2 and 24 h. The incubation of fibroblasts with TEGDMA even at subtoxic concentrations quickly decreased the intracellular glutathione level to 30–50% of controls within the first 2–6 hours. However, no simultaneous adverse effect on cell viability was found. Longer incubation periods up to 24 h caused a regulatory reincrease at TEGDMA concentrations ≤ 2.5 mM, whereas higher concentrations resulted in a continuous depletion of glutathione concentration concomitant with a significant decrease of cell viability. Because glutathione plays an important role in protection and detoxification processes as well in the regulation of cell death, the early and extensive depletion of the intracellular glutathione pool due to TEGDMA may significantly contribute to the cytotoxic potency of this compound. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res (Appl Biomater) 63: 746–751, 2002
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To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer–target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.
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Aptamers are nucleic acids that can bind to various molecules. Because they have some features that are lacking in antibodies, aptamers could serve as alternatives to antibodies. For the purpose of biosensing, we focused on aptamers that undergo structural changes on binding to their target molecules. We constructed an aptamer-based bound/free (B/F) separation system that uses a designed aptamer named the "capturable aptamer". The capturable aptamer changes its structure upon recognizing its target molecule thereby exposing a specific single-strand region. The oligonucleotide that is complementary to this exposed region, named the "capture DNA" is immobilized on a support. This design permits the exclusive capture by the capture DNA of the aptamer bound to its target, and subsequent removal of any unbound aptamer and contaminants by B/F separation. The removal of unbound contaminants or aptamers results in highly sensitive detection at similar levels to those achievable by sandwich-based immunoassay. We describe the construction of a thrombin-detection system by using a capturable aptamer, and we discuss the potential of capturable aptamers in clinical diagnostics.
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Debaryomyces hansenii
Homeostasis
Osmotic shock
Intracellular pH
Strain (injury)
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Aptamers have emerged as research hotspots of the next generation due to excellent performance benefits and application potentials in pharmacology, medicine, and analytical chemistry. Despite the numerous aptamer investigations, the lack of comprehensive data integration has hindered the development of computational methods for aptamers and the reuse of aptamers. A public access database named AptaDB, derived from experimentally validated data manually collected from the literature, was hence developed, integrating comprehensive aptamer-related data, which include six key components: (i) experimentally validated aptamer-target interaction information, (ii) aptamer property information, (iii) structure information of aptamer, (iv) target information, (v) experimental activity information, and (vi) algorithmically calculated similar aptamers. AptaDB currently contains 1350 experimentally validated aptamer-target interactions, 1230 binding affinity constants, 1293 aptamer sequences, and more. Compared to other aptamer databases, it contains twice the number of entries found in available databases. The collection and integration of the above information categories is unique among available aptamer databases and provides a user-friendly interface. AptaDB will also be continuously updated as aptamer research evolves. We expect that AptaDB will become a powerful source for aptamer rational design and a valuable tool for aptamer screening in the future. For access to AptaDB, please visit http://lmmd.ecust.edu.cn/aptadb/.
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When aptamers first emerged almost two decades ago, most were RNA species that bound and tagged or inhibited simple target ligands. Very soon after, the 'selectionologists' developing aptamer technology quickly realized more potential for the aptamer. In recent years, advances in aptamer techniques have enabled the use of aptamers as small molecule inhibitors, diagnostic tools and even therapeutics. Aptamers are now being employed in novel applications. We review, herein, some of the recent and exciting applications of aptamers in cell-specific recognition and delivery.
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