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Nanoencapsulation method for high selectivity sensing of hydrogen peroxide inside live cells.

ABSTRACT: Reactive oxygen species (ROS) are ubiquitous in life and death processes of cells (Finkel, T.; Holbrook, N. J. Nature 2000, 408 (6809), 239-247), with a major role played by the most stable ROS, hydrogen peroxide (H(2)O(2)). However, the study of H(2)O(2) in live cells has been hampered by the absence of selective probes. Described here is a novel nanoprobe ("nanoPEBBLE") with dramatically improved H(2)O(2) selectivity. The traditional molecular probe, 2',7'-dichlorofluorescin (DCFH), which is also sensitive to most other ROS, was empowered with high selectivity by a nanomatrix that blocks the interference from all other ROS (hydroxyl radical, superoxide, nitric oxide, peroxynitrite, hypochlorous acid, and alkylperoxyl radical), as well as from enzymes such as peroxidases. The blocking is based on the combination of multiple exclusion principles: time barrier, hydrophobic energy barrier, and size barrier. However, H(2)O(2) sensitivity is maintained down to low nanomolar concentrations. The surface of the nanoprobe was engineered to address biological applications, and the power of this new nanoPEBBLE is demonstrated by its use on RAW264.7 murine macrophages. These nanoprobes may provide a powerful chemical detection/imaging tool for investigating biological mechanisms related to H(2)O(2) or other species, with high spatial and temporal resolution.

SUBMITTER: Kim G

PROVIDER: S-EPMC2838456 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Nanoencapsulation method for high selectivity sensing of hydrogen peroxide inside live cells.

Kim Gwangseong G Lee Yong-Eun Koo YE Xu Hao H Philbert Martin A MA Kopelman Raoul R

Analytical chemistry 20100301 6

Reactive oxygen species (ROS) are ubiquitous in life and death processes of cells (Finkel, T.; Holbrook, N. J. Nature 2000, 408 (6809), 239-247), with a major role played by the most stable ROS, hydrogen peroxide (H(2)O(2)). However, the study of H(2)O(2) in live cells has been hampered by the absence of selective probes. Described here is a novel nanoprobe ("nanoPEBBLE") with dramatically improved H(2)O(2) selectivity. The traditional molecular probe, 2',7'-dichlorofluorescin (DCFH), which is a ...[more]

PMID: 20163178

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Project description:The regulatory mechanisms by which hydrogen peroxide (H2O2) modulates the activity of transcription factors in bacteria (OxyR and PerR), lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4) and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1) are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1) synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii) stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii) cytoplasm-nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and (iv) DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M(-1) s(-1) and ≥1.3 × 10(3) M(-1) s(-1) were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for highly specific effects on gene regulation that depend on the cell type and on signals received from the cellular microenvironment.

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Nanoencapsulation method for high selectivity sensing of hydrogen peroxide inside live cells.

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Nanoencapsulation method for high selectivity sensing of hydrogen peroxide inside live cells.

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