The syntheses, properties, and biological applications of the Peroxysensor family, a new class of fluorescent probes for hydrogen peroxide, are presented. These reagents utilize a boronate deprotection mechanism to provide high selectivity and optical dynamic range for detecting H 2 O 2 in aqueous solution over similar reactive oxygen species (ROS) including superoxide, nitric oxide, tert-butyl hydroperoxide, hypochlorite, singlet oxygen, ozone, and hydroxyl radical. Peroxyresorufin-1 (PR1), Peroxyfluor-1 (PF1), and Peroxyxanthone-1 (PX1) are first-generation probes that respond to H 2 O 2 by an increase in red, green, and blue fluorescence, respectively. The boronate dyes are cell-permeable and can detect micromolar changes in H 2 O 2 concentrations in living cells, including hippocampal neurons, using confocal microscopy and two-photon microscopy. The unique combination of ROS selectivity, membrane permeability, and a range of available excitation/ emission colors establishes the potential value of PR1, PF1, PX1, and related probes for interrogating the physiology and pathology of cellular H 2 O 2 .

Engineering efficient, directional electronic communication between living and nonliving systems has the potential to combine the unique characteristics of both materials for advanced biotechnological applications. However, the cell membrane is designed by nature to be an insulator, restricting the flow of charged species; therefore, introducing a biocompatible pathway for transferring electrons across the membrane without disrupting the cell is a significant challenge. Here we describe a genetic strategy to move intracellular electrons to an inorganic extracellular acceptor along a molecularly defined route. To do so, we reconstitute a portion of the extracellular electron transfer chain of Shewanella oneidensis MR-1 into the model microbe Escherichia coli. This engineered E. coli can reduce metal ions and solid metal oxides ∼8× and ∼4× faster than its parental strain. We also find that metal oxide reduction is more efficient when the extracellular electron acceptor has nanoscale dimensions. This work demonstrates that a genetic cassette can create a conduit for electronic communication from living cells to inorganic materials, and it highlights the importance of matching the size scale of the protein donors to inorganic acceptors.cytochrome c | nanobioelectronics | synthetic biology | iron reduction | living-nonliving interfaces

Hydrogen peroxide (H2O2) is a potent small-molecule oxidant that can exert a diverse array of physiological and/or pathological effects within living systems depending on the timing and location of its production, accumulation, trafficking, and consumption. To help study the chemistry and biology of this reactive oxygen species (ROS) in its native cellular context, we now present a new method for monitoring local, subcellular changes in H2O2 levels by fluorescence imaging. Specifically, we have exploited the versatility of the SNAP-tag technology for site-specific protein labeling with small molecules on the surface or interior of living cells with the use of boronate-capped dyes to selectively visualize H2O2. The resulting SNAP-Peroxy-Green (SNAP-PG) probes consist of appropriately derivatized boronates bioconjugated to SNAP-tag fusion proteins. Spectroscopic measurements on the SNAP-PG constructs confirm their ability to detect H2O2 with specificity over other biologically relevant ROS. Moreover, these hybrid small-molecule/protein reporters can be used in live mammalian cells expressing SNAP-tag fusion proteins directed to the plasma membrane, nucleus, mitochondria, and endoplasmic reticulum. Imaging experiments using scanning confocal microscopy establish organelle-specific localization of the SNAP-tag probes and their fluorescence turn-on in response to changes in local H2O2 levels. This work provides a general molecular imaging platform for assaying H2O2 chemistry in living cells with subcellular resolution.