We report the redox mediation of glucose oxidase (GOx) in a
self-assembled structure of cationic poly(allylamine) modified by ferrocene (PAA-Fc) and anionic GOx deposited
electrostatically layer-by-layer on
negatively charged alkanethiol-modified Au surfaces. Successive
PAA-Fc and GOx layers were deposited
by alternate immersion of the thiol-modified Au in the respective
polyelectrolyte and enzyme solutions.
The uptake of thiol, redox polymer, and GOx on the surface was
monitored by quartz crystal microbalance.
Cyclic voltammetry shows nearly ideal surface waves of ferrocene
in the polymer with charge independent
of sweep rate; the redox surface concentration was obtained from
integration of the ferrocene/ferricinium
voltammetric peaks. The redox charge increases in step with the
number of PAA-Fc layers deposited.
Enzyme catalysis for the oxidation of β-d-glucose
was achieved with a multilayer PAA-Fc/GOx assembly,
with each GOx layer contributing equally to the catalytic response.
Only a small fraction of the active
assembled GOx molecules are “electrically wired” by the ferrocene
polymer although all of the enzyme
could be oxidized by soluble ferrocenesulfonate when added to
solution.
A new strategy to build caged-compounds is presented. The approach is based on heterolytic photocleavage of a metal-ligand bond in a coordination compound. A ruthenium polypyridine complex, containing the neurocompound 4-amino pyridine (4AP) is used as the core of the phototrigger. The biomolecule is released by irradiation with visible light (>480 nm). The liberated 4AP promotes the activation of a leech neuron by means of blocking its K+ channels. The syntesis, characterization, and the inherent advantages of this method are discussed.
We introduce a new caged glutamate, based in a ruthenium bipyridyl core, that undergoes heterolytic cleavage after irradiation with visible light with wavelengths up to 532 nm, yielding free glutamate in less than 50 ns. Glutamate photorelease occurs also efficiently following two-photon (2P) excitation at 800 nm, and has a functional cross section of 0.14 GM.
We describe neurobiological applications of RuBi-Glutamate, a novel caged-glutamate compound based on ruthenium photochemistry. RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one-or two-photon excitation. It has high quantum effi ciency and can be used at low concentrations, partly avoiding the blockade of GABAergic transmission present with other caged compounds. Two-photon uncaging of RuBi-Glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics. With laser beam multiplexing, two-photon RuBi-Glutamate uncaging can also be used to depolarize and fi re pyramidal neurons with single-cell resolution. RuBi-Glutamate therefore enables the photoactivation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single cell, or even single spine, precision.
The uptake of glucose oxidase (GOx) onto a polycationic redox polymer (PAA-Os)-modified surface, by adsorption from dilute aqueous GOx solutions, was followed by the quartz crystal microbalance (QCM) and shows double exponential kinetics. The electrochemistry of the layer-by-layer-deposited redox-active polymer was followed by cyclic voltammetry in glucose-free solutions, and the enzyme catalysis mediated by the redox polymer was studied in beta-D-glucose-containing solutions. AFM studies of the different layers showed the existence of large two dimension enzyme aggregates on the osmium polymer for 1 microM GOx and less aggregation for 50 nM GOx solutions. When the short alkanethiol, 2,2'-diaminoethyldisulfide was preadsorbed onto gold, a monoexponential adsorption law was observed, and single GOx enzyme molecules could be seen on the surface where the enzyme was adsorbed from 50 nM GOx in water.
The synthesis and characterization of a series of ruthenium bis(bipyridine) complexes where the inorganic moiety acts as a photolabile protecting group is described. Complexes of the type [Ru(bpy)2L2]+ where bpy = 2,2'-bipyridine and L = butylamine, gamma-aminobutyric acid, tyramine, tryptamine, and serotonin were studied by nuclear magnetic resonance, cyclic voltammetry, and electronic absorption spectroscopy. In all cases, ligands are coordinated by the amine group. The complexes are stable in water for several days and deliver one molecule of ligand upon irradiation with visible light (450 nm). These properties make them suitable for their use as biological caged compounds.
Octahedral Ru(II) polypyridyl complexes constitute a superb platform to devise photoactive triggers capable of delivering entire molecules in a reliable, fast, efficient and clean way. Ruthenium coordination chemistry opens the way to caging a wide range of molecules, such as amino acids, nucleotides, neurotransmitters, fluorescent probes and genetic inducers. Contrary to other phototriggers, these Ru-based caged compounds are active with visible light, and can be photolysed even at 532 nm (green), enabling the use of simple and inexpensive equipment. These compounds are also active in the two-photon regime, a property that extends their scope to systems where IR light must be used to achieve high precision and penetrability. The state of the art and the future of ruthenium polypyridyl phototriggers are discussed, and several new applications are presented.
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