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The effects of film morphology and surface termination on the direct electron transfer of horse heart cytochrome c on boron-doped ultrananocrystalline (B-UNCD) and microcrystalline (B-MCD) diamond thin-film electrodes were investigated. Quasi-reversible, diffusion-controlled cyclic voltammetric responses were observed on oxygen-terminated (atomic O/C ~0.015), but not hydrogen-terminated (atomic O/C ~0.02) diamond thin films. The effect of the surface termination was the same for both the nanostructured B-UNCD film with sp2-bonded carbon atoms in the grain boundaries and the well faceted B-MCD film with micron-sized grains and largely devoid of sp2 carbon. Stable cyclic voltammetric i-E curves were recorded with cycling for both oxygen-terminated films indicating the absence of protein denaturation and electrode fouling. The peak currents increased linearly with the square root of the scan rate and the protein concentration; both indicative of a reaction rate limited by semi-infinite linear diffusion of the protein. Similar heterogeneous electron-transfer rate constants were observed for oxygen-terminated B-UNCD (3.48 (± 1.25) × 10-3 cm/s) and B-MCD films (2.38 (± 0.72) × 10-3 cm/s). The results clearly reveal that the oxygen-terminated surface is more active for electron-transfer with this soluble redox protein than is the hydrogen-terminated surface. The film morphology does not influence the diffusion-controlled response of the redox protein.

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We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID) imaging microscopy. A triazole-substituted BIPS (TzBIPS) is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP) and a far-red absorbing merocyanine (MC) state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C12-TzBIPS) is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP) has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz) within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.

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An optically controlled Ca(2+)-chelator 1 was developed to mimic natural calcium oscillations. Compound 1, a spiroamido-rhodamine derivative of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), underwent cycles of reversible transitions between a colorless closed state and a fluorescent open form. The closed-state exhibited a high affinity for Ca(2+) (K(d): 509 nM) with excellent selectivity over Mg(2+) (K(d): 19 mM). The open isomer had a 350-fold lower Ca(2+) affinity (K(d): 181 µM), while the Mg(2+) affinity was not significantly affected (K(d): 14 mM).

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We report on the design and performance of a thin-layer electrochemical cell optimized for use with optically transparent film electrodes in combination with UV/vis and IR transmission spectroscopic measurements. The cell allows for measurements under both aerobic and anaerobic conditions. The direct, unmediated electron transfer, as assessed by the current transient, and the corresponding optical response observed for the Fe(CN)(6)(3-/4-) couple were in good agreement with theoretical predictions for voltammetry and optical absorption by an analyte confined in a thin layer. Chronoamperometric and spectroscopic measurements of Fe(CN)(6)(3-/4-) on gold mesh electrode revealed fast kinetics strongly influenced by the electrolyte concentration. Maximal apparent rates exceeding 2 s(-1) in 1 M KCl were observed optically. The direct kinetic and thermodynamic behavior of cytochrome c was compared with several electrode materials using the cell. The results showed heme ligand-dependent changes in the protein-electrode interactions. Mid-UV/visible spectral changes upon redox transitions in native cytochrome c and its cyanide derivative, as well as dissociation of the ferrous cytochrome c-CN complex, are reported.

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Given the useful electroanalytical properties of carbon electrodes, having an optically transparent material could lead to new measurement paradigms that combine electrochemical and spectroscopic measurements.

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A new analytical spectroelectrochemical methodology is reported on that utilizes an optically transparent boron-doped diamond thin film. The film was deposited on undoped Si by microwave-assisted chemical vapor deposition using a 4-h growth with a 0.5% CH4/H2 source gas mixture and 2 ppm B2H6 added for boron doping. The thin-film electrode possessed a transparency of 40-60% in the mid- and far-IR regions of the electromagnetic spectrum. The physical, electrical, optical, and electrochemical properties of the electrode were characterized by scanning electron microscopy, Raman spectroscopy, X-ray diffraction, four-point probe electrical resistance measurements, IR spectroscopy, and cyclic voltammetry. The film's electrochemical behavior was evaluated using both aqueous (Fe(CN)(6)3-/4-, methyl viologen, Ru(NH3)(6)3+/2+, and IrCl(6)2-/3-) and nonaqueous (ferrocene) redox systems. The film exhibited a low and stable background current and a nearly reversible voltammetric response for all these redox systems. The diamond/Si optically transparent electrode (OTE) and a thin-layer transmission cell were used to record the spectroelectrochemical response for 10 mM Fe(CN)(6)3-/4- in 1 M KCl. Difference IR spectra (oxidized minus reduced), recorded at various applied potentials, showed that the CN vibrational mode at 2039 cm-1 for Fe(CN)(6)4- reversibly shifted to 2116 cm-1 upon oxidation to Fe(CN)(6)3-, as expected. Difference IR spectra (oxidized minus reduced) were also recorded for 20 mM ferrocene in 0.1 M TBABF4/CH3CN. A shift of the C-H bending mode of the cyclopentadienyl ring from 823 to 857 cm-1 occurred upon oxidation of ferrocene to ferricenium. The key finding from the work is that the diamond OTE provides sensitive, reproducible, and stable spectroelectrochemical responses for aqueous and nonaqueous redox systems in the mid- and far-IR.

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