skip to main content

Explore Research Products in the NSF-PAR

  • NSF Public Access
  • Search Results
  • Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic structures
Title: Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic structures
Zero-mode waveguides (ZMW) have the potential to be powerful confinement tools for studying electron transfer dynamics at single molecule occupancy conditions. Flavin mononucleotide contains an isoalloxazine chromophore, which is fluorescent in the oxidized state (FMN) while the reduced state (FMNH 2 ) exhibits dramatically lower light emission, i.e. a dark-state. This allows fluorescence emission to report the redox state of single FMN molecules, an observation that has been used previously to study single electron transfer events in surface-immobilized flavins and flavoenzymes, e.g. sarcosine oxidase, by direct wide-field imaging of ZMW arrays. Single molecule electron transfer dynamics have now been extended to the study of freely diffusing molecules using fluorescence measurements of Au ZMWs under single occupancy conditions. The Au in the ZMW serves both as an optical cladding layer and as the working electrode for potential control, thereby accessing single molecule electron transfer dynamics at μM concentrations. Consistent with expectations, the probability of observing single reduced molecules increases as the potential is scanned negative, E appl < E eq , and the probability of observing emitting oxidized molecules increases at E appl > E eq . Different single molecules exhibit different electron transfer properties as reflected in the position of E eq and the distribution of E eq among a population of FMN molecules. Two types of actively-controlled electroluminescence experiments were used: chronofluorometry experiments, in which the potential is alternately stepped between oxidizing and reducing potentials, and cyclic potential sweep fluorescence experiments, analogous to cyclic voltammetry, these latter experiments exhibiting a dramatic scan rate dependence with the slowest scan rates showing distinct intermediate states that are stable over a range of potentials. These states are assigned to flavosemiquinone species that are stabilized in the special environment of the ZMW nanopore.  more » « less
Award ID(s):
1404744
NSF-PAR ID:
10055673
Author(s) / Creator(s):
; ; ; ;
Date Published:
Journal Name:
Faraday Discussions
Volume:
184
ISSN:
1359-6640
Page Range / eLocation ID:
101 to 115
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ( , Chemical Science)
    null (Ed.)
    The ability of zero-mode waveguides (ZMW) to guide light into subwavelength-diameter nanoapertures has been exploited for studying electron transfer dynamics in zeptoliter-volume nanopores under single-molecule occupancy conditions. In this work, we report the spectroelectrochemical detection of individual molecules of the redox-active, fluorogenic molecule flavin mononucleotide (FMN) freely diffusing in solution. Our approach is based on an array of nanopore-confined recessed dual ring electrodes, wherein repeated reduction and oxidation of a single molecule at two closely spaced annular working electrodes yields amplified electrochemical signals. We have articulated these structures with an optically transparent bottom, so that the nanopores are bifunctional, exhibiting both nanophotonic and nanoelectrochemical behaviors allowing the coupling between electron transfer and fluorescence dynamics to be studied under redox cycling conditions. We also investigated the electric field intensity in electrochemical ZMWs (E-ZMW) through finite-element simulations, and the amplification of fluorescence by redox cycling agrees well with predictions based on optical confinement effects inside the E-ZMW. Proof-of-principle experiments are conducted showing that electrochemical and fluorescence signals may be correlated to reveal single molecule fluctuations in the array population. Cross-correlation of single molecule fluctuations in amperometric response and single photon emission provides unequivocal evidence of single molecule sensitivity. 
    more » « less
  2. ; ; ; ; ; ; ( , Nanoscale Horizons)
    null (Ed.)
    Intercalating ds-DNA/RNA with small molecules can play an essential role in controlling the electron transmission probability for molecular electronics applications such as biosensors, single-molecule transistors, and data storage. However, its applications are limited due to a lack of understanding the nature of intercalation and electron transport mechanisms. We addressed this long-standing problem by studying the effect of intercalation on both the molecular structure and charge transport along the nucleic acids using molecular dynamics simulations and first-principle calculations coupled with Green’s function method, respectively. The study on anthraquinone and anthraquinone-neomycin conjugate intercalation into short nucleic acids reveals some universal features: 1) the intercalation affects the transmission by two mechanisms: a) inducing energy levels within the bandgap and b) shifting the location of the Fermi energy with respect to the molecular orbitals of the nucleic acid, 2) the effect of intercalation was found to be dependent on the redox state of the intercalator: while oxidized anthraquinone decreases, reduced anthraquinone increases the conductance, and 3) the sequence of intercalated nucleic acid further affects the transmission: lowering the AT-region length was found to enhance the electronic coupling of the intercalator with GC bases, hence yielding an increase of more than four times in conductance. We anticipate our study to inspire designing intercalator-nucleic acid complexes for potential use in molecular electronics via creating a multi-level gating effect. 
    more » « less
  3. ; ; ; ; ; ; ( , Chemistry – A European Journal)
    Abstract

    Two wide‐band‐capturing donor‐acceptor conjugates featuring bis‐styrylBODIPY and perylenediimide (PDI) have been newly synthesized, and the occurrence of ultrafast excitation transfer from the1PDI* to BODIPY, and a subsequent electron transfer from the1BODIPY* to PDI have been demonstrated. Optical absorption studies revealed panchromatic light capture but offered no evidence of ground‐state interactions between the donor and acceptor entities. Steady‐state fluorescence and excitation spectral recordings provided evidence of singlet‐singlet energy transfer in these dyads, and quenched fluorescence of bis‐styrylBODIPY emission in the dyads suggested additional photo‐events. The facile oxidation of bis‐styrylBODIPY and facile reduction of PDI, establishing their relative roles of electron donor and acceptor, were borne out by electrochemical studies. The electrostatic potential surfaces of the S1and S2states, derived from time‐dependent DFT calculations, supported excited charge transfer in these dyads. Spectro‐electrochemical studies on one‐electron‐oxidized and one‐electron‐reduced dyads and the monomeric precursor compounds were also performed in a thin‐layer optical cell under corresponding applied potentials. From this study, both bis‐styrylBODIPY⋅+and PDI⋅−could be spectrally characterizes and were subsequently used in characterizing the electron‐transfer products. Finally, pump–probe spectral studies were performed in dichlorobenzene under selective PDI and bis‐styrylBODIPY excitation to secure energy and electron‐transfer evidence. The measured rate constants for energy transfer,kENT, were in the range of 1011 s−1, while the electron transfer rate constants,kET, were in the range of 1010 s−1, thus highlighting their potential use in solar energy harvesting and optoelectronic applications.

     
    more » « less
  4. ; ; ; ( , Physical Chemistry Chemical Physics)
    null (Ed.)
    Of particular interest in radiation-induced charge transfer processes in DNA is the extent of hole localization immediately after ionization and subsequent relaxation. To address this, we considered double stranded oligomers containing guanine (G) and 8-oxoguanine (8OG), i.e. , ds(5′-GGG-3′) and ds(5′-G8OGG-3′) in B-DNA conformation. Using DFT, we calculated a variety of properties, viz. , vertical and adiabatic ionization potentials, spin density distributions in oxidized stacks, solvent and solute reorganization energies and one-electron oxidation potential ( E 0 ) in the aqueous phase. Calculations for the vertical state of the -GGG- cation radical showed that the spin was found mainly (67%) on the middle G. However, upon relaxation to the adiabatic -GGG- cation radical, the spin localized (96%) on the 5′-G, as observed in experiments. Hole localizations on the middle G and 3′-G were higher in energy by 0.5 kcal mol −1 and 0.4 kcal mol −1 , respectively, than that of 5′-G. In the -G8OGG- cation radical, the spin localized only on the 8OG in both vertical and adiabatic states. The calculated vertical ionization potentials of -GGG- and -G8OGG- stacks were found to be lower than that of the vertical ionization potential of a single G in DNA. The calculated E 0 values of -GGG- and -G8OGG- stacks are 1.15 and 0.90 V, respectively, which owing to stacking effects are substantially lower than the corresponding experimental E 0 values of their monomers (1.49 and 1.18 V, respectively). SOMO to HOMO level switching is observed in these oxidized stacks. Consequently, our calculations predict that local double oxidations in DNA will form triplet diradical states, which are especially significant for high LET radiations. 
    more » « less
  5. ; ; ( , Biology)
    NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV0) of the opening transition. While the ΔV0 characteristic of the oxidized enzyme is as large as −88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to −34 and −18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email