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1. Redox cycling-based detection of phenazine metabolites secreted from Pseudomonas aeruginosa in nanopore electrode arrays

   https://doi.org/10.1039/D0AN02022B  

   Do, Hyein; Kwon, Seung-Ryong; Baek, Seol; Madukoma, Chinedu S.; Smiley, Marina K.; Dietrich, Lars E.; Shrout, Joshua D.; Bohn, Paul W. (January 2021, The Analyst)

   null (Ed.)

   The opportunistic pathogen Pseudomonas aeruginosa ( P. aeruginosa ) produces several redox-active phenazine metabolites, including pyocyanin (PYO) and phenazine-1-carboxamide (PCN), which are electron carrier molecules that also aid in virulence. In particular, PYO is an exclusive metabolite produced by P. aeruginosa , which acts as a virulence factor in hospital-acquired infections and is therefore a good biomarker for identifying early stage colonization by this pathogen. Here, we describe the use of nanopore electrode arrays (NEAs) exhibiting metal–insulator–metal ring electrode architectures for enhanced detection of these phenazine metabolites. The size of the nanopores allows phenazine metabolites to freely diffuse into the interior and access the working electrodes, while the bacteria are excluded. Consequently, highly efficient redox cycling reactions in the NEAs can be accessed by free diffusion unhindered by the presence of bacteria. This strategy yields low limits of detection, i.e. 10.5 and 20.7 nM for PYO and PCN, respectively, values far below single molecule pore occupancy, e.g. at 10.5 nM 〈 n pore 〉 ∼ 0.082 per nanopore – a limit which reflects the extraordinary signal amplification in the NEAs. Furthermore, experiments that compared results from minimal medium and rich medium show that P. aeruginosa produces the same types of phenazine metabolites even though growth rates and phenazine production patterns differ in these two media. The NEA measurement strategy developed here should be useful as a diagnostic for pathogens generally and for understanding metabolism in clinically important microbial communities. 

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2. Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic structures

   https://doi.org/10.1039/c5fd00072f  

   Zaino, Lawrence P.; Grismer, Dane A.; Han, Donghoon; Crouch, Garrison M.; Bohn, Paul W. (January 2015, Faraday Discussions)

   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. 

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3. Single-molecule spectroelectrochemical cross-correlation during redox cycling in recessed dual ring electrode zero-mode waveguides

   https://doi.org/10.1039/C7SC02250F  

   Han, Donghoon; Crouch, Garrison M.; Fu, Kaiyu; Zaino III, Lawrence P.; Bohn, Paul W (January 2017, 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. 

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4. Voltage Modulation of Nanoplasmonic Metal Luminescence from Nano-Optoelectrodes in Electrolytes

   https://doi.org/10.1021/acsnano.3c01491  

   Zhao, Yuming; Xiao, Chuan; Mejia, Elieser; Garg, Aditya; Song, Junyeob; Agrawal, Amit; Zhou, Wei (May 2023, ACS Nano)

   Metallic nanostructures supporting surface plasmon modes can concentrate optical fields, and enhance luminescence processes from the metal surface at plasmonic hotspots. Such nanoplasmonic metal luminescence contributes to the spectral background in surface-enhanced Raman spectroscopy (SERS) measurements and is helpful in bioimaging, nano-thermometry, and chemical reaction monitoring applications. Despite increasing interest in nanoplasmonic metal luminescence, little attention has been paid to investigating its dependence on voltage modulation. Also, the hyphenated electrochemical surface-enhanced Raman spectroscopy (EC-SERS) technique typically ignores voltage-dependent spectral background information associated with nanoplasmonic metal luminescence due to limited mechanistic understanding and poor measurement reproducibility. Here, we report a combined experiment and theory study on dynamic voltage-modulated nanoplasmonic metal luminescence from hotspots at the electrode-electrolyte interface using multiresonant nanolaminate nano-optoelectrode arrays. Our EC-SERS measurements under 785 nm laser excitation demonstrate that short-wavenumber nanoplasmonic metal luminescence associated with plasmon-enhanced electronic Raman scattering (PE-ERS) exhibits a negative voltage modulation slope (up to ≈30 % V-1) in physiological ionic solutions. Furthermore, we have developed a phenomenological model to intuitively capture plasmonic, electronic, and ionic characteristics at the metal-electrolyte interface to understand the observed dependence of the PE-ERS voltage modulation slope on voltage polarization and ionic strength. The current work represents a critical step toward the general application of nanoplasmonic metal luminescence signals in optical voltage biosensing, hybrid optical-electrical signal transduction, and interfacial electrochemical monitoring. 

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5. Electrochemistry at single molecule occupancy in nanopore-confined recessed ring-disk electrode arrays

   https://doi.org/10.1039/C6FD00062B  

   Fu, Kaiyu; Han, Donghoon; Ma, Chaoxiong; Bohn, Paul W. (January 2016, Faraday Discuss.)

   Electrochemical reactions at nanoscale structures possess unique characteristics, e.g. fast mass transport, high signal-to-noise ratio at low concentration, and insignificant ohmic losses even at low electrolyte concentrations. These properties motivate the fabrication of high density, laterally ordered arrays of nanopores, embedding vertically stacked metal–insulator–metal electrode structures and exhibiting precisely controlled pore size and interpore spacing for use in redox cycling. These nanoscale recessed ring-disk electrode (RRDE) arrays exhibit current amplification factors, AF RC , as large as 55-fold with Ru(NH 3 ) 6 2/3+ , indicative of capture efficiencies at the top and bottom electrodes, Φ t,b , exceeding 99%. Finite element simulations performed to investigate the concentration distribution of redox species and to assess operating characteristics are in excellent agreement with experiment. AF RC increases as the pore diameter, at constant pore spacing, increases in the range 200–500 nm and as the pore spacing, at constant pore diameter, decreases in the range 1000–460 nm. Optimized nanoscale RRDE arrays exhibit a linear current response with concentration ranging from 0.1 μM to 10 mM and a small capacitive current with scan rate up to 100 V s −1 . At the lowest concentrations, the average pore occupancy is 〈 n 〉 ∼ 0.13 molecule establishing productive electrochemical signals at occupancies at and below the single molecule level in these nanoscale RRDE arrays. 

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