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1. 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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2. Self-induced redox cycling coupled luminescence on nanopore recessed disk-multiscale bipolar electrodes

   https://doi.org/10.1039/c5sc00433k  

   Ma, Chaoxiong ; Zaino III, Lawrence P. ; Bohn, Paul W. ( January 2015 , Chemical Science)

   We present a new configuration for coupling fluorescence microscopy and voltammetry using self-induced redox cycling for ultrasensitive electrochemical measurements. An array of nanopores, each supporting a recessed disk electrode separated by 100 nm in depth from a planar multiscale bipolar top electrode, was fabricated using multilayer deposition, nanosphere lithography, and reactive-ion etching. Self-induced redox cycling was induced on the disk electrode producing ∼30× current amplification, which was independently confirmed by measuring induced electrogenerated chemiluminescence from Ru(bpy) 3 2/3+ /tri- n -propylamine on the floating bipolar electrode. In this design, redox cycling occurs between the recessed disk and the top planar portion of a macroscopic thin film bipolar electrode in each nanopore. Electron transfer also occurs on a remote (mm-distance) portion of the planar bipolar electrode to maintain electroneutrality. This couples the electrochemical reactions of the target redox pair in the nanopore array with a reporter, such as a potential-switchable fluorescent indicator, in the cell at the distal end of the bipolar electrode. Oxidation or reduction of reversible analytes on the disk electrodes were accompanied by reduction or oxidation, respectively, on the nanopore portion of the bipolar electrode and then monitored by the accompanying oxidation of dihydroresorufin or reduction of resorufin at the remote end of the bipolar electrode, respectively. In both cases, changes in fluorescence intensity were triggered by the reaction of the target couple on the disk electrode, while recovery was largely governed by diffusion of the fluorescent indicator. Reduction of 1 nM of Ru(NH 3 ) 6 3+ on the nanoelectrode array was detected by monitoring the fluorescence intensity of resorufin, demonstrating high sensitivity fluorescence-mediated electrochemical sensing coupled to self-induced redox cycling. 

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3. Electrochemical Zero-Mode Waveguide Studies of Single Enzyme Reactions

   https://doi.org/10.1109/NANO.2018.8626402  

   Han, D. ; Kwon, S.-R. ; Fu, K. ; Crouch, G.M. ; Bohn, P.W. ( July 2018 , 2018 IEEE 18th International Conference on Nanotechnology (IEEE-NANO))

   null (Ed.)

   Because electron transfer reactions are fundamental to life processes, such as respiration, vision, and energy catabolism, it is critically important to understand the relationship between functional states of individual redox enzymes and the macroscopically observed phenotype, which results from averaging over all copies of the same enzyme. To address this problem, we have developed a new technology, based on a bifunctional nanoelectrochemical-nanophotonic architecture - the electrochemical zero mode waveguide (E-ZMW) - that can couple biological electron transfer reactions to luminescence, making it possible to observe single electron transfer events in redox enzymes. Here we describe E-ZMW architectures capable of supporting potential-controlled redox reactions with single copies of the oxidoreductase enzyme, glutathione reductase, GR, and extend these capabilities to electron transfer events where reactive oxygen species are synthesized within the  100 zL volume of the nanopore. 

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4. Multifunctional nanopore electrode array method for characterizing and manipulating single entities in attoliter-volume enclosures

   https://doi.org/10.1063/5.0101693  

   Baek, Seol ; Cutri, Allison R. ; Han, Donghoon ; Kwon, Seung-Ryong ; Reitemeier, Julius ; Sundaresan, Vignesh ; Bohn, Paul W. ( November 2022 , Journal of Applied Physics)

   Structurally regular nanopore arrays fabricated to contain independently controllable annular electrodes represent a new kind of architecture capable of electrochemically addressing small collections of matter—down to the single entity (molecule, particle, and biological cell) level. Furthermore, these nanopore electrode arrays (NEAs) can also be interrogated optically to achieve single entity spectroelectrochemistry. Larger entities such as nanoparticles and single bacterial cells are investigated by dark-field scattering and potential-controlled single-cell luminescence experiments, respectively, while NEA-confined molecules are probed by single molecule luminescence. By carrying out these experiments in arrays of identically constructed nanopores, massively parallel collections of single entities can be investigated simultaneously. The multilayer metal–insulator design of the NEAs enables highly efficient redox cycling experiments with large increases in analytical sensitivity for chemical sensing applications. NEAs may also be augmented with an additional orthogonally designed nanopore layer, such as a structured block copolymer, to achieve hierarchically organized multilayer structures with multiple stimulus-responsive transport control mechanisms. Finally, NEAs constructed with a transparent bottom layer permit optical access to the interior of the nanopore, which can result in the cutoff of far-field mode propagation, effectively trapping radiation in an ultrasmall volume inside the nanopore. The bottom metal layer may be used as both a working electrode and an optical cladding layer, thus, producing bifunctional electrochemical zero-mode waveguide architectures capable of carrying out spectroelectrochemical investigations down to the single molecule level. 

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5. Fully inkjet-printed multilayered graphene-based flexible electrodes for repeatable electrochemical response

   https://doi.org/10.1039/d0ra04786d  

   Pandhi, Twinkle ; Cornwell, Casey ; Fujimoto, Kiyo ; Barnes, Pete ; Cox, Jasmine ; Xiong, Hui ; Davis, Paul H. ; Subbaraman, Harish ; Koehne, Jessica E. ; Estrada, David ( October 2020 , RSC Advances)

   null (Ed.)

   Graphene has proven to be useful in biosensing applications. However, one of the main hurdles with printed graphene-based electrodes is achieving repeatable electrochemical performance from one printed electrode to another. We have developed a consistent fabrication process to control the sheet resistance of inkjet-printed graphene electrodes, thereby accomplishing repeatable electrochemical performance. Herein, we investigated the electrochemical properties of multilayered graphene (MLG) electrodes fully inkjet-printed (IJP) on flexible Kapton substrates. The electrodes were fabricated by inkjet printing three materials – (1) a conductive silver ink for electrical contact, (2) an insulating dielectric ink, and (3) MLG ink as the sensing material. The selected materials and fabrication methods provided great control over the ink rheology and material deposition, which enabled stable and repeatable electrochemical response: bending tests revealed the electrochemical behavior of these sensors remained consistent over 1000 bend cycles. Due to the abundance of structural defects ( e.g. , edge defects) present in the exfoliated graphene platelets, cyclic voltammetry (CV) of the graphene electrodes showed good electron transfer ( k = 1.125 × 10 −2 cm s −1 ) with a detection limit (0.01 mM) for the ferric/ferrocyanide redox couple, [Fe(CN) 6 ] −3/−4 , which is comparable or superior to modified graphene or graphene oxide-based sensors. Additionally, the potentiometric response of the electrodes displayed good sensitivity over the pH range of 4–10. Moreover, a fully IJP three-electrode device (MLG, platinum, and Ag/AgCl) also showed quasi-reversibility compared to a single IJP MLG electrode device. These findings demonstrate significant promise for scalable fabrication of a flexible, low cost, and fully-IJP wearable sensor system needed for space, military, and commercial biosensing applications. 

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