Production and electrical characterization of the reflectin A2 isoform from Doryteuthis (Loligo) pealeii

Abstract

Cephalopods have recently emerged as a source of inspiration for the development of novel functional materials. Within this context, a number of studies have explored structural proteins known as reflectins, which play a key role in cephalopod adaptive coloration in vivo and exhibit interesting properties in vitro. Herein, we report an improved high-yield strategy for the preparation and isolation of reflectins in quantities sufficient for materials applications. We first select the Doryteuthis (Loligo) pealeii reflectin A2 (RfA2) isoform as a "model" system and validate our approach for the expression and purification of this protein. We in turn fabricate RfA2-based twoterminal devices and employ both direct and alternating current measurements to demonstrate that RfA2 films conduct protons. Our findings underscore the potential of reflectins as functional materials and may allow a wider range of researchers to investigate their properties.Cephalopods (squid, octopuses, and cuttlesh) are well known for their sophisticated neurophysiology, complex behavior, and stunning camouage displays.1-6 Recently, these animals have drawn signicant attention as sources of novel materials for optical systems, 7-10 biomedical technologies, 11-15 and bioelectronic devices. [16][17][18][19][20] Within this context, a number of literature reports have investigated the properties of unique structural proteins known as reectins, [7][8][9][10]14,[16][17][18][21][22][23][24] which are found in cephalopod skin cells (i.e. leucophores, iridophores, and chromatophores). [25][26][27][28][29][30] In vivo, reectins in general have been shown to play important roles in cephalopod adaptive coloration by serving as components of optically-active ultrastructures, including layered stacks of membrane-enclosed platelets in iridophores, 25,26 membrane-bound arrangements of spherical microparticles in leucophores, 27,28 and interconnected networks of pigment granules in chromatophores. 29,30 In vitro, the Doryteuthis (Loligo) pealeii reectin A1 (RfA1) isoform has found applications in recongurable infrared camouage coatings that are actuated by chemical and mechanical stimuli, 7,8 proton-conducting lms with electrical gures of merit rivaling those of some articial analogues, 16-18 and biocompatible substrates that support the proliferation and differentiation of neural stem cells.14 Overall, reectins' fascinating properties have provided a strong impetus for their continued exploration from both fundamental and applied perspectives.Herein, we describe an improved methodology for the production of difficult-to-handle reectins in quantities sufficient for materials applications. We rst select the Doryteuthis (Loligo) pealeii reectin A2 (RfA2) isoform as a "model" system for electrical characterization and validate a new high-yield strategy for the expression and purication of this precipitation-prone protein. We subsequently fabricate and characterize two-terminal devices for which RfA2 thin lms constitute the active layer. We in tu...

Neutralization of a Distributed Coulombic Switch Precisely Tunes Reflectin Assembly

“…These thin films have optical properties that are reversibly sensitive to both hydration and pH [22][23][24][29][30][31][32] . In a non-optical context, reflectin-based thin films have also been investigated as proton conductors and transistors, as well as substrates for neural cell growth [33][34][35][36] .…”

Protochromic Devices from a Cephalopod Structural Protein

“…The general morphology and simplified chemical activation mechanism for a squid iridophore are shown in Figure A. The cell's plasma membrane forms a periodic arrangement, which is comprised of membrane‐enclosed platelets (i.e., lamellae) from proteins called reflectins that alternate with deep invaginations (i.e., folds) into the cellular interior . This ultrastructure effectively constitutes a biological Bragg reflector, wherein the membrane‐enclosed platelets function as the high refractive index material and the externally accessible invaginations function as the de facto low refractive index material .…”

“…For our approach, we were conceptually encouraged by (1) prior demonstrations that the neurally controlled influx/efflux of ions and water alters the dimensions and optical properties of single iridophore platelets (lamellae) and (2) the report that acetic acid‐induced injection of protons into RfA1‐based coatings modulates their thickness and reflectance in vitro . Furthermore, our device design leveraged recent advances in bioprotonics, wherein PdH x electrodes exchange protons with cephalopod‐derived proton conductors, such as maleic chitosan and RfA1 (note that the chemical injection of protons via acid vapor, while highly effective, is impractical for interfacing with external electronics) . Thus, in lieu of a single protein‐based platelet, our device incorporated a thin film of RfA1 as the color‐changing active component, and in lieu of a surrounding ion‐permeable membrane, our device incorporated a PdH x electrode as the conductive actuating component (the ion‐blocking gold electrode functioned as a stable reference) (Figure B).…”

“…We began our experiments by fabricating the desired color‐changing bioelectronic devices according to the scheme in Figure S1 (Supporting Information). In our approach, we adapted protocols previously validated for the fabrication of RfA1‐based protonic devices . First, as the bottom electrode, we deposited a chromium (Cr) adhesion layer followed by a palladium (Pd) layer onto a silicon dioxide/silicon (SiO 2 /Si) substrate via electron‐beam evaporation.…”

“…In turn, as the top electrode, we deposited an Au layer directly onto the Pd‐ and RfA1‐modified surface, again via electron‐beam evaporation (note that a layered, low‐temperature deposition strategy mitigated damage to the protein film, yielding pristine devices like the one shown in Figure S2, Supporting Information). Finally, for the electrical measurements, we converted the electron‐injecting Pd contacts to proton‐injecting PdH x contacts through exposure to hydrogen gas . The overall procedure furnished devices with a sandwich‐type PdH x /RfA1/Au architecture.…”