TOBACCO MOSAIC VIRUS BIOTEMPLATED ELECTROCHEMICAL BIOSENSOR Hadar Ben-Yoav 1* , Adam D. Brown 2 , Ekaterina Pomerantseva 1 , Deanna L. Kelly 3 , James N. Culver 2 , and Reza Ghodssi 1* 1 MEMS Sensors and Actuators Laboratory, Department of Electrical and Computer Engineering, Institute for Systems Research, 2 Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Maryland, USA 3 Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland, USA ABSTRACT This work demonstrates the first utilization of virus molecules as nano-scale biotemplates assembled on an electrochemical biosensor, allowing for an 8-times increased signal and an improved biosensing performance of 9.5-fold. The versatile and inexpensive biological Tobacco mosaic virus was integrated as a high aspect ratio, low footprint, low-cost, easy to genetically functionalize, nanostructured three-dimensional scaffold for the synthesis of novel multifunctional electrodes. The biotemplated scaffold allows for an increased surface area resulting in higher electrochemical currents, better signal-to-noise ratio and improved sensitivity when incorporated into miniaturized biosensors. INTRODUCTION Electrochemical Biosensors Electrochemical biosensors are based on a bioelectrochemical interaction process, where electrochemical species are consumed or generated. Most electrochemical measurements detect oxidation/reduction of the product generated by biological conversion of the analyte undergoing redox reactions on the sensor's surface and solely respond to electro-active species. Electrochemical measurements are classified according to the variable being measured: amperometry, potentiometry and conductometry [1, 2]. In recent decades, nanostructured materials have received much attention due to unique properties they offer as robust platforms for electronic and optical signal transduction. One of its major contributions is the design of a new generation of miniaturized biosensing devices. Furthermore, biofunctional nanoparticles can produce a synergistic effect between catalytic activity, conductivity, and biocompatibility to accelerate the signal transduction, leading to the quick development of stable, specific, selective and sensitive biosensors in different fields. An important point besides the biosensing applicability of nanomaterials is the use of manipulation techniques for their integration in fabrication techniques [3]. By integrating with electrochemical sensor fabrication, these materials provide interesting properties such as increased surface area. The augmented surface area leads to higher signal-to-noise ratio, increased sensitivity and dynamic range, short distances for mass transport and charge transfer as well as their ability to create complex nano-bio-architectures that allow volume change and unique selective biological functionality. Nanomaterials allow the development of new architectures applied in electrochemical sensing and biosensing devices [4, 5]. For example, Ye and colleagues presented a multi-walled carbon nanotube (MWNT)-based electrochemical biosensor for glucose detection [5]. The higher surface area of the well-aligned MWNT generated higher electrochemical currents that were correlated to a highly sensitive sensor. Tobacco mosaic virus Biological nanomaterials provide a versatile and cost effective solution for the fabrication of high surface area nanoarchitectures. One category of these biological nanostructures is plant and bacteria virus particles consisting of macromolecular assemblies of nucleic acid packaged by many copies of coat proteins. These molecules display some unique advantages as they show exceptional stability in a wide range of temperatures and pH values, in addition to their surface-exposed functional groups, self- assembly and tunability [6]. Among the available plant viruses [7], Tobacco mosaic virus (TMV) is the most extensively studied filamentous plant structure for nanoscale applications. The TMV virion is a rigid rod consisting of about 2,130 identical coat protein subunits stacked in a helix around a single strand of plus sense RNA, forming a 4 nm diameter channel through the 300 nm long virion axis. Properties of the TMV system that make it particularly useful as a self- assembling macromolecular template for nanomaterials include: 1) its known three-dimensional structure [8]; 2) a wealth of biophysical information on its self-assembly characteristics [9]; 3) the availability of creating novel virus structures and surfaces via established molecular techniques [10]; 4) a wide range of existing coat protein variants with diverse assembly properties [11], and 5) the ability to easily purify large quantities of virus and coat protein from infected plants. TMV-structured metal and metal-oxide nanowires have been synthesized using several techniques [12, 13], transport properties have been studied [14], and potential applications in nano-scale devices have been investigated through proof-of-concept demonstrations [15]. Previous work with engineered mutations of the TMV has resulted in enhanced particle coatings and templates that can be readily integrated into microfabricated devices and has established a novel patterning process. Efficient templates for metallic coatings have been achieved through the introduction of one (TMV-1cys) or two (TMV-2cys) cysteine residues within the coat protein open reading frame. Cysteines are amino acids with thiol groups that show enhanced metal binding properties based on strong, covalent-like interactions. One and two-step electroless plating methods have been used for the fabrication of TMV-2cys- based wires coated with gold, silver and palladium clusters that show more uniform coating compared to the wild-type virus [16]. Additionally, the rod-shaped viruses can be directionally attached to various surfaces and coated to create high aspect ratio nickel, cobalt and platinum materials (TMV-1cys). Alternative pathways were explored for patterning the viral molecules in microfabricated electrodes as well as controlled environments, utilizing nucleic acid hybridization [17]. Recent work in our team has focused upon the development of novel inorganic structures using the TMV and their application in energy storage devices. A simple and versatile approach for the selective patterning of both metal-coated and uncoated TMV using lift-off processing has been developed [18]. The high aspect ratio of the coated TMV in addition to its robustness was utilized in the development of high surface area nickel-zinc [19] and Li-ion microbatteries [20, 21]. By the integration of high aspect ratio biotemplated TMV scaffold in electrode fabrication process, high surface area electrochemical sensors can be realized improving the overall biosensing performance.