title: A self-referencing biosensor for real-time monitoring of physiological ATP transport in plant systems.
abstract: The objective of this study was to develop a self-referencing electrochemical biosensor for the direct measurement of ATP flux into the extracellular matrix by living cells/organisms. The working mechanism of the developed biosensor is based on the activity of glycerol kinase and glycerol-3-phosphate oxidase. A stratified bi-enzyme nanocomposite was created using a protein-templated silica sol gel encapsulation technique on top of graphene-modified platinum electrodes. The biosensor exhibited excellent electrochemical performance with a sensitivity of 2.4±1.8 nA/µM, a response time of 20±13 s and a lower detection limit of 1.3±0.7 nM. The self-referencing biosensor was used to measure exogenous ATP efflux by (i) germinating Ceratopteris spores and (ii) growing Zea mays L. roots. This manuscript demonstrates the first development of a non-invasive ATP micro-biosensor for the direct measurement of eATP transport in living tissues. Before this work, assays of eATP have not been able to record the temporally transient movement of ATP at physiological levels (nM and sub-nM). The method demonstrated here accurately measured [eATP] flux in the immediate vicinity of plant cells. Although these proof of concept experiments focus on plant tissues, the technique developed herein is applicable to any living tissue, where nanomolar concentrations of ATP play a critical role in signaling and development. This tool will be invaluable for conducting hypothesis-driven life science research aimed at understanding the role of ATP in the extracellular environment.
authors.affiliation: Agricultural and Biological Engineering Department, University of Florida, Gainesville, USA; Food Engineering Department, Universidad del Valle, Cali, Colombia.
authors.forename: Diana C
authors.initials: DC
authors.lastname: Vanegas
authors.affiliation: Department of Molecular Biosciences, University of Texas, Austin, USA.
authors.forename: Greg
authors.initials: G
authors.lastname: Clark
authors.affiliation: Department of Molecular Biosciences, University of Texas, Austin, USA.
authors.forename: Ashley E
authors.initials: AE
authors.lastname: Cannon
authors.affiliation: Department of Molecular Biosciences, University of Texas, Austin, USA.
authors.forename: Stanley
authors.initials: S
authors.lastname: Roux
authors.affiliation: Department of Mechanical Engineering, University of Colorado, Denver, USA.
authors.forename: Prachee
authors.initials: P
authors.lastname: Chaturvedi
authors.affiliation: Agricultural and Biological Engineering Department, University of Florida, Gainesville, USA. Electronic address: emclamor@ufl.edu.
authors.forename: Eric S
authors.initials: ES
authors.lastname: McLamore
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism*; D001692:Biological Transport; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004800:Enzymes, Immobilized / Q000378:metabolism; D004867:Equipment Design; D005991:Glycerol Kinase / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism; D008958:Models, Molecular; D018517:Plant Roots / Q000502:physiology; D064028:Tracheophyta / Q000502:physiology*; D003313:Zea mays / Q000502:physiology*
chemical_list: D004800:Enzymes, Immobilized; D000255:Adenosine Triphosphate; D005993:Glycerolphosphate Dehydrogenase; C098831:glycerol-3-phosphate oxidase; D005991:Glycerol Kinase
keywords: ATP; Biosensor; Graphene; Nanoplatinum; Plant physiology; Self-referencing