title: Survey of Redox-Active Moieties for Application in Multiplexed Electrochemical Biosensors.
abstract: Recent years have seen the development of a large number of electrochemical sandwich assays and reagentless biosensor architectures employing biomolecules modified via the attachment of a redox-active "reporter." Here we survey a large set of potential redox reporters in order to determine which exhibits the best long-duration stability in thiol-on-gold monolayer-based sensors and to identify reporter "sets" signaling at distinct, nonoverlapping redox potentials in support of multiplexing and error correcting ratiometric or differential measurement approaches. Specifically, we have characterized the performance of more than a dozen potential reporters that are, first, redox active within the potential window over which thiol-on-gold monolayers are reasonably stable and, second, are available commercially in forms that are readily conjugated to biomolecules or can be converted into such forms in one or two simple synthetic steps. To test each of these reporters we conjugated it to one terminus of a single-stranded DNA "probe" that was attached by its other terminus via a six-carbon thiol to a gold electrode to form an "E-DNA" sensor responsive to its complementary DNA target. We then measured the signaling properties of each sensor as well as its stability against repeated voltammetric scans and against deployment in and reuse from blood serum. Doing so we find that the performance of methylene blue-based, thiol-on-gold sensors is unmatched; the near-quantitative stability of such sensors against repeated scanning in even very complex sample matrices is unparalleled. While more modest, the stability of sensors employing a handful of other reporters, including anthraquinone, Nile blue, and ferrrocene, is reasonable. Our work thus serves as both to highlight the exceptional properties of methylene blue as a redox reporter in such applications and as a cautionary tale-we wish to help other researchers avoid fruitless efforts to employ the many, seemingly promising and yet ultimately inadequate reporters we have investigated. Finally, we hope that our work also serves as an illustration of the pressing need for the further development of useful redox reporters.
authors.forename: Di
authors.initials: D
authors.lastname: Kang
authors.affiliation: Dipartimento di Scienze e Tecnologie Chimiche, University of Rome , Tor Vergata, Via della Ricerca Scientifica, 00133, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Ricci
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, Baltimore County , Baltimore, Maryland 21250, United States.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: White
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Plaxco
mesh_terms: D000880:Anthraquinones / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000379:methods*; D004566:Electrodes; D005296:Ferrous Compounds / Q000737:chemistry; D006046:Gold / Q000737:chemistry*; D000075163:Metallocenes / Q000737:chemistry; D008751:Methylene Blue / Q000737:chemistry; D010078:Oxazines / Q000737:chemistry; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D000880:Anthraquinones; D004277:DNA, Single-Stranded; D005296:Ferrous Compounds; D000075163:Metallocenes; D010078:Oxazines; D013438:Sulfhydryl Compounds; C008619:Nile Blue; D006046:Gold; D004247:DNA; D008751:Methylene Blue; C004998:ferrocene