Label free redox capacitive biosensing Flávio C. Bedatty Fernandes a , Márcio S. Góes a , Jason J. Davis b,1 , Paulo R. Bueno a,n a Instituto de Química, Universidade Estadual Paulista, CP 355, 14800-900 Araraquara, São Paulo, Brazil b Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK article info Article history: Received 15 May 2013 Received in revised form 19 June 2013 Accepted 20 June 2013 Available online 2 July 2013 Keywords: Capacitance spectroscopy Immunosensors Biosensor Redox capacitance abstract A surface confined redox group contributes to an interfacial charging (quantifiable by redox capacitance) that can be sensitively probed by impedance derived capacitance spectroscopy. In generating mixed molecular films comprising such redox groups, together with specific recognition elements (here antibodies), this charging signal is able to sensitively transduce the recognition and binding of specific analytes. This novel transduction method, exemplified here with C-reactive protein, an important biomarker of cardiac status and general trauma, is equally applicable to any suitably prepared interfacial combination of redox reporter and receptor. The assays are label free, ultrasensitive, highly specific and accompanied by a good linear range. & 2013 Elsevier B.V. All rights reserved. 1. Introduction The electrochemical interrogation of biorecognition at man- made interfaces constitutes a potentially powerful means of establishing highly effective, cheap and portable diagnostic assays (Berggren et al., 2001; Davis and Tkac, 2009; Estrela et al., 2010; White et al., 2012; Xu et al., 2013; Eckermann et al., 2010; Bueno and Gabrielli, 2008). Within these, impedimetric tools, based on Electrochemical Impedance Spectroscopy (EIS), constitute potent, label free, and ultrasensitive probes of antibody–antigen or pep- tide–protein interactions on suitably modified electrode surfaces (Lisdat and Schäfer, 2008; Bryan et al., 2013; Johnson et al., 2012; Daniels and Pourmand, 2007; K’Owino and Sadik, 2005; Bryan et al., 2012). In its simplest and most common modality, the formation of bioaffinity complexes retards the interfacial electron transfer kinetics associated with a solution phase redox probe. The quantification of this “charge transfer resistance” (R ct ) is, then, the transducing signal used to quantify target analyte concentration across, typically, nanomolar to picomolar concentration limits (Lisdat and Schäfer, 2008; Daniels and Pourmand, 2007; Rodriguez et al., 2005; Bogomolova et al., 2009). In recent years there has been progress in moving such assays to the analysis of real clinical samples (Bryan et al., 2013; Johnson et al., 2012; Bryan et al., 2012). The use of R ct does, however, require the application of a redox probe to the analytical solution prior to analysis, and a subsequent fitting of data to an “equivalent circuit”. In recent work we have introduced a detailed capacitance analysis of pure dielectric and redox active molecular films and, in particular, electroactive monolayer capacitance spectroscopy, EMCS (Bueno et al., 2012a, 2012b; Goes et al., 2012). We show herein that the redox capacitance of a faradaic probe confined within a molecular film is a sensitive function of protein biomarker concentration when that film is additionally capable of selectively recruiting the target from solution. In principle, a number of electrochemical techniques might be utilised, in each case resulting in interrogating the probe output as a function of its local environment and any deliberately engi- neered local binding events (White et al., 2012; Elliott et al., 1986; Liu et al., 2008; Sumner and Creager, 2001). For example, voltam- metric methods have been proposed where the current response to an applied voltage is reported to detect antigen binding when the probe is tethered to the antibody or aptamer receptor (Johnson et al., 2012; Rodriguez et al., 2005; Darwish et al., 2012a, 2012b; Gooding and Darwish, 2012; Liu et al., 2008). Although analytically simple, these assays require the pre-synthesis of a receptor–redox conjugate and, in the case of aptamers, a predictable target induced conformational change (a change very often highly dependent on solution composition) (Rodriguez et al., 2005; Liu et al., 2008; Darwish et al., 2012a, 2012b; Gooding and Darwish, 2012; Liu et al., 2008). We have recently shown that the EIS derived complex capaci- tance signal can be used to generate an interfacial charging signal that arises solely from the redox activity of a confined group (its redox capacitance, C r ) and depends very sensitively of its electro- static environment. We start here by noting that C r of a surface confined electroactive film is not a common electrostatic capacitance Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.06.043 n Corresponding author. Tel.: +55 16 3301 9642; fax: +55 16 3322 2308. E-mail addresses: jason.davis@chem.ox.ac.uk (J.J. Davis), prbueno@iq.unesp.br (P.R. Bueno). 1 Tel.: +44 1865 275914. Biosensors and Bioelectronics 50 (2013) 437–440