Label-free RNA aptamer-based capacitive biosensor for the detection of C-reactive proteinw Anjum Qureshi, Yasar Gurbuz, Saravan Kallempudi and Javed H. Niazi* Received 11th March 2010, Accepted 5th July 2010 DOI: 10.1039/c004133e In this study, we report a novel aptamer-based capacitive label-free biosensor for monitoring transducing aptamer–protein recognition events, based on charge distribution under the applied frequency by non-Faradaic impedance spectroscopy (NFIS). This approach to capacitive biosensors is reported for the first time in this study, is reagent-less in processing and is developed using gold interdigitated (GID) capacitor arrays functionalized with synthetic RNA aptamers. The RNA atpamers served as biorecognition elements for C-reactive protein (CRP), a biomarker for cardiovascular disease risk (CVR). The signal is generated as a result of the change in relative capacitance occurring as a result of the formation of an RNA–CRP complex on GID capacitors with the applied AC electrical frequency (50–350 MHz). The dispersion peak of the capacitance curve was dependent on the CRP concentration and tends to shift toward lower frequencies, accompanied by the increase in relaxation time due to the increased size of the aptamer–CRP complex. The dissociation constant (K d ) calculated from the non-linear regression analysis of the relative capacitance change with the applied frequency showed that strong binding of CRP occurred at 208 MHz (K d = 1.6 mM) followed by 150 MHz (K d = 4.2 mM) and 306 MHz (K d = 3.4 mM) frequencies. The dynamic detection range for CRP is determined to be within 100–500 pg ml 1 . Our results demonstrates the behavior of an RNA–protein complex on GID capacitors under an applied electric field, which can be extended to other pairs of affinity biomolecules as well as for the development of electrical biosensor systems for different applications, including the early diagnosis of diseases. 1. Introduction A biosensor is a device designed to detect or quantify bio- molecules and they have been widely used as a powerful analytical tool in medical diagnostics, in the food industry and in environmental, security and defence research. Biosensors can detect proteins, nucleic acids DNA sequences and can monitor antigen–antibody interactions. In principle, they are generally fabricated by immobilizing a biological receptor material on the surface of a suitable transducer that converts the biochemical signal into quantifiable electronic signals. However, there are several shortcomings as these classical biosensors composed of recognition molecules often derived of living cells, such as enzymes, receptors, and anti- bodies. 1–3 The main disadvantage of using antibodies is their instability due to irreversible denaturation under external environmental perturbations. Therefore, alternative routes are continuously sought after in order to develop stable biosensors with synthetic biorecognition elements. Novel synthetic molecules such as aptamers can fill the gaps associated with biomolecules derived of living cells. Aptamers are short, single-stranded DNA or RNA oligonucleotides that can bind to their targets and offer specific properties, which favor them as new biorecognition elements for biosensors. 4 The different nature of these nucleic-acid recognition elements and their protein targets, and the unique properties of aptamers indicate great promise for designing innovative sensing protocols. 4 Most aptamer-based biosensors reported to date rely on standard sandwich-type bioaffinity assays connected to a common enzyme, 5 fluorophore, 6 or nanoparticle tracer. 7 Recently, electrochemical aptamer-based biosensors (Faradaic type) have been reported on different platforms to detect specific proteins as disease biomarkers. 5,8–10 The electrochemical- based detection process required the help of electron transfer mediators and the participation of additional substrates. In some cases, long incubation times are required due to the slow diffusion of analyte through an unstirred layer to form the immunocomplex. These disadvantages limit greatly their application in disease diagnosis. Here, we describe a novel aptamer-based capacitive label- free biosensor for monitoring transducing aptamer–CRP recognition events based on charge distribution under the applied frequency by NFIS using capacitors made of two electrodes for ground and signal in the absence of redox mediators. NFIS is an effective method for probing bio- molecular binding events such as ligand–target interactions. The measuring principle of these sensors is based on simple changes in dielectric properties, charge distribution, and conductivity changes when a ligand–target complex formed Faculty of Engineering and Natural Sciences, Sabanci University, Orhanli, Tuzla 34956, Istanbul, Turkey. E-mail: javed@sabanciuniv.edu; Fax: +90 (216) 483 9550; Tel: +90 (216) 483 9000 Extn. 9879 w This article was submitted as part of an issue to coincide with Faraday Discussion 149: Analysis for Healthcare Diagnostics and Theranostics. 9176 | Phys. Chem. Chem. Phys., 2010, 12, 9176–9182 This journal is  c the Owner Societies 2010 PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics Published on 20 July 2010. Downloaded by Sabanci University on 07/12/2016 08:05:53. View Article Online / Journal Homepage / Table of Contents for this issue