Full Paper Detection of C Reactive Protein (CRP) in Serum by an Electrochemical Aptamer-Based Sandwich Assay Sonia Centi, a * Laura Bonel Sanmartin, a Sara Tombelli, a Ilaria Palchetti, a Marco Mascini a, b a Dipartimento di Chimica, Universita ` degli Studi di Firenze, Polo Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy b Istituto Nazionale di Biostrutture e Biosistemi, Viale delle medaglie doro 305, 00136, Roma, Italy *e-mail: sonia.centi@unifi.it Received: November 11, 2008 Accepted: December 8, 2008 Abstract A disposable electrochemical assay involving magnetic particles and carbon-based screen-printed electrodes (SPCEs) was developed for the detection of C Reactive Protein (CRP). CRP is a plasma protein and is among the most expressed proteins in acute phase inflammation cases, being a known biomarker for inflammatory states. The assay was based on a sandwich format in which a RNA aptamer was coupled to a monoclonal antibody and alkaline phosphatase (AP) was used as enzymatic label. After the sandwich assay, the modified magnetic beads were captured by a magnet on the surface of a graphite working electrode and the electrochemical detection was thus achieved through the addition of the AP substrate (a-naphthyl-phosphate) and a-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV). The parameters influencing the different steps of the assay were optimized in order to reach the best sensitivity and specificity. With the optimized conditions, the assay was applied to the analysis of CRP free serum and serum samples. Keywords: Aptamers, Biosensors, Magnetic beads, C reactive proteins, Electrochemical transduction, Serum DOI: 10.1002/elan.200804560 1. Introduction Detection and quantification of C-reactive protein (CRP) in an easy, cheap, and fast way can improve clinical diagnostics in order to prevent serious inflammatory states. CRP is a protein present in plasma and is one of the most expressed proteins in acute phase inflammation cases, being a known biomarker for inflammatory states [1]. CRP is organized in a two-chain form, but it can adapt a circular form constituted by five equal subunits [2]. Each subunit binds to two calcium ions, interacts by intermolecular noncovalent salt bridges [3] and comprises 224 amino acids with a molecular weight of approximated 25 kDa [4, 5]. The CRP reference concentration of healthy subjects is < 5 mg/L in serum [6] and the clinical range of interest is 1 – 500 mg/L. High levels of CRP are related with a large clinical state of diseases [7] such as cardiovascular diseases [8, 9] and bacterial infections [7, 10]. The detection of CRP levels is of extreme importance because of the clinical role of CRP. There are several assays to detect CRP levels in human fluids. Their denomination is connected with the CRP detection range. According to the Food and Drug Administration, the assays might be qualified as Conventional C reactive protein (CRP), High sensitivity CRP (hsCRP) and Cardiac C reactive protein (cCRP) [11]. The assays with the aims to detect the level of inflammatory damage are denominated conventional and detect CRP levels in the range 5 – 10 mg/L [11]. The hsCRP assays detect CRP concentrations equal to 0.2 mg/L. These assays have the aim to predict the risk of a possible disease related with CRP levels. The cCRP assays are designed to be an independent auxiliary prognostic test in populations with stable coronary diseases or possible future development of cardiovascular accidents. The levels of detection are the same for hsCRP concentrations [11]. The techniques normally used to detect CRP are radial immunodiffusion (RID), radioimmunoassay (RIA), immu- nonephelometry (IN), immunoturbidimetry (IT), immuno- fluorescence, immunochemiluminescence and standard en- zyme immunoassay (as ELISA). The limit of detection of these assays ranges between 0.1 and 0.2 mg/L. In the last years several biosensors for CRP detection have been published. Many of them employ antibodies as biorecognition elements [12 – 18], whereas few papers are based on the use of aptamers [19, 20]. Aptamers are single- stranded DNA or RNA oligonucleotides generated by an in vitro selection process called SELEX (systematic evolution of ligands by exponential enrichment). It is possible by the SELEX process to identify RNA/DNA molecules from a very large population of random sequence oligomers (DNA or RNA libraries), which bind to the target molecule with very high affinity and specificity. Aptamers have been selected against a wide number of diagnostically relevant marker molecules, among these cancer-associated proteins 1309  2009 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Electroanalysis 2009, 21, No. 11, 1309 – 1315