Detection of C Reactive Protein (CRP) in Serum by an Electrochemical Aptamer‐Based Sandwich Assay

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.

Voltammetric aptasensors for protein disease biomarkers detection: A review

“…However, this classification "signal-on" and "signal-off", is also often employed in the sandwich assays (Centi et al, 2007(Centi et al, , 2009Zhang et al, 2007Zhang et al, , 2009 or label-free aptasensors, when an increase or decrease of the signal is observed after the formation of the aptamer-target complex (Cheng et al, 2007;Degefa and Kwak, 2008;Meirinho et al, 2015;Rodríguez and Rivas, 2009).…”

“…Some works have reported the use of the sandwich-format aptasensor for the detection of different proteins, namely thrombin (Centi et al, 2007;Ding et al, 2010;Kang et al, 2008;Zhao et al, 2011a), C-reactive protein (CRP) (Centi et al, 2009) and PDGF Zhang et al, 2007Zhang et al, , 2009 . Centi et al (2007) developed a labelled "signal-on" electrochemical aptasensor for the detection of thrombin in complex matrices using the sandwich assay with magnetic particles as solid supports.…”

“…However, in this case a biotinylated aptamer with affinity for CRP immobilized onto streptavidin magnetic beads (capture probe) and a biotinylated monoclonal antibody anti-CRP conjugated with alkaline phosphatase (AP) (detection probe) were used. Again very low detection limits (2 nM) in serum samples were obtained (Centi et al, 2009). A similar sandwich strategy was designed by Zhang et al (2007Zhang et al ( , 2009 to detect the proteins PDGF-BB and PDGF, respectively.…”

An electrochemical immunosensor for ochratoxin A determination in wines based on a monoclonal antibody and paramagnetic microbeads

“…We have also developed immunosensors based on a quartz-crystal microbalance [35], and molecularly imprinted polymers for the screening of the mycotoxin by fluorescence detection in an automated flowing system [16]. In this work, we report an improved electrochemical immunosensor for determination of OTA in wine samples, in which competitive immunoassays are performed using paramagnetic microbeads (MBs) as solid support, providing a relative large number of binding sites and faster assay kinetics, with strategies similar to those we have used in sensitive electrochemical aptasensors to C-reactive protein [36] and OTA [22].…”

Aptamer‐Based Inhibition Assay for the Electrochemical Detection of Tobramycin Using Magnetic Microparticles

“…Mascini group has explored several ELISA format assays for the detection of thrombin [11,12] and C reactive protein [13] by transferring the MPs, where the recognition event takes place, to a screen-printed electrode with a magnetic block in the bottom and further electrochemical detection of an enzymatic product.…”