Detection of folic acid protein in human serum using reduced graphene oxide electrodes modified by folic-acid

Highlights

• The use of electrophoretically deposited rGO for electrochemical sensing is proposed.

• The integration of folic acid allows for the sensing of folic acid proteins.

• A detection limit of 1 pM for folic acid proteins was achieved.

• The possibility to use this sensor interface in human serum is possible.

Abstract

The detection of disease markers is considered an important step for early diagnosis of cancer. We design in this work a novel electrochemical sensing platform for the sensitive and selective detection of folic acid protein (FP). The platform is fabricated by electrophoretic deposition (EPD) of reduced graphene oxide (rGO) onto a gold electrode and post-functionalization of rGO with folic acid. Upon FP binding, a significant current decrease can be measured using differential pulse voltammetry (DPV). Using this scheme, a detection limit of 1 pM is achieved. Importantly, the method also allows the detection of FP in serum being thus an appealing approach for the sensitive detection of biomarkers in clinical samples.

Introduction

The use of biosensors for the early diagnosis of diseases has become widely accepted (Arya and Bhansali, 2011, Yildiz Uludag and Tothill, 2012). These sensors are based on the detection of disease markers (e.g. proteins overexpressed in blood and serum) and provide a point-of-care diagnosis that is rapid and cheap with appropriate specificity and sensitivity. However, biomarkers are present at very low concentrations in the early stage of cancer and necessitate the use of devices with extremely high sensitivity. One way to overcome some of the current limitations of biomarker sensors is through the use of nanostructures, which can be integrated into sensing platforms (Perfézou et al., 2012).

Next to nanoparticles, graphene-based sensors have shown promise for the sensitive and selective detection of key biomarker proteins (Kim et al., 2013, Myung et al., 2011, Qu et al., 2011, Xu et al., 2011). Myung et al. (2011) showed for example that the increased surface-to-volume ratio significantly helped in lowering the detection limits to 1 pM for the target biomarkers. A reduced graphene oxide (rGO)-based field effect transistor (rGO-FET) was reported by Kim et al. (2013) to detect protein–protein interactions down to femtomolar level with a dynamic range of over 6-orders of magnitude. In most cases, the detection is based on an immunoassay platform, where specific antibodies are immobilized onto the sensing transducer to capture selectively the biomarkers. The binding activity of the antibody is largely linked to the way it is immobilized onto the surface, with the possibility of losing in binding capacity due to a degradation of the protein over time. Thus, the development of simple analysis systems without the need of biomolecules’ immobilization on the sensor’s surface remains challenging. Furthermore, to obtain clinically relevant results, it is essential to perform the tests in human serum samples. The main difficulty of using serum as the analysis medium is the high non-specific interaction between the sensor’s surface and the serum proteins. A number of strategies have been developed to reduce nonspecific binding of clinical serum samples; such as the incorporation of ethylene glycol units onto the sensing surface (Ayela et al., 2007).

We develop in this work novel electrochemical sensing matrixes for the selective and sensitive detection of folic acid protein (FP) (Fig. 1). FP, also known as the folate receptor, is a kind of tumor-associated antigen, which is over-expressed in many human epithelial-derived tumors. Levels of FP in metastic diseases can increase to 22 pM (Eichner et al., 1978). Given that human serum is free of FP, detection of FP in serum serves as an early stage cancer diagnostic step. However, the low concentration (picomolar range) of this biomarker necessitates the use of ultrasensitive detection methods. Current serum FP detection strategies consist of using different analytical methods (Table 1), such as quartz crystal microbalance (QCM) (Henne et al., 2006), different electrochemical (Castillo et al., 2013, Maiyalagan et al., 2013, Wang et al., 2014, Wu et al., 2009) and optical approaches (Ahmad et al., 2015, Jiang and Wang, 2014, Li et al., 2014, Wu et al., 2009, Zhao et al., 2014). However, only a handful of these methods exhibited the required sensitivity for real time sensing of biological samples.

We show in this paper that an electrochemical transducer, prepared through electrophoretic deposition (EPD) of reduced rGO onto gold electrode and post-functionalized with folic acid as ligand, allows for the picomolar sensing of folic acid proteins (FP) in an easy manner (Fig. 1). It is now well established, that FP, which can be isolated from bodily liquid, including blood, urine and from culture media of folate-receptor-positive tumor cell lines, binds with high affinity in a 1:1 stoichiometry to folic acid (Anthony, 1996). We will also demonstrate that the developed interface allows for the detection of FP in serum, being thus adapted to the sensitive detection of biomarkers in clinical samples.