Sensors and Actuators B 209 (2015) 645–651 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical jo ur nal home page: www.elsevier.com/locate/snb Capacitive aptamer–antibody based sandwich assay for the detection of VEGF cancer biomarker in serum Anjum Qureshi a,∗∗ , Yasar Gurbuz b , Javed H. Niazi a,∗ a Sabanci University Nanotechnology Research and Application Center, Orta Mahalle 34956, Tuzla, Istanbul, Turkey b Facutly of Engineering and Natural Science, Sabanci University, Orhanli 34956, Tuzla, Istanbul, Turkey a r t i c l e i n f o Article history: Received 21 September 2014 Received in revised form 4 December 2014 Accepted 9 December 2014 Available online 16 December 2014 Keywords: VEGF Aptamer Cancer biomarker Biosensor Capacitance a b s t r a c t In this paper, we report on in vitro anti-VEGF aptamer selection and the development of a capacitive aptamer–antibody based sandwich assay for sensitive detection of vascular endothelial growth factor- 165 (VEGF) in human serum. The assay design involves capturing of VEGF protein through two epitopes, one with anti-VEGF aptamer and the other with the antibody. The capacitive sensor was functionalized with anti-VEGF aptamer which first captures the VEGF protein followed by sandwiching with antibody conjugated magnetic beads (MB-Abs). Changes in capacitance was measured using non-Faradaic elec- trochemical impedance spectroscopy (nFIS) at different AC electrical frequencies (50–300 MHz). The sandwich assay format exhibited enhanced capacitive signal with respect to concentration with a detec- tion range 5 pg mL -1 to 1 ng mL -1 of VEGF protein in human serum. Use of anti-VEGF aptamers enabled chemical stability on sensors during and after the chemical coupling on sensors. This work demonstrated the successful application of on-chip aptamer–antibody based sandwich assays for detection of target proteins in real serum sample for early cancer diagnosis. © 2014 Elsevier B.V. All rights reserved. 1. Introduction VEGF is a signaling protein, also used as a serum biomarker for a number of human diseases, including cancer [1,2], rheumatoid arthritis [3], psoriasis [4], and proliferating retinopathy [5]. VEGF potently promotes angiogenesis for vascular development, making it an attractive target for controlling angiogenic factor that plays a pivotal role in tumor growth and metastasis [6,7]. The abnormally fast growth and division of tumors prompts the over-expression of VEGF because of supply of more nutrients and oxygen, resulting in the induction of tumor lymphatic-vessels and the metastasis of cancer [8]. Patients with breast cancer have serum VEGF concentra- tion range, from 18 to 328 pg/mL, whereas in healthy individuals, it is from 1 to 177 pg/mL, therefore an assay must be able to measure these concentration ranges [9]. Existing detecting methods such as immunohistochemistry (IHC) [10], enzyme-linked immunosor- bent assays (ELISAs) [11] and fluorescence in situ hybridization (FISH) techniques [12] require sophisticated instrumentation that are complicated, expensive and time consuming. Therefore, it ∗ Corresponding author. Tel.: +90 216 483 9879; fax: +90 216 483 9885. ∗∗ Corresponding author. Tel.: +90 216 483 9000/2441. E-mail addresses: anjum@sabanciuniv.edu (A. Qureshi), javed@sabanciuniv.edu, javedkolkar@gmail.com (J.H. Niazi). is imperative to develop a rapid, sensitive, label-free, and cost- effective method for the detection of VEGF in blood/serum from the suspected patients for early diagnosis. Alternative new routes are ever demanding for developing suit- able biosensors for disease diagnosis, such as by using synthetic biorecognition elements mainly ssDNA or RNA aptamers. Aptamers can effectively bind to their target biomarker proteins and offer spe- cific properties of stability and resistance to denaturation, which favor them as new class of biorecognition elements for biosensors [13]. Aptamers can be selected through an in vitro selection process known as systematic evolution of ligands by exponential enrich- ment (SELEX) which yields synthetic nucleic acid ligands specific to desired target species from complex libraries [14]. The unique properties of aptamers enable designing innovative sensing pro- tocols through interfacing them with electrochemical, optical or mass-sensitive transducing approaches [15–17]. Recently, several aptamer-based biosensors (aptasensors or aptamer beacons) [18] have been used for probing binding-induced conformational changes in aptamers and monitor their interaction with targets by means of color changes [19,20] or electron transfer [21,22]. However, these methods are limited by sensor size, sensitivity or complexity of assays. For example, colorimetric or fluorescence quenching methods require collecting a large amount of particles to induce a discernable color change or fluorescent signals. Whereas Faradiac principle based electrochemical sensing http://dx.doi.org/10.1016/j.snb.2014.12.040 0925-4005/© 2014 Elsevier B.V. All rights reserved.