SCH727965 Traditional methods for the detection of ER includ

Traditional methods for the detection of ERα include enzyme-linked immunosorbent assay (ELISA) [22], Western blotting [4] and immunohistochemistry [11]. Nevertheless, these methods usually present some shortcomings. For example, immunohistochemistry and Western blotting provide typically semiquantitative results [19,23] while ELISA can produce quantitative data but is time-consuming and expensive [27]. Electrochemical biosensors represent a clear current alternative to conventional methods in the clinical field because of their simplicity of use and inherent high selectivity and sensitivity. Moreover, they are able to provide accurate quantitative determinations using cost-effective instrumentation with pocket-size dimensions, ideal for integration in point-of-care (POC) devices [1,14]. Despite these excellent features only one electrochemical DNA sensor has been reported for the determination of ERα [27]. This biosensor involves the protection of DNA duplex from Exonuclease III (Exo III)-mediated SCH727965 by specific binding of ERα to its DNA response elements and allowed quantification of ERα in the concentration range of 0.5 to 100nM with a detection limit of 0.38nM (25.1ngmL). The electrochemical DNA sensor was applied to interrogate the ERα levels in nuclear extracts from different breast cancer cell lines but the electrode surface modification protocol was long (16.5h) and the total assay time took 19h [27,28]. To the best of our knowledge, no electrochemical immunosensor has been so far reported for the determination of ERα. This paper describes the first electrochemical immunosensor for the selective and sensitive determination of this clinically relevant biomarker. The proposed design implies the use of functionalized magnetic beads (MBs) which have demonstrated to constitute powerful tools to construct electrochemical immunosensors contributing to improve their bioanalytical performance in terms of sensitivity, reduced assay time and minimization of matrix effects which are essential to perform determinations in complex matrices [20,26]. In this particular case, the developed magnetoimmunosensor involves a sandwich configuration using two sheep anti-human ERα antibodies. The capture antibody was immobilized onto carboxylic acid-modified magnetic beads (HOOC-MBs) whereas the biotinylated detector antibody was labeled with a streptavidin-HRP conjugate. The electrochemical monitoring of the affinity reactions was carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) using hydroquinone (HQ) as electron transfer mediator and H2O2 as HRP substrate. The applicability of the developed disposable magnetoimmunosensor was evaluated by determining the target ERα protein in cell lysates and spiked human serum as well as by in vitro monitoring of the ERα status receptor in cancer cells. With the merits of simplicity, quick response, high sensitivity, easy operation and low cost, the proposed method can overcome disadvantages of traditional methods and may have a positive impact in the diagnosis and follow-up of breast cancer.
Materials and methods
Results and discussion The immunoassay fundamentals as well as the electrochemical reactions involved in the development of the magnetoimmunosensing platform for ERα biomarker determination are schematically displayed in Fig. 1. As can be observed, all the immunoreactions involved in the sandwich-type assay occurred on the MBs surface. After magnetically trapping the MBs bearing the immunoconjugates on the working electrode surface, amperometric detection of the HRP enzymatically catalyzed reduction current generated in the presence of the HQ/H2O2 redox system was carried out.
Conclusions In this work, the first electrochemical immunosensor for the determination of ERα protein is reported. The novel magnetoscaffold involves the use of a sandwich immunosensing configuration, HOOC-MBs and amperometric detection at SPCEs using the HQ/HRP/H2O2 system. The magnetoimmunosensor exhibits an excellent analytical performance in terms of sensitivity, selectivity and reliability of the measurements in complex biological samples. These attractive analytical characteristics, which bring relevant improvements with respect to commercial ELISA spectrophotometric kits in terms of simplicity and assay time, allow the accurate determination of ERα in spiked human serum and raw cell lysates. Moreover, this is the first time that an electrochemical biosensor is applied to monitor this hormonal receptor status in intact breast cancer cells thus making it a promising tool to be implemented in useful low-cost devices designed for diagnostic and therapeutic action in breast cancer.