Electrochemical sensor for rapid determination of fibroblast growth factor receptor 4 in raw cancer cell lysates

Abstract

The first electrochemical immunosensor for the determination of fibroblast growth factor receptor 4 (FGFR4) biomarker is reported in this work. The biosensor involves a sandwich configuration with covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic microcarriers (HOOC-MBs) and amperometric detection at disposable carbon screen-printed electrodes (SPCEs). The biosensor exhibits a great analytical performance regarding selectivity for the target protein and a low LOD of 48.2 pg mL-1. The electrochemical platform was successfully applied for the determination of FGFR4 in different cancer cell lysates without any apparent matrix effect after a simple sample dilution and using only 2.5 μg of the raw lysate. Comparison of the results with those provided by a commercial ELISA kit shows competitive advantages by using the developed immunosensor in terms of simplicity, analysis time, and portability and cost-affordability of the required instrumentation for the accurate determination of FGFR4 in cell lysates.

Fig 1. Schematic representation of the amperometric sensor for FGFR4 determination using magnetic immunocarriers and a sandwich format.

Fig 2. Dependence of the amperometric responses obtained with the developed immunosensor with the BDAb concentration (a), and the number of steps carried out to perform the immunoassay (b).

Results are shown in the absence (white bars) or in the presence of 5,000 pg mL^-1 FGFR4 (grey bars) together with the corresponding S/B ratio ($•$). 2(A), two sequential steps involving 30 min incubation of the CAb-MBs in a mixture solution containing FGFR4 and BDAb, and 30 min incubation in the Strep-HRP solution; 2(B) two steps involving 30 min incubation of the Cab-MBs with FGFR4 solution, followed by a 30 min incubation step in a mixture solution containing BDAb and Strep-HRP. Error bars estimated as triple of the standard deviation (n = 3).

Fig 3. Dependence of the amperometric response measured with the developed magnetic immunocarriers-based sensor with the concentration of FGFR4 standards.

Error bars estimated as triple of the standard deviation (n = 3).

Fig 4. Evaluation of the selectivity of the developed immunosensor for FGFR4.

Amperometric signals measured in the absence (white bars) and in the presence of 2,500 pg mL^-1 FGFR4 (grey bars) and the corresponding S/B ratio ($•$) in the absence (1) and in the presence of 10 ng mL^-1 TNFα (2), 200 ng mL^-1 human p53 (3), 5 mg mL^-1 BSA (4), 5 mg mL^-1 hemoglobin (5) and 1 mg mL^-1 human IgG (6). Error bars estimated as triple of the standard deviation (n = 3).

Fig 5. Analysis of FGFR4 in cell lysates by Western Blot (10 μg) and amperometric traces recorded with the developed immunosensor (2.5 μg).