An optimization of blocking agents for designing reliable electrochemical biosensors for ovarian cancer

Abstract

Here, we report development of an electrochemical biosensor for ovarian cancer diagnosis using an optimized blocking agent for reliable performance of the device. The sensitivity, specificity and reproducibility of the device are negatively impacted by non-specific binding of molecules present in sample - proteins, DNA, RNA, small drug molecules, hormones etc, other than the analyte of interest bind to biorecognition molecule on the biosensor surface. Electrochemical biosensor was fabricated by functionalizing carbon screen printed electrodes with citrate-reduced gold nanoparticles followed by immobilization of 5′-amine modified ssDNA probe against miRNA 204 (ovarian can biomarker). To avoid or minimize non-specific binding, blocking reagent was optimized. Blocking reagents and methods are typically chosen in an empirical manner since a single standardized procedure has not been determined suitable for all applications. This paper presents the commonly used blocking buffers with varying molecular weights to minimize the non-specific binding process on the surface of the electrode. Here, three different blocking buffers are used - Bovine serum albumin (BSA; MW: 66430.3 kDa), Gelatin (MW: 40 kDa), and Polyethylene Glycol (PEG MW 4 kDa and 6 kDa). Additionally, different surfactants like tween20, Tritonx100, and HEPES buffer are used for preparation of blocking agents. Further, to evaluate the performance of different blocking agents, the sample of miRNA-204 is spiked in 0.01 M Phosphate buffer saline (PBS) and fetal bovine serum (FBS) were tested using chroamperometry. The difference in the saturation current was recorded for both curves obtained for 0.01 M PBS and FBS. Optimum blocking buffer 1% Gelatin in Tween20 was found to be giving negligible nonspecific binding for DNA based ovarian cancer biosensor.