Surface architecture-based electrochemical biosensors work on a complex structure that is created to capture probes. A small defect in the architecture can cause an error in probe-target interaction. In such electrochemical sensors, the primary assumption is that the sensor has an ideal architecture. Nevertheless, these sensors drive the detection of analytes as the focus is on the probe-target interaction; and often, the underlying surface properties due to the resultant architecture are neglected while analyzing the detection phenomenon. Hence, the assessment of the dependence of the sensing process on the non-idealities present is required to achieve reliable measurements.The sensor response depends on the size, shape, and surface attachment of nanoparticles [1-4]. In most of the sensing methods, the use of high-density nanoparticles causes polydispersity and defects due to orientations on the electrode surface that conceal the efficacy of these factors in biosensing [5,6]. In this work, gold electrodes were fabricated using photolithography, RIE, and evaporation techniques, with the intent to achieve a more reliable surface structure. The screen-printed carbon electrodes (SPCE) are also considered; however, it is noted that the surface roughness and effective surface area in SPCEs depend on the polishing process and hence vary from electrode to electrode. For gold electrodes, although defects are still present in the microfabrication process, the surface was assumed to be less distinct than other electrodes because of the bulk processing. The inter-electrode variation of fabricated electrodes without any surface modification has also been taken into consideration in this work. These electrodes were functionalized using Au nanoparticles and then modified using DTSP (dithiobis(succinimidyl propionate)) self-assembled monolayer SAM and cortisol antibodies to make them selective towards cortisol molecules. The enhancement in the electroactive surface area and sensitivity was observed for Au nanoparticle (AuNP) functionalized electrodes prepared by drop casting, aerosolized and plasma assisated aerosolized methods.There are various methods available to detect the analyte using electrochemical immunosensing include competitive, sandwich, and labeled immunoassay [7,8]. Nanoparticles are used as labels or as an underlying layer to bind biorecognition elements for biosensing. Due to the strong affinity of Au surface with amino groups and mercapto groups, AuNPs facilitate the conjugation of biological ligands [9]. Cortisol is a vital stress hormone, and its level in many physiological functions such as fat mobilization for metabolism, immune suppression, cardiovascular disease, autoimmune disorders, infectious diseases, and mental illness makes it an important biomarker in health-monitoring [10].The electrochemical immunosensor composed of AuNPs on the activated Screen-Printed carbon electrode (SPCE) surface as a current collector, along with biotin-conjugated cortisol antibody covalently cross-linked on the surface of the nanoparticles using DTSP. The sulfur binding of DTSP is attached to the surface of AuNPs. Further, the amine group of the antibodies binds to the DTSP ligand. This self-assembled monolayer (SAM) of antibody on the nano-substrate provides irreversible binding sites for the cortisol present in any given sample. This irreversible binding of the protein structures limits ion transport to the sensor surface. Considering NPs as a fundamental layer in surface modification, this work focuses on the NP distribution on the sensing surface. In this work, the correlation of NP distribution with electroactive surface area, separation potential of oxidation and reduction peak, and size of NPs is achieved. Three methods of deposition of three different sizes of AuNPs as 20nm, 40nm, and 60nm were chosen as drop-casting, aerosol deposition, and plasma-assisted aerosolized deposition.In this work, microfabricated silicon-based Au electrodes are used. The AuNPs were deposited on the working area of these electrodes to improve their performance and have been achieved by increasing the active surface area using nanoparticle (NP)-functionalized electrodes as they exhibit higher electric field intensities [11]. Plasma-assisted NP functionalized electrodes displayed higher electrochemical performance. This improved electrochemical response is due to plasma-assisted surface activation of NPs. However, as the enhanced NP coverage was observed in plasma-assisted NP deposition, this might also be the reason for the enhancement of the electrochemical properties, electroactive surface area, and sensitivity. Figure 1
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