Electrochemical detection or analysis of bio-molecules is an effective technique in laboratories due to its simplicity over conventional optical approaches. The technique is usually performed using three-electrode voltammetry composed of a working, counter and reference electrode that are made of noble metals. Graphite electrodes are major alternatives for resource-limiting experimental conditions such as point-of-care diagnostics and rapid screening, which would require disposable electrodes. To further reduce the cost of graphite-based electrodes for personal hormone-level monitoring in which several sampling activities are needed each day in months, pencil leads are proposed as an electrode material in this paper.

Competitive-based enzyme immunoassay (EIA) is a method for the quantification of hormones, which is performed in a 96-well plate where the plate surface is coated with a capturing antibody in charge of hormone binding. Target hormones in the sample and artificial hormones conjugated with a peroxidase compete for limited binding sites on the plate surface. Peroxidase is an enzyme catalyzing the oxidation by hydrogen peroxide of 3, 3', 5, 5'-Tetramethylbenzidine (TMB), an electro-active redox dye indicator. The low-level hormone in the sample would lead to a high signal of oxidized TMB due to the large presence of the conjugated peroxidase, and vice versa. In this paper, the use of pencil leads allows a direct dip-in process to measure electrochemical signals from tested solution in a 96-well plate.

Our work presents a portable enzyme immunoassay system that contains an electrochemical voltammetry composed of a three-electrode sensor module, potentiostat and data acquisition. The devices are intended for generic employment in the quantification of cortisol, a steroid hormone and indicator of human stress level. The three-electrode sensors were developed based on mechanical pencil-leads. We also report the preparation of Ag/AgCl reference electrodes, for the first time, fabricated by directly casting and chemically modifying silver epoxy on pencil-lead electrodes to form Ag/AgCl layers.

The electrochemical properties of the redox indicator, TMB, were studied by cyclic voltammetry. The enzymatic activity of peroxidase was evaluated to indicate its linear characteristics in terms of time. The amperometric responses of varying concentration of cortisol samples (3200, 800, 400, 200, and 0 pg/ml) were obtained by our system. The results are consistent to the mechanism of competitive-based EIA in which a low amount of oxidized TMB should be measured due to a low amount of conjugated peroxidase molecules captured by the antibody is in presence if the cortisol level of a test sample is high. The non-linear relationship between the peak current values and cortisol concentration of samples at low and high concentration range was demonstrated indicating diffusion-limited reactions. The electrochemical enzyme immunoassay was for the first time performed using the pencil-lead based sensors as presented in this paper. The results promise that the proposed electrochemical assay is able to quantify hormone levels and serve as an ultra-low-cost biosensing system for point-of-care diagnostics and personal health monitoring.