Advanced electrochemical biosensor for anticancer inhibitor screening

In today's world, cancer stands as a primary cause of death in the most developed nations, with projected increases in mortality rates in the years ahead. Hence, there is an urgent need to enhance research efforts in developing anti-cancer drugs. This study aims on refining the process of screening anti-cancer drugs. It involves immobilizing the enzyme Lactate Dehydrogenase (LDH) on mesoporous silica supports to create an enzyme-based device capable of efficiently testing potential anti-cancer drugs by assessing their ability to inhibit enzyme activity. The enzyme was immobilized on SBA-15 and functionalized using two different organosilanes, APTES and GPTMS. Various ratios of these organosilanes (3:1, 1:1, 1:3) were tested to identify the optimal combination that maximizes biocatalyst activity. The synthesized and functionalized supports underwent comprehensive analyses, including nitrogen physisorption at 77 K to evaluate specific surface area and pore distribution, FTIR for surface chemistry characterization, identification of surface groups involved in forming covalent bonds between support and enzyme during immobilization, CO and ammonia adsorption to assess surface acidity, and zeta potential analysis. Immobilization on the support showed the best performance with a 1:1 ratio of the two organosilanes, resulting in a residual biocatalyst activity of 15.3% compared to free enzyme. The time of enzyme-support contact was explored as a critical parameter for immobilization, with 3 hours proving optimal and resulting in a residual biocatalyst activity of 34.7% compared to free enzyme. To assess the enhanced stability of immobilized enzyme with respect to free enzyme, activity tests were conducted in the presence of denaturing agents such as alkaline pH. Kinetic evaluations were conducted both with and without two well-known inhibitors, NHI-2 and galloflavin. Also repeatability test were conducted in presence of the inhibitors, and allowed to confirm the inhibition reversibility. Computational modeling using BioVia Discovery Studio confirmed the findings from kinetic studies regarding the interaction between inhibitors and the enzyme. Reusability tests of the enzyme post-exposure to inhibitors showed comparable residual activity to that observed without inhibitors, confirming the reversible nature of both galloflavin and NHI-2 inhibitors. Electrochemical tests were performed using a three-electrode cell, with a glassy carbon electrode modified by titanium deposition as the working electrode, Ag|AgCl as the reference electrode, and platinum as the counter electrode. These tests allowed chronoamperometric measurements, monitoring current changes linked to NADH oxidation as NADH concentration in solution varied. This verified a NADH oxidation potential of 1.26 V from previous linear sweep voltammetry assessments. A calibration curve constructed from current density variations against NADH concentrations established the instrument's sensitivity at 0.0015 μA/(μM cm) and determined its limit of detection. Tests conducted in the presence of interferents assessed their impact on current variations, which were no longer exclusively tied to NADH oxidation. Finally, chronoamperometric tests conducted with free and immobilized enzymes, with and without inhibitors, corroborated results obtained through traditional methods like UV-Vis spectrophotometry. This validation underscores the potential for developing this electrochemical biosensor device for drug screening purposes.