Preparation of a Mesoporous Biosensor for Human Lactate Dehydrogenase for Potential Anticancer Inhibitor Screening

Cancer is the second leading cause of death worldwide, with a dramatic impact due to the acquired resistance of cancers to used chemotherapeutic drugs and treatments. The enzyme lactate dehydrogenase (LDH-A) is responsible for cancer cell proliferation. Recently the development of selective LDH-A inhibitors as drugs for cancer treatment has been reported to be an efficient strategy aiming to decrease cancer cell proliferation and increase the sensitivity to traditional chemotherapeutics. This study aims to obtain a stable and active biocatalyst that can be utilized for such drug screening purposes. It is conceived by adopting human LDH-A enzyme (hLDH-A) and investigating different immobilization techniques on porous supports to achieve a stable and reproducible biosensor for anticancer drugs. The hLDH-A enzyme is covalently immobilized on mesoporous silica (MCM-41) functionalized with amino and aldehyde groups following two different methods. The mesoporous support is characterized by complementary techniques to evaluate the surface chemistry and the porous structure. Fluorescence microscopy analysis confirms the presence of the enzyme on the support surface. The tested immobilizations achieve yields of >= 80%, and the best retained activity of the enzyme is as high as 24.2%. The optimal pH and temperature of the best immobilized hLDH-A are pH 5 and 45 degrees C for the reduction of pyruvate into lactate, while those for the free enzyme are pH 8 and 45 degrees C. The stability test carried out at 45 degrees C on the immobilized enzyme shows a residual activity close to 40% for an extended time. The inhibition caused by NHI-2 is similar for free and immobilized hLDH-A, 48% and 47%, respectively. These findings are significant for those interested in immobilizing enzymes through covalent attachment on inorganic porous supports and pave the way to develop stable and active biocatalyst-based sensors for drug screenings that are useful to propose drug-based cancer treatments.