Immobilization of Proline Dehydrogenase on Functionalized Silica Mesoporous Nanomaterial Towards Preparation of a Novel Thermostable Enzyme Biosensor

Abstract

In this report, a thermostable electrochemical biosensor for sensitive determination of L-proline in whole blood was prepared by immobilization of proline dehydrogenase within n-Propylamine functionalized mobile crystalline material-41 doped with iron oxide nanoparticles. By combining of magnetic mesoporous silica with proline dehydrogenase and using voltammetry analysis, a proline biosensor was fabricated on glassy carbon electrode pre-modified by poly cysteine supported beta cyclodextrin. The magnetic mesoporous silica networks having a high surface area (362 m2 g−1) exhibited excellent properties for entrapment of proline dehydrogenase. The prepared sensor was used as an electrochemical detector for selective detection of L-proline at physiological pH. The results show that the oxidation of L-proline is ir-reversible and controlled by diffusion. Using differential pulse voltammetry, the oxidation peak current was proportional to L-proline concentration in the range of 1–100 mM and 1 μM–1 nM, while the low limit of quantification was 1 nM under physiological pH. For proof-of-technology, the biosensor was used for the detection of L-proline in biological samples such as whole blood and human blood serum of neonates. It is demonstrated that engineered biosensor provides a rapid and sensitive method for determination of L-proline in neonatal blood serum samples. The current response of electrocatalytic oxidation of L-Pro was maintained in the analytical solution temperature up to 310 °K. The applied approach led to better resistance to temperature and pH inactivation in comparison to the free enzyme. The method is a potential tool for diagnostic of neonatal hyperprolinemia. Taking a good facet of the biosensor into account, it was implemented for the detection of the L-Pro in normal cell (L929) and various cancer cell lines such as gastric cancer cell-CAT 3, colon cancer cell-HCT, colon cancer cell-SW, and breast cancer cell-MCF7. Finally, this sensor was used to determine of L-Pro in unprocessed blood samples.