Nanomaterial-based Novel Methods For The Assay Of Enzyme Activity

Almost all biochemical reactions take place under the catalysis of enzymes. The anomalism of enzyme activity will seriously disrupt normal metabolic activities, which will be closely related with many diseases. So the assay of enzyme activity has attracted considerable research interest, and a variety of biosensors have been developed for many kinds of enzymes. Nanomaterials, due to their unique properties, are able to improve the function of biosensors when incorporated with biological elements. In this dissertation, the author has fabricated two novel biosensors to assay the activity of purine nucleoside phosphorylase and indoleamine 2,3-dioxygensae based on nanoparticles, which may have significant values for applications in clinical diagnosis.1. Sensing purine nucleoside phosphorylase activity by using silver nanoparticlesPurine nucleoside phosphorylase is a ubiquitous enzyme of purine metabolism that functions in the salvage pathway. In this chapter, a novel biosensor for assay of purine nucleoside phosphorylase activity is reported. Due to the facilitation of sliver nanoparticles to the electron transfer reactivity of guanosine and guanine, the electrochemical response of these species can clearly reveal the activity of the enzyme PNP. Meanwhile, on the basis of the finding that guanosine and guanine can induce silver nanoparticles to different agglomeration degrees, we have also developed a rapid UV-vis spectroscopy assay method for PNP activity. This work may show excellent sensitivity, reliability and specificity, and may avoid the utilization of coupled enzymes or radiochemical reagents, which is good enough for clinical applications.2. Sensing indoleamine 2,3-dioxygensae activity by using platinum nanoparticlesIndoleamine 2,3-dioxygensae is an intracellular heme-containing enzyme that catalyzes the cleavage of the pyrrole ring of L-tryptophan and the insertion of two oxygen atoms into the organic substrate to afford N-formylkynurenine. A new strategy to fabricate electrochemical biosensor is reported in this chapter based on the selective combination of enzyme catalysis and electrocatalysis, thus an electrochemical method to assay the activity of indoleamine 2,3-dioxygensae is proposed. Specifically, only when IDO-catalyzed oxidation process has occurred, platinum nanoparticles can be immobilized onto the electrode surfaces, electrochemically catalyzing the reduction of H2O2 to produce electrochemical signals. Under optimized conditions, IDO activity can be assayed in the range of 6.24 to 400 U/mL with a detection limit of 6.24 U/mL. The proposed biosensor shows high sensitivity, acceptable reliability, and can be used for the investigation of the enzymatic inhibition by inhibitors as well as the screen of the enzymatic activity in complex matrix such as serum samples.