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Discipline: Chemistry and Chemical Sciences
Subcategory: Chemistry (not Biochemistry)
Abrahan Alvarado Aguilar - Universidad del Turabo
Co-Author(s): Ileana Gonzalez, Universidad del Turabo, Gurabo, Puerto Rico
Kidneys remove waste by-products of metabolism in the body. One of such products is urea, which is formed from the degradation of proteins. Urea can build up because of kidney failure and can cause various medical conditions such as fluid retention, fatigue, insomnia, among others. Our work is focused on creating a bio device that is able to detect urea levels in the blood and break it down into less harmful by-products. For this, we suggest the use of urease which could break down urea into ammonia, and then oxidize it into Nitrogen using Pt nanoparticles embedded into the membrane interface. The enzyme is protected from degradation in a membrane like structure called a nanodisc. We carried out the synthesis of two types of nanodiscs (called NDP and NDM) using Membrane Scaffold Protein, two different types of membrane lipids (1,2Dipalmitoyl phosphatidylcholine for the NDP sample, and 1,2Dimyristoyl-rac-glycero-3-phosphocholine for the NDM sample.) and urease in 0.1M PBS buffer at a pH of 7.4 and also pH 9. The nanodisc self-assemble after 24 hours at 37o C by removing the detergent used to dissolve the lipids. At this point, we tested the activity of the urease using a Urea Activity Assay Kit quantified the urease activity, which measures the absorbance at 670nm. After obtaining the absorbance of each sample, we were able to determine the urease units per L with the Berthelot method. We obtained 23.7 units/L for the NDM sample, and 23.9 units/L for the NDP sample. The urease sample had 14.6 units/L. For determining the size of the nanodiscs, first Dynamic Light Scattering (DLS) was used to determine the hydrodynamic size of the nanodisc particles in the solution as well as to determine if there are different sized populations in the solution. We were able to find particles of around the 12-15nm range which correspond to the nanodisc size. We also found a large amount of particles of 1,000nm size which we attributed to the buffer salts themselves. Atomic Force Microscopy was used to image the topography of the surface of the nanodiscs. As far as the future of the project is concerned, we will focus on the electrochemical properties of the nanodiscs in order to determine the amount of ammonia formed thanks to the breakdown of urea by the urease, and then record the oxidation of such ammonia by the Pt nanoparticles in Cyclic Voltammetry and Chronoamperometry.
Funder Acknowledgement(s): This work was supported by funded by NIH/NIGMS - Award Number P20GM103475-13 | Hosted by HPCf, UPR.
Faculty Advisor: Ileana Gonzalez,
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