Exploration of the Diabetic Urinary Proteome via Hydrogel Nanotechnology and Proof of Principle of a Non-Invasive A1c Assay
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Harpole, Michael George
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Abstract
An urgent need exists to develop non invasive diagnostic tests to monitor chronic health conditions such as diabetes. Urine is an attractive biofluid that holds untapped potential for diagnostics because of its availability, ease of collection, and the urine proteome’s reflection of the body’s physiologic state. Nevertheless, important challenges related to urinary protein biomarkers have not been met in the past: low abundance and perishability of protein biomarkers, and masking of low abundant proteins by highly abundant proteins. In order to address these issues hydrogel nanoparticles loaded with organic dyes, never used before in protein chromatography, were developed that in a short amount of time can capture, concentrate, and preserve low abundance proteins present in urine. In this work, dye loaded hydrogel nanoparticles were applied to capture and concentrate the glycated form of hemoglobin (A1C), a recognized blood biomarker for diabetes that has not previously been measured in urine. A dye chemistry was identified that harvested A1C from urine with high affinity. Anecdotally, western blot analysis showed the presence of A1C in the urine of one diabetic patient. A1C was not detectable in the urine without the use of nanoparticles. The work herein provides preliminary data and procedures for further development of a urinary test to monitor A1C in the urine of diabetic patients. Moreover, to gain a better understanding of the urinary proteome, urine processed by dye loaded nanoparticles was analyzed by liquid chromatography mass spectrometry (LC/MS-MS) to identify low abundance, labile urinary proteins along with highly abundant proteins. The analysis yielded 1226 proteins from 2ml of urine, supporting the hypothesis that dye loaded hydrogel nanoparticles are essential for developing diagnostic tests that are sensitive and non-invasive at the same time.
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This document has been embargoed until June 1, 2020
Keywords
Nanotechnology, Urinary proteome, Diabetes, Glycated hemoglobin, A1c, Hydrogel nanoparticles