Alexandra Asgarian's GOx Aptamer Selection Project (2018)

Anti-Glucose Oxidase Aptamer for the Improvement of Diabetes

Introduction and Background

Significant advancements have been made to increase the ease and painlessness of testing one’s glucose levels. Yet, readings of glucose levels can range with a 20% margin of error (Spero 2013). Furthermore, diabetes test strips vary in price from sixteen cents to two dollars due to the delicacy and expensive research necessitated to produce these strips. When the price is high and not entirely trustworthy, consumers often turn to wholesale to buy large amounts of expired test strips to save money. Since diabetes necessitates daily monitoring, up to ten times a day, new methods must be applied to decrease cost and increase a test strip’s lifespan. Given the severity of diabetes and the extreme consequences of improper management, glucose concentration results should be held to a higher degree of precision and accuracy, which can be accomplished by creating an inhibitory aptamer against glucose oxidase (imaged in figure 1).

Test strips for glucometers report glucose levels by glucose oxidase interacting with glucose in the blood to emit an electric current. As shown in figure 2, the enzyme layer would contain glucose oxidase and the carbon layer would consist of the electrodes transmitting the electric current. The intensity of the electric current emitted then equates to the glucose concentration reported (Gebel 2012). The focus of this project is to increase the longevity of glucose oxidase within the strip by finding a glucose oxidase inhibitor. Given that glucose oxidase is an enzyme, extreme temperatures can decrease the sensitivity of glucose oxidase resulting in inaccurate results. Its decrease in sensitivity is often due to side reactions within the test strip prior to readings and the importance of maintaining appropriate conditions so the enzyme will not expire. With the utilization of a glucose oxidase inhibitor, the glucose oxidase within the test strip could remain inactive until activated by the removal of the aptamer targeted against glucose oxidase by either a change in voltage or pH, which would be induced by the glucometer thereby increasing longevity (Boucher 2015).

An aptamer is a nucleic acid with a high binding affinity to a target that can range from an ion to a cell (Stovall, 2016). The SELEX methodology will be utilized to select species with higher affinity from a highly diverse pool. This process is then repeated several times in order to retrieve the tightest binders to the target and then undergoes Sanger Sequencing to determine the sequence of the bound pool, resulting in the discovery of an aptamer. For this research, glucose oxidase (imaged in figure 1) was immobilized on streptavidin magnetic beads, but due to poor results, the partitioning method was altered to the filter-based method which is more cost efficient and is ideal for the size of the target (160 kDa).

Current progress is focusing on trouble-shooting this round of aptamer selection. This is due to a lack of DNA bands being presented in all three washes. Through the process of aptamer selection, an aptamer will be found against glucose oxidase and act as an inhibitor thereby slowing the metabolic activities of the enzyme and extending the expiration date of glucose test strips so that it is not affected by outside factors, such as temperature.

Click here for Final Report

References

Boucher, K. (2015, December 1). Klayme-Shane Boucher's GOx Aptamer Selection - Aptamer Stream. Retrieved April 03, 2018, from https://sites.google.com/site/friaptamerstream/projects-1/glucose-oxidase-gox- aptamer-development/klayme-shane-boucher-s-gox-aptamer-selection

Gebel, E. (2012, July). Anatomy of a Test Strip. Retrieved April 03, 2018, from http://www.diabetesforecast.org/2012/jul/anatomy-of-a-test-strip.html

Glucose Oxidase with FAD in Red. May 2006. PDB-101: Molecular Explorations through Biology and Medicine, pdb101.rcsb.org/motm/77. Accessed 1 Dec. 2018.

Hernandez, L. I., Machado, I., Schafer, T., & Hernandez, F. J. (2015). [Photograph found in Aptamers Overview: Selection, Features and Applications]. Retrieved April 3, 2018, from http://benthamscience.com/journals/current-topics-in-medicinal-chemistry/volume/15/issue/12/page/1066

Kaur, Kal. "A Guide to Understanding Blood Glucose Monitoring Sensors." AZoSensors. Last modified July 6, 2012. Accessed November 4, 2018. https://www.azosensors.com/article.aspx?ArticleID=30.

Merle, G., Lopes, J. H., Colson, F., & Barralet, J. E. (2016, March 30). Engineering mediatorless glucose oxidase microbioreactor. Retrieved April 03, 2018, from https://www.frontiersin.org/10.3389/conf.fbioe.2016.01.01518/event_abstract

Spero, D. (2013, September 18). Why Do Test Strips Cost So Much? Retrieved April 03, 2018, from https://www.diabetesselfmanagement.com/blog/why-do-test-strips-cost-so-much/

Stovall, G. (2016). RNA Bead-Based Background. World Health Organization. (2017, November). Diabetes. Retrieved April 03, 2018, from http://www.who.int/mediacentre/factsheets/fs312/en/