Rachel Arredondo's GOx Aptamer Project (2017)

Aptamer Against Glucose Oxidase for Glucometer Modification to Aid Diabetic Patients

Introduction and Background

Annually, about 1.4 million Americans are diagnosed with diabetes (ADA, 2017). Diabetes is a disease in which the body is unable to regulate its blood sugar levels due to a lack or misuse of insulin, causing the levels to rise uncontrollably. Diabetic patients use glucometers to measure their glucose levels in their blood to attempt to regulate their sugar intake. Glucometers use test strips which the patients put a drop of their blood onto and it calculates the blood-glucose level. These test strips have a sensor which utilizes the enzyme glucose oxidase by observing the amount of glucose produced by the enzyme to determine glucose concentration (Yanez, 2013). Although some test strips use other enzymes, such as glucose dehydrogenase, the ones that utilize glucose oxidase seem to have a wider range in aiding patients with diabetic complications, such as rapid fluctuation in glucose levels (Bilen, 2009). A focus on the enzyme glucose oxidase could be a key element into glucometer modification.

Glucose oxidase (GOx) is an enzyme that is found on fungal surface tissues that catalyzes the oxidation of glucose into hydrogen peroxide. As shown in Figure 2, glucose oxidase is a dimer that is made up of two identical 80kDa subunits and two non-covalently bound flavin adenine dinucleotides (FAD) (Wong, 2008). Glucose oxidase is highly negative, similar to RNA. The use of glucose oxidase in glucometers has made the measuring process of glucose within the body easier and cheaper than the utilization of other enzymes. However, glucometers are currently relying on disposable strips, which is costly for the patient who is spending upwards of 750 dollars a year just on test strips. It is proposed that a way to inhibit the enzyme and turn it “off” temporarily, while stored in the test strips, will allow for longer usage of glucometers and more accurate results. An aptamer against glucose oxidase will allow this inhibition to be possible.

An aptamer is an oligonucleotide, or RNA sequence that binds to a target with high specificity to either enhance or inhibit it. An aptamer has major benefits in drug development over antibodies, including shorter time for development and cost efficiency. This aptamer against glucose oxidase would be for a diagnostic application to help diabetic patients in their daily measurements of glucose in their blood.

In vitro bead-based aptamer selection against glucose oxidase is a way to find an aptamer to temporarily inhibit glucose oxidase to store within glucometer stips. The process, shown in Figure 3, is performed using a bead-based method, in which the target is bound to the beads and RNA at specific settings that suit the target. The target is then turned into DNA for the ability to amplify, and transcribed back to RNA for purification. This gives the template for the next round, and the process is repeated for increased stringency and higher binding affinity, until the product is ready to be assayed to officially find the aptamer.

A round of aptamer selection against glucose oxidase has not been completed yet. Problems within the first round, such as lack of amplification, led to many troubleshooting tests. The tests have concluded successfully, and the round was continued. Once a round of selection has been completed, a few more rounds and an assay will need to be completed before an aptamer is found. When an aptamer is found, it will be applied onto glucometer test strips, making them reusable and more affordable.

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References

"Statistics About Diabetes." American Diabetes Association. N.p., n.d. Web. Sept. 2017. http://www.diabetes.org/diabetes-basics/statistics/?utm_source=Offline&utm_medium=Print&utm_content=statistics&utm_campaign=CON.

"Glucose Meter Fundamentals and Design." Freescale Semiconductor, Inc, Yanez, Jan.2013. Web. Sept. 2017. .

Bilen, H. "Journal of Medical Engineering & Technology." Taylor and Francis Online. N.p., 09 July 2009. Web. Sept. 2017. < http://www.tandfonline.com/doi/full/10.1080/03091900600861590?scroll=t op&needAc cess=true>.

Wong, Chun Ming, Kwun Hei Wong, and Xiao Dong Chen. "Glucose Oxidase: Natural Occurrence, Function, Properties and Industrial Applications." SpringerLink. Springer-Verlag, 11 Mar. 2008. Web. Sept. 2017.

Figure 2: Goodsel, David. "PDB-101: Glucose Oxidase." PDB-101: Glucose Oxidase. N.p., May 2006. Web. Sept. 2017.

Figure 3: Huang, Yukun, Xiujuan Chen, Yu Xia, Shijia Wu, Nuo Duan, Xiaoyuan Ma, and Zhouping Wang. "Selection, Identification and Application of a DNA Aptamer against Staphylococcus Aureus Enterotoxin A." Analytical Methods. The Royal Society of Chemistry, 11 Dec. 2013. Web. Sept. 2017.