Hoyeon Kim's GOx Aptamer Selection Project (2018)

RNA Aptamer Against Glucose Oxidase for the Enhancement of the Glucometer Test Strips

Introduction and Background

The Centers for Disease Control and Prevention (CDC) released that as of 2015, about 9.4% of the United States populations had diabetes (CDC, 2017). Diabetes–a group of metabolic diseases resulting in hyperglycemia, or an excess of sugar in the bloodstream–can lead to long- term failure of different organs like the kidneys, nerves, and heart. Diabetes, in general, is characterized by the lack of insulin secretion, which is necessary for the body to properly use glucose for energy. Although Type 2 diabetes is more common and marked by insulin deficiency that occurred later in their lives, Type 1 and Type 2 are both incurable (American Diabetes Association, 2013). Therefore, diabetic patients must rely on glucometers for continuous regulation and treatment of their conditions.

Those affected by the many types of diabetes rely heavily on glucometers to analyze the blood glucose levels in many different settings like hospitals and homes; therefore, it is crucial that these devices work properly (Tonyushkina et al., 2009).

One type of blood glucose meters uses electrochemical test strips in Figure 1 that contains enzyme electrodes with reagents like GOx, for the patient to measure his/her blood glucose level at home (Kumar, 2015). Typically, the patient would retrieve a small sample of his/her blood and place it on the disposable strip for the glucometer to read. Then the enzyme, as depicted in Figure 2, within the detector reacts with the glucose from the blood sample to produce gluconic acid, which also reacts with ferrocyanide chemical to create a current to be read (Kumar, 2015). However, the enzyme that drives the mechanism of the disposable test strips and the glucometer is extremely sensitive to its environment, which could lead to a malfunction. Upon finding an inhibitory aptamer against glucose oxidase, one of the main enzymatic reactions currently used in glucometers (Tonyushkina et al., 2009), these devices could become more stable. This inhibitory aptamer will bind to GOx and prevent the enzyme from denaturing at high temperatures (over 60 oC) or reacting when it is not in use. Once the drop of blood is placed onto the surface and the mechanism begins, the current produced will shake the aptamer off the enzyme for a fully functioning glucose oxidase.

Humans use GOx commonly in their everyday lives by applying its technology in glucometers and biosensors to measure the presence of certain chemicals and food to prolong shelf life (Wong et al., 2008), thus finding an inhibitory aptamer against this target is crucial to the advancement of medical technologies.

Figure 1: Glucose Oxidase Test Strip (Kumar, 2015)

Figure 2: GOx (GOD in the figure) Test Strip Amperometric Measurement Mechanism (Kumar, 2015)

Aptamers, oligonucleotide sequences, have high affinity and specificity to different targets. These sequences are used in diagnostic assays to manage and treat diseases. As a result of their stability and lower cost benefits, aptamers are developing in order to replace antibodies in many of the applications (Jayasena, 1999). Aptamers are easier to produce through chemical synthesis–reducing the time and cost of production. they are also easier to use in a laboratory setting with their ability to restore their original forms at optimal temperatures (Toh et al., 2015). The focus of this project lies in the diagnostics application over the detection of glucose concentration in those with diabetes using an aptamer against glucose oxidase.

The aptamer selection against glucose oxidase will begin under the in vitro setting with the Streptavidin magnetic beads and the N71 RNA pool. Using the idea of evolution, the SELEX method creates an environment in which through iterative rounds, the binding species becomes more and more specific. This process is briefly illustrated in Figure 2. The selection process is performed for multiple rounds in order to discern the RNA sequence with the highest binding affinity to the particular target (in this case glucose oxidase).

Currently, the first round of the experiment has been in progress and the agarose gel for ccPCR has been visualized. This step shows how many cycles are necessary for optimal amplification of the ssDNA pool. With this, the inhibitory aptamer will prevent the GOx enzyme from reacting in any way until necessary; thus, the glucometers and the strips used at home will become more stable.

Figure 3: The Aptamer Selection Process. From a large pool of RNA, the active, sequences are bound to the beads and are placed through reverse transcription into ssDNA for amplification, after which the DNA is transcribed back to RNA and collected for another round of selection (Mok and Li, 2008).

Click here for Final Report

REFERENCES AND CITATIONS

Diagnosis and Classification of Diabetes Mellitus. (2013). Diabetes Care, 36(Supplement 1), S67. https://doi.org/10.2337/dc13-S067

Enzymes Glucose Oxidase. (n.d.). Retrieved April 16, 2018, from http://indogulfgroup.com/AE%20-%20Enzymes%20-%20Glucose%20Oxidase.asp

Jayasena, S. D. (1999). Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics. Clinical Chemistry, 45(9), 1628–1650.

Mok, W., & Li, Y. (2008). Recent Progress in Nucleic Acid Aptamer-Based Biosensors and Bioassays. Sensors, 8(11), 7050–7084. https://doi.org/10.3390/s8117050

Kumar, A. M. (2015, January 10). Bio-Resource: How does glucometer or Glucose monitoring device work? Retrieved December 5, 2018, from http://technologyinscience.blogspot.com/2015/01/how-does-glucometer-or-glucose.html

National Diabetes Fact Sheet, 2011. (n.d.), 12. National Diabetes Statistics Report, 2017. (n.d.), 20.

Toh, S. Y., Citartan, M., Gopinath, S. C. B., & Tang, T.-H. (2015). Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay. Biosensors and Bioelectronics, 64, 392–403. https://doi.org/10.1016/j.bios.2014.09.026

Tonyushkina, K., & Nichols, J. H. (2009). Glucose Meters: A Review of Technical Challenges to Obtaining Accurate Results. Journal of Diabetes Science and Technology (Online), 3(4), 971–980.

WHO | Diabetes mellitus. (n.d.). Retrieved September 24, 2018, from http://www.who.int/mediacentre/factsheets/fs138/en/

Wong, C. M., Wong, K. H., & Chen, X. D. (2008). Glucose oxidase: natural occurrence, function, properties and industrial applications. Applied Microbiology and Biotechnology, 78(6), 927–938. https://doi.org/10.1007/s00253-008-1407-4