Amukthamalyada Kunduru's GOx Aptamer Project

Diabetes is common disease that affects over 382 million people worldwide as of 2013. Statistics show that in the United States 9.3% of the population are diagnosed with diabetes.[5] Diabetes is a metabolic disorder that occurs when the pancreas is not able to produce enough of the hormone insulin or the body is not able to respond to the insulin produced. Since insulin is required for glucose to be able to enter cells in the body, a lack of insulin produced or a lack of response to insulin results in glucose building up in the blood stream. Because of this, diabetics have high blood glucose levels. This disease can be extremely detrimental to a diabetic’s health if not carefully monitored through glucometers. For example, if not carefully monitored, diabetes can lead to diabetic nephropathy otherwise known as the diabetic kidney disease. However, glucometers, instruments that are currently used to measure blood glucose levels, can result in inaccurate readings giving false highs and lows. This can either be due to the environment, such as temperature and altitude, or errors that patients make themselves, such as incorrect hand washing.[1]

Glucose Oxidase (GOx), when it was found, soon became the heart of biosensors responsible for monitoring blood glucose levels. The enzyme is typically found in fungi and in honey.[8] GOx is a dimeric protein that oxidizes glucose into glucolactone, in the process producing hydrogen peroxide as seen in Figure 2.[5] Glucose is a difficult molecule to quantify. On the other hand, hydrogen peroxide is relatively easy to measure from blood samples. The minor role that glucose oxidase played soon became a pivotal process that helped effectively maintain sugar levels.

As can be seen in figure 1, GOx is a glycoprotein. It has carbohydrates chains attached to the protein subunits. It is also categorized as a dimer protein, which means that its structure consists of two non-covalently bound macromolecules. It has a molecular weight of 160 kDa.[4] Furthermore, the red region inside the protein, the FAD cofactors, is where oxidation reactions take place.[5] Since it is a nucleic acid binding region, an aptamer could possibly be able to bind there as well.

Aptamers are molecules of nucleic acids that have a high specificity for certain targets. Aptamers have various functions that can be beneficial. Since they are highly specific molecules, it would be easy to deliver certain drugs to a specific target. Furthermore, they are also good with diagnostics. They can help detect a detrimental molecule, for example, an aptamer that detects an HIV virus. This allows for doctors to better treat their patients. They are also inexpensive to produce.

Recently, research has begun on aptamer-based biosensors, aptasensors. Since aptamers are small, chemically stable molecules that are flexible in their structures and show high selectivity, they have shown to contain board applications in medicine.[6] These aptasensors can be characterized as electrochemical or optical.[7] Aptasensors are being preferred over bioassay methods such as the ELISA (enzyme-linked immunosorbent assay) due to the fact that smaller molecules can be more easily detected, modifying aptamers during immobilization can be done without affecting affinity, and aptamers can be denatured and regenerated multiple times over.[6] For diabetics, aptasensors can be an answer to more accurate readings.

In the past year, researchers from Xinyang Normal University have come up with a scheme to create a type of electrochemical aptasensor called an ECL (electrochemiluminescence) aptasensor.[9] In their scheme, the researchers created an electrochemical film that bound the primary aptamers. The aptamer was then exposed to the analyte, which bound to the aptamers. The analyte was then sandwiched between the primary aptamer and a secondary aptamer tagged with glucose oxidase. Oxidation occurred when glucose was exposed to the glucose oxidase and created H2O2, hydrogen peroxide. This allowed luminol-H2O2 system allowed for greater detection of PDGF-BB (platelet derived growth factor BB). Their scheme is similar to my application in which an aptamer for GOx will bind GOx to and electrochemical film. GOx will be exposed to glucose, which will trigger an oxidation reaction. A measurement of H2O2 can help determine the amount of glucose that was exposed to the aptasensor.

Finding an aptamer for GOx can help diabetics get more accurate blood glucose readings to help them control their disease.

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Citations