Amanda Martinez's GOx Aptamer Project

The American Diabetes Association said that 9.3% of the American population had diabetes in 2012. This means that there is a high likelihood of knowing at least one person who has diabetes. Type two diabetes is the most common form and typically leads to hyperglycemia when the pancreas fails to secrete enough insulin. Hyperglycemia is the term used to describe high blood glucose while hypoglycemia means low blood glucose. The cells require glucose in order to generate ATP, a form of energy, through a pathway called glycolysis in the liver and muscle. The glucose is unable to reach the cells without the help of insulin, a messaging molecule that tells the body to store it for later use. In addition, insulin resistance can occur within the cells that make it difficult to recognize the insulin molecules signaling for too much glucose in the bloodstream.

Hyperglycemia leads to an array of problems when the body is depleted of ATP. Figure 2 illustrates the pancreas when type two diabetes occurs. The goal of this project is to create a more efficient diagnostic tool to detect blood glucose levels by using an aptamer biosensor. The device would provide a numeric value similar to that of a glucometer indicating if the patient is hypo- or hyperglycemic.

The target for this research project is glucose oxidase (GOx), an enzyme that catalyzes the breakdown of glucose into glucolactone and converts oxygen into hydrogen peroxide (Goodsell, 2006). GOx is currently used in glucometers that test for the blood glucose concentration using the invasive method of finger pricking to obtain a sample of blood. GOx can be found in both fungi and honey (Goodsell, 2006). The hydrogen peroxide that it makes as a byproduct is used against bacteria and as a natural preservative, respectively. The active site where glucose binds is located in a deep pocket above the FAD (flavin-adenine dinucleotide) (“Glucose Oxidase”). The molecular weight of GOx is 160,000 Da. The target is a dimer meaning that it is composed of two polypeptide chain subunits attached through disulfide bonds (“Glucose Oxidase”).

One diagnostic development that has used GOx as a target is a biosensor as dental implant to detect blood glucose levels (Lu, 2015). The motivation for this development is similar to the motivation for this project. The biosensor was developed as a way to minimize the invasive approach to checking blood glucose levels. The results proved that biosensor was sensitive to glucose in the in vitro cell lines, which was a good sign. According the researchers, further experimentation on the engineering of that biosensor is to be done. Another diagnostic development that also sparked the interest in this project is a smart tattoo that uses (GOx)- and Apo-GOx-based glucose sensors which don’t appear to involve aptamers (Srivastava).

The results showed that the sensor was sensitive to detecting the amount glucose present. In addition, the research combated inflammation to the implant site with anti-inflammatory medication, which was made to mimic a site on a human body in the in vitro cell line. The researchers further emphasized that a tattoo-like sensor would detect glucose levels 24/7 which would make something like an artificial pancreas effective at treating diabetes with minimal work on the patients’ side.

Currently the labs working on the two projects mentioned above are: the Graduate Institute of Mechanical and Electrical Engineering at the National Taipei University of Technology in Taiwan, the Department of Biosciences and Bioengineering at the Indian Institute of Technology Bombay, Powai, Mumbai, India and the Biomedical Engineering Department at Texas A&M University, College Station, Texas. It does not appear that either lab has produced the target recombinantly.

An aptamer is a highly specific nucleic acid with a strong attraction for binding to a target. Aptamers are made of single-stranded DNA or RNA molecules. They are small molecules, which is good for getting into a variety of places in the body. The strong affinity for binding makes it great to attach to certain targets as well as its high specificity leading to a very exact location of where it binds. They are relatively affordable in comparison to their counterparts in medicine antibodies. An aptamer can be modified to bind to the target and signal when a certain molecule is present. An example of an aptamer being used for a biosensor is the research done on the detection of salmonella, a food-associated disease (Ma, 2014). The researchers found that their method was specific, fast and had the possibility for real-world application. A possible application as an alternative to creating a tattoo-based sensor using aptamers could be using the GOx aptamer to improve the accuracy of glucometers.

The target GOx has deep pockets, which may be useful to as a binding site for an aptamer. No data was found on the stability of the target at a particular buffer. The ideal condition of a successful aptamer would involve a pH close to the human body. The end goal of this project is to create an aptamer that can function in a tattoo-like ink that penetrates the skin. From the research papers found on this topic, it does not appear an aptamer has been developed for this target. There are no results at this moment to conclude here. A modified aptamer would bind to GOx to signal the presence of hydrogen peroxide, which would indicate the amount of glucose present in the bloodstream. This method would improve the diagnostic approach already in place by allowing a 24/7 detection of glucose levels as well as a possibility of developing a mechanism in which an aptamer and artificial pancreas could work in harmony. Figure 3A below indicates the process of selecting for a high affinity aptamer.