Millions of people worldwide are affected by diabetes mellitus, a leading cause of death in the United States. This disease develops as a result of either disrupted insulin production (type 1) or altered insulin absorption (type 2), leading to increased blood glucose levels over long periods of time. To combat this disease, patients must closely monitor their blood glucose (BG) levels. Change in glucose levels can cause severe complications, such as cardiovascular damage, nerve degeneration, and vision damage, if left uncontrolled. Therefore, each patient requires access to reliable sensors to collect data to determine BG levels at any given time. While commercial sensors are widely available, they are quite expensive due to their high production cost, expensive glucose sensing materials, and the materials used to hide the sensor from the immune system. As more patients are diagnosed with conditions requiring access to reliable glucose sensors, the need for less expensive sensors dramatically increases.
The goal of this project is to explore different ways of creating low cost and reliable sensors. Most diabetes patients use one of two categories of sensors: single use test strips or continuous glucose monitors (CGMs). Both types of sensor are explored and improved upon with affordability being the central goal of this project. Furthermore, a standard metric for identifying if the sensor would be applicable for real patient use was identified as “within 15% of reference value if ≥ 100 mg/dL or 15 mg/dL of reference value if ≤ 100 mg/dL.”1
Glucose Enzyme Molecule
Nearly all glucose sensors on the market employ the same technology for sensing glucose levels in a sample; the sensors use an enzyme to convert the concentration of glucose into an electrical signal (Figure 1) that is interpreted by standard electronics. The electronics used range from simple devices that measure the resistance of the sensor to more complicated ones that characterize the relationship between voltage and current applied to the sensor. The enzyme turns the glucose molecule and an oxygen (O2) molecule into gluconic acid and hydrogen peroxide (H2O2).
Instead of using the typical oxygen, commercial sensors utilize electrons in this reaction. As more electrons are consumed, the electrical resistance of the material increases. Therefore, a simple resistance measurement can determine the glucose concentration in the solution.
This enzyme is an apt tool for determining the BG levels in patients. However, it is very difficult to use in integrated sensors. The difficulty lies in binding the enzyme to a solid material from which charges can be donated, since the enzyme most commonly exists in liquid solutions. The process of embedding enzymes in a conductive material is called immobilization. Developing new methods for immobilization is a primary research topic for developing glucose sensors that are lower cost, more accurate, or generating novel ideas altogether due to the high level of difficulty of embedding enzymes.
Molecular Imprinting Technology
Molecular imprinting (MIP) is a technique that can potentially mimic the glucose binding site in the enzyme. The theory is that while a polymer is forming from the building of monomer groups, another molecule can be embedded in the polymer solution. Afterwards, the molecules that were on the surface of the polymer can be removed, forming a binding site that is the exact shape of the molecule used.2 Therefore, this molecule is referred to as the template molecule.
This technology and process can also be used for glucose sensing (Figure 2). Glucose can be used as the template molecule for molecular imprinting, but the correct polymer must be used. To sense glucose levels, a conductive polymer is needed because the electrical characteristics change when glucose binds to the surface. Polyaniline (PANI) was chosen as it is a conductive polymer that can be easily synthesized for a low cost. Therefore, a glucose sensor could be created using polyaniline as the polymer and glucose as the template molecule.
Reusable MIP Test Strips
In this research project, MIP is used to create low-cost test strips for measuring the glucose concentration of a solution. The challenge of this research is creating a method which measures glucose precisely in a wide linear range. The range at which a patient with diabetes mellitus might measure their BG is between 40-500 mg/dL or 2-30 mmol/L. Designing a sensor which linearly represents this range is a significant challenge due to decreased accuracy at extremely low and high glucose levels.
One problem with using PANI is that the material is not physically stable when its dimensions are small. When the sensor is fabricated, only a very thin film of polyaniline is synthesized. Therefore, our process is modified to include the addition of small paper strips to the solution. The polyaniline solution is soaked into the paper substrate, giving it increased physical stability while maintaining similar electrical characteristics. Then to measure the concentration of glucose, electrodes can be connected on either side of the PANI soaked paper to measure the conductivity of the material.
GOx Ink Continuous Glucose Monitor
In addition to the test strips, we developed a flexible ink to sense blood glucose levels; interestingly, this can also be used in a variety of applications like smart tattoos.
To create this ink, the fabrication process of the test strip sensor is modified. Instead of adding glucose as a template molecule, the aforementioned enzyme is added to be immobilized in the PANI solution. Paper strips are not added as a substrate for the material because the PANI will be either embedded in the skin or deposited on a material. The ink could also be soaked into another substrate if desired.
The challenge with creating the ink is that the PANI should not fully form until it is ready to be deposited on another surface; once the monomers are linked together and the enzyme is embedded, it cannot be dissolved again. So, the ink must be stored for long periods of time in a simple, monomer form. Then, when ready to use, the monomers would knit together to create the final glucose sensing. The hydrogen peroxide product from the enzyme’s reaction can be used to build the polymer. This allows the ink monomers to be linked together by simply soaking in a glucose solution. After the ink is saturated, the sensor is ready to react to glucose solutions.
MIP Sensor Fabrication
Fabricating the MIP sensor requires three groups of steps. The first and second are the preparation of the aniline solution and the oxidant solution. The third step is the combination of the two solutions, and the preparation of the strips (Figure 3).
Hydrochloric acid (HCl) and Ammonium Persulfate (APS) are used in the reaction to bind the monomers together.
Glucose Sensing Ink Fabrication
Creating the ink is quite similar to creating the test strips. The main difference is that instead of imprinting the polymer with glucose, the enzyme is immobilized in the polymer due to its large size. In addition, HCl and APS are not used because the ink should lie dormant until it is ready to be used.
First, similar to the test strip fabrication process, the aniline monomer solution is created with the addition of the enzyme, instead of glucose as used in strip fabrication. Unlike the fabrication of the test strip, no oxidant solution is created because the hydrogen peroxide from the enzyme reaction is used. However, we create a solution that is 20% saturated with glucose for use in the enzyme. Next, after depositing the monomer solution onto a surface such as the skin, the glucose solution is added to the surface. After several hours, the monomers will be fully linked and the sensor is ready. Figure 4 gives a visual representation of how this process works.
MIP IS USED TO CREATE LOW-COST TEST STRIPS FOR MEASURING THE GLUCOSE CONCENTRATION OF A SOLUTION
Glucose Test Strips
The test strips were very successful in correctly identifying the glucose solution. Figure 5 shows that a linear range is observed with increasing resistance as more glucose is added. The R2 value of the results indicates the linearity of the data. The closer the number is to 1, the better. In our case, this indicates great linearity.
Glucose Sensing Ink
Testing is still in progress for the ink, although some challenges have been identified. One of these being that the enzyme used to sense the glucose levels is very sensitive to pH and temperature levels. This affects the rate at which hydrogen peroxide is produced. The significance of this finding is that polyaniline synthesis is highly dependent on the rate of production, so the pH and temperature of the solution should be optimized. Using a fully formed polymer and breaking it apart might allow the enzyme to become immobilized.
A successful experiment was done that proves the process described above can work to synthesize polyaniline. However, experiments on ink performance in response to glucose are still in progress.
THIS IS A GOOD STEP TOWARD ACHIEVING NONINVASIVE GLUCOSE MEASUREMENT
There is a large potential for usage of the improved MIP glucose test strips. Because of the very high linearity of the test strips, this shows the viability for replacing the current commercial strips. As a result, this could lead to huge savings in cost for patients. Because these strips also do not use the expensive enzyme, this leads to a much longer shelf life and greater ease of use in a variety of climate conditions.
As for the glucose sensing ink, this is a good step toward achieving noninvasive glucose measurement, a milestone goal for diabetes patients. This project demonstrates the large potential that this solution has and proves that the concept could work if more research is conducted on the ink’s performance.
I would like to thank Dr. Jun Kameoka for his support and guidance throughout this research project. In addition, I would like to thank Onder Dincel, Zheyuan Chen, and Ting-Yen Chi for their guidance and advice during this project. Special thanks go out to the Texas A&M Undergraduate Research Scholars program for their guidance through the research process.
Christopher Evan Wright ‘19
Christopher Evan Wright ‘19 is an electrical engineering major with a minor in mathematics from Katy, Texas. Christopher’s research project was conducted in the 2018–2019 Undergraduate Research Scholar’s program under the guidance of Dr. Jun Kameoka. Christopher hopes to attend graduate school a few years after working in industry. Afterwards, he hopes to continue to develop medical devices to help patients lead happier, healthier lives.