Hannah Hospital's GOx Aptamer Project (2017)

Aptamer​ ​Selection​ ​against​ ​Glucose​ ​Oxidase to​ ​Improve​ ​the​ ​Efficiency​ ​of​ ​Biofuel​ ​Cells

Introduction

Many of the devices and vehicles used today, such as laptops, cars, planes, and phones are powered by Lithium batteries(Chambers 2010). The processes of mining and purifying metal catalysts, such as copper and aluminum used in lithium batteries are expensive and not environmentally friendly, given that the manufacturing requires lots of energy and uses harmful chemicals(Atanassov 2007). Also, the lithium used in lithium batteries is not very abundant in nature and scientists are concerned that they may run out (Nitta 2015). These metal catalysts are required in order to perform the oxidation-reduction reactions to generate electricity within the lithium battery. These lithium batteries are effective in producing energy, however, there are possible alternatives to this form of energy cells.

A far less expensive and more eco-friendly battery option currently being researched is the biofuel cell. Biofuel cells work as a battery, however, they operate differently than most batteries in that the convert sugar(glucose) into electricity. Biofuel cells use enzyme catalysts to generate this electricity rather than metal catalysts, as are used in most batteries. A biofuel cell consists of an anode(oxidation), cathode,(reduction), and a circuit. The anode surface contains an enzyme to oxidize glucose to produce hydrogen peroxide and electrons, thus generating current. The electrons are transferred from the anode through the circuit to the cathode. The cathode surface is coated with an enzyme to receive the electrons(Atanassov 2007). In a project done by Dmitri Ivnitski, of the University of New Mexico, a biofuel cell was created such that glucose oxidase was used as the enzyme on the anode. Glucose, the source of the chemical energy of the biofuel cell, would bind to the active site of glucose oxidase, thus oxidizing glucose and producing hydrogen peroxide and electrons. These electrons will travel from anode and through the circuit, thus generating a current and producing electricity, to the cathode, where electrons are accepted.

The target protein used in the application for this project will be glucose oxidase. Glucose Oxidase(GOx) catalyzes the reaction of glucose to hydrogen peroxide. This enzyme is found on the fungal tissue surface to prevent infection from bacteria and in honey to act as a natural preservative. GOx from the common fungi Aspergillus niger is a dimer, consisting of two identical primers each weighing 80 kDa, resulting in a total of 160 kDa. However, for the purposes of this project and experiment, human GOx will be utilized. GOx is not only being used by scientists to create a biofuel cell, but it is also being used in glucometers to measure glucose levels of diabetes patients. The glucometer uses GOx to catalyze the reaction of glucose to hydrogen peroxide to indirectly measure the glucose levels in the patient. GOx has been produced recombinantly from Aspergillus niger expressed in yeast Pichia pastoris(Meng 2014).

Biofuel cells have many different possible applications. For example, scientists are working to use biofuel cells to power devices requiring implantable power, such as pacemakers. The implantable devices could use the large source of glucose in the bloodstream to generate electricity through a series of oxidation-reduction reactions(Atanassov 2007). These devices wouldn’t require any external chemicals or alien power input, but only sugars in the form of glucose, which are in typically in high abundance in the blood. Another application for biofuel cells is transportation and energy generation. The world’s largest power source is fossil fuels, nonrenewable resources whose emissions have very harmful implications to the environment. These fossil fuels are used to generate energy to operate vehicles and other forms of transportation. If biofuel cells could be utilized to operate within transportation devices, fossil fuels could be replaced by carbohydrates(an abundant and renewable resource) as the main fuel source, an abundant and renewable resource could be used to replace them(Davis 2007). A fuel that is glucose and sugar based would not only be better for the environment, but it would also be a lot more economical than gasoline and fuels. Another possible application for biofuel cells is generation of power and energy from wastewater treatment. This application is phenomenal because it not only removes waste, which is detrimental to the environment and wellbeing of biological populations in the surrounding area, but it also uses the organic saccharide groups in the waste to generate electricity that can be used for many different applications(Davis 2007).

Biofuels can be very beneficial in that they are more economical, better for the environment, and safer. Typical batteries use lithium or precious metals, such as gold or platinum, as catalysts in order to generate electricity. Enzymes are a lot less expensive to use on a large scale in devices than lithium and transition metals, the d-block of the periodic table. Biofuel cells are also a lot better for the environment. The transition metals, composing the more typically used batteries today are a lot less abundant in nature. Lithium ion batteries are expensive, and a lack of lithium and some of the transition metals may one day become an issue(Nitta 2015). Using glucose or other very renewable and available organic inputs as chemical energy to generate electricity, would make the availability of electric energy a lot more probable in the the future. Not only are biofuel cells more economical and more green, but also a lot safer than the common battery or fuel cell. A fuel cell uses hydrogen and chemicals like methanol and ethanol to directly generate electricity. Glucose is a lot less flammable than hydrogen or methanol, and thus less likely to ignite or catch fire. This safety characteristic would be very beneficial in the case that a biofuel cell were used in a car, because, upon collision, a car with a biofuel cell would be less likely to explode compared to a car with a battery or fuel cell.

Aptamers can be help improve the efficiency of biofuel cells. Aptamers are short oligonucleotides that bind to target proteins with very high specificity. Aptamers can be found through many rounds of the selection process. In this project, the specific aptamer will be found by starting with a large pool of RNA sequences and after each round of selection, increasing the stringency to which the available RNA pool binds to GOx. Then, taking the bound and unbound RNA samples, reverse transcribing them into DNA. Then precipitating the DNA using an Ethanol Precipitation procedure and then, amplifying the DNA by ccPCR and then lsPCR. Then transcribing the DNA back into RNA and performing Ethanol Precipitation again. Then, purifying the RNA using a PAGE. That encompasses one round of selection. To start the next round of selection, take the RNA sample from the PAGE and purify it. Increase the stringency of the washes for the next round by increasing buffer volume or by decreasing the protein concentration from 200 pmol to something smaller.

Aptamers have many different possible functions. They can serve as signalling molecules, reporting the presence of a certain enzyme or as inhibitors by binding to the active site of an enzyme and inhibiting its function. This use of an aptamer has been used in many experiments, for example in a project by Yusuke Kaida at Kurume University School of Medicine, Kurume, Japan. In this project, an aptamer was used to inhibit the function of AGEs, which are toxic compounds found in commonly eaten foods (Kaida 2013). The application for my project regards biofuel cells using GOx as the enzyme catalyzing the oxidation of glucose to generate electricity. Therefore, in order to produce a biofuel cell which more efficiently generates and stores energy, the main goal of the project will be to select for an aptamer that inhibits the function of the GOx in the biofuel cell, so that whenever the device is not in use, the state of the enzyme can be preserved.

Many of the applications for biofuel cells listed earlier in the paper would be successful in their use, given that the biofuel cell is properly functioning. Any and all devices or tools containing biofuel cells as their battery should be effective in the energy they produce. Therefore, consider the fact that biofuel cells contain enzymes. The structure of the enzyme is fragile and can denature with change in environment such as temperature or pH(Becktel 1987). To preserve the state of the glucose oxidase enzyme and minimize the period it could denature, the goal of this project to select for an aptamer that binds to GOx. Aptamers have many benefits over antibodies, which are proteins that bind to other molecules. For example, aptamers are stable at room temperature, whereas antibodies can denature very easily. Aptamers, which are composed of oligonucleotides, are also a lot smaller than antibodies. Aptamers can also be produced on a larger scale and more economically since they are produced in vitro whereas antibodies are produced in vivo.

This aptamer, when bound to the active site of the enzyme would inhibit its function. Thus, whenever the aptamer is bound to GOx, no hydrogen peroxide will produce, thus no donating of electrons at the anode or accepting of electrons at the cathode to generate a current, and thus no electricity. The aptamer could be handled in such a way that whenever the application for the biofuel cell, whether it be a car or a laptop, etc. , is not operating and is at rest, the aptamer is bound to the GOx. Whenever the device is in use, the aptamer is not bound to the target. This function of the aptamer not binding to the target could be managed by creating a mechanism such that whenever the key in the ignition, is turned and the device is turned on, an optimum voltage or pH is initiated to cause the aptamer to detach itself from the GOx. This mechanism would make the device containing the biofuel cell more effective by preserving the enzyme and would make the device cheaper, since GOx bound to the anode surface in the biofuel cell wouldn’t have to be replaced as often, in order to preserve the function of the device. Therefore, by using an aptamer to bind to the active site of the GOx at the anode of biofuel cell, the shelf life of this enzyme shall be greatly increased.

Glucose Oxidase is negatively charged and so are aptamers. RNA is negatively charged due to the phosphate backbone. Therefore, due to repulsion of molecules of similar charge, it is difficult to select for aptamers for GOx. However, this barrier between the protein and the RNA due to similar negative charges can be bridged by the use of a divalent salt, Calcium Chloride. As far as stability goes, GOx is stable in phosphate buffer around 5-6 pH. The phosphate buffer that was made had 5.8 pH and was made of 0.1M Sodium Phosphate, monobasic and 0.1M Sodium Phosphate, dibasic.Since the conditions of the aptamer and GOx will be similar to that of a battery, the buffer and the pH of the selection reactions need to reflect that. A project completed by Bertrand Reuillard, Caroline Abreu, etc. of the University of Grenoble Alpes in France noticed that their glucose biofuel cell exhibited its maximum power density when used in a phosphate buffer at around a pH of 5 (Reuillard 2015). Therefore, for this project a phosphate buffer will be used.The recipe for a phosphate buffer pH 5.8 contains 92 mL monobasic Sodium Phosphate with a 0.1 M concentration, 8 mL dibasic Sodium Phosphate with a 0.1 M concentration, and 100 mL diH20(Man 2014). According to Apta-Index, no aptamer for GOx has been found yet. However, an aptamer for GOx is underway in order to improve the efficiency of Biofuel cells in a variety of their possible applications. So far, I am on round 1 of selection. Problems arose with ccPCR in regards to amplification and contamination. However, after troubleshooting, I was advised to reperform the selection round at Target Immobilization.

Click Here for Final Report

References

Atanassov, Apblett, Banta, Brozik, Barton, Cooney, Liaw, Mukerjee, Minteer. (2007) “Enzymatic Biofuel Cells.” The Electrochemical Society. 28-31.

Barton, Gallaway, Atanassov. (2004) “Enzymatic Biofuel Cells for Implantable and Microscale Devices.” ACS Publications: Chemistry Reviews. 104: 4867–4886.

Becktel, Schellman. (1987) “Protein stability curves.” Wiley Online Library: Biopolymers. 26: 1859- 1877.

Chambers. (2010) “The Path to Lithium Batteries: Friend or Foe?.” Clean Technology.

Davis, Higson. (2007) “Biofuel cells- Recent advances and applications.” Elsevier: Biosensors and Bioelectronics. 22: 1224-1235.

Heller. (2004) “Miniature biofuel cells.” Publishing: Physical Chemistry Chemical Physics. 6: 209-216.

Ivnitski, Branch, Atanassov, Apblett. (2006) “Glucose oxidase anode for biofuel cell based on direct electron transfer.” Elsevier: Electrochemistry Communications. 8: 1204-1210.

Kaida, Fukami, Matsui, Higashimoto, Nishino, Obara, Nakayama, Ando, Toyonaga, Ueda, Takeuchi, Inoue, Okuda, Yamagishi. (2013) “DNA Aptamer Raised Against AGEs Blocks the Progression of Experimental Diabetic Nephropathy.” American Diabetes Association. 62: 9.

Man. (2014) “Biological Buffers: pH Range and How to Prepare Them.” G-Biosciences.

Meng, Zhao, Yang, Zhang, Hao. (2014) “Production and characterization of recombinant glucose oxidase from Aspergillus niger expressed in Pichia pastoris.” Society for Applied Microbiology. 58: 393-400.

Nitta, Wu, Lee, Yushin. (2015) “Li-ion battery materials: present and future.” Sciencedirect: Materials Today. 18: 252–264

Reuillard, Abreu, Lalaoui, Goff, Holzinger, Ondel, Buret, Cosnier. (2015) “One-year stability for a glucose/oxygen biofuel cell combined with pH reactivation of the laccase/carbon nanotube biocathode.” Elsevier: Bioelectrochemistry. 106: 73-76.

Yoo, Youn-Lee. (2010) “Glucose Biosensors: An Overview of Use in Clinical Practice” Sensors. 10: 4558–4576.

Liu, Y. (2005) “A low-cost biofuel cell with pH- dependent power output based on porous carbon as matrix”. Pub Met, 17: 494004