Design
Our plan was to create a biosensor that could be used as an affordable yet accurate and reliable diagnostic tool for oncologic applications using bioengineering methods. After carefully reviewing the latest scientific literature on biomarkers, we concluded that, given the evidence, the best option would be the assessment of the Phosphorylation Level of Mitogen Activated Protein Kinases (MAP Kinases).
As their name implies, kinases, phosphorylate specific groups, and by an extend control the activity and function, of biomolecules consisting a cascade crucial for cellular processes such as proliferation, differentiation, death and overall survival, utilizing a phosphate group originating from Adenosine Triphosphate (ATP). Under normal conditions, MAPs are activated by other kinases in response to a cellular signal by alteration of the phosphorylation level, however in phenomena that lead to oncogenesis and eventually cancer, due to alterations in the physiology of the cell, their phosphorylation state is deregulated. Thereby, by measuring said state in a tissue, it is plausible to assess the possibility of cancer occurrence.
We considered kinase phosphorylation as a valuable biomarker for cancer as they provide significant evidence for the alteration of cell physiology and oncogenesis. There are many existing proteomic methods to assess such metrics such as western blot and mass spectrometry, however each one of them had a combination of shortcomings that abstract their commercial application and scaling including amongst others high cost of operation and reagents, duration and personnel training. Therefore we began considering and looking for alternatives that would address said issues in order to streamline early, affordable and precise cancer diagnosis. Having searched through the scientific literature, we found out that such device would probably come in form of a biosensor like setup, able to recognise the phosphorylated kinases.
In order for the assessment to be successful we needed to find the right method to detect and measure the phosphorylation state, or in other words kenome. Detection of kinases molecules for the profiling of the sample can be done using engineered antibodies that bind to them. For the sake of making the method time efficient and economical, we have opted to proceed with micro-wells to study the reaction that leads to the detection. On the walls of each micro-well of an array, affimers, a more efficient alternative to antibodies that are smaller, have a wider range of target molecules and a faster production rate that does not require animals, are placed and bind to target kinases. After washing the unbound molecules, enzymes remove phosphate and reactants that illuminate once in contact with it thus granting the ability for quantification.
Build
Once we had accumulated the necessary information, we proceeded with applying our research to a device that would eventually be the diagnostic tool. Our build was based on an existing patent designed for the detection of SARS-CoV-2 using ACE-2 receptors co-developed by our Principal Investigator. The biosensor is primarily consisted of two components, one physical/biological were the reactions take place and one electronic were the detection and user interface happens and the reaction as well as all the parameters are controlled. Those two components are connected to each other via gold electrodes to minimize resistance to the current and all the implications that it would result such as degradation of biomolecules due to temperature and inaccurate results. The electrodes’ surface needed to be clean to minimize contamination and maximize durability and accountability. After cleaning, L-Cysteine were applied to the electrodes for the bonding of massive biomolecules to the gold electrodes via their thiol groups. Binding to the L-Cysteine layer are the affimeres used for the detection of kinases, after proper preparation. Those affimeres are specific to the kinases that we want to detect and alter the physicochemical characteristics of the solution when bound to them. The biosensor utilizes the alteration of the electrochemical properties of the solution to sense the presence as well as the state of the reaction. We also used Wi-Fi and Bluetooth connectivity in order to implement Internet of Things protocols thus making the process more efficient and scalable.
Test
To test the biosensor, we will collect and test blood samples from a population of 30 patients and 5 healthy donors under the guidelines set by our bioethics committee approved trial protocol, since human biological material raises special bioethical and legal concerns. Although the biosensor can, under conditions, be used for specimens originating from any tissue, for our experiments we have optimized the device for blood as it is the most easily extractable and manageable tissue whilst maintaining diagnostic value. Sampling will be carried out by medically trained personnel at the controlled and hygienic facilities of “Errikos Dynan” hospital in Athens, Greece and handling will be a responsibility of trained members of our team originating from the health and life sciences sector. Our protocol binds the team to treat the donors as well as handle the samples and the generated data with the highest respect. Additionally, we grant the participants the ability to recall their participation as well as the right to be forgotten. Should any breach to the guideline of the protocol occur, testing will pause and the case will be examined and if needed the project will be terminated prematurely.
Learn
As was expected, the team had faced some setbacks in several aspects of the project. One of our most significant challenges was fundraising for the project as well as sourcing the affimers and other reactants for the build. We believe that such a situation was faced due to the saturation of the sector leading to greater competition for supplies as well as the dire financial state many institutions and other parties are facing.
Additionally, we allocated significant resources and consideration towards the handling of the biological material as we intended to follow Good Laboratory Practice in accordance with bioethical and biosafety standards in order to protect the identity of the donors, the health and safety of our members and the wider community and the integrity of our experiments.
In the event that our observations do not match with the expectations originating from our theoretical background, we will have to retrospect on all our processes and likely points of error. We will also review our theoretical background and plan of action in order to assess any possible shortcomings or oversights.
