- Title
- Developing an electrochemical aptasensor platform for detection of extracellular vesicles
- Creator
- Mohamed Amin, Zarinah
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2024
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Lung cancer is a leading cause of cancer-related deaths worldwide. The 5-year survival rate is only 10 to 20%. Due to its insidious nature, the onset often remains undetected until it reaches an advanced stage and is no longer treatable. As widely recognised and often lamented, one reason for the high mortality rate is late diagnosis. Early detection can improve the quality of a patient’s life and allow early treatment. The current methods for diagnosis are less than satisfactory and present a research gap for innovation, or at least improvement. Conventional methods such as bronchoscopy, computed tomography scans (CT) and tissue biopsy are tedious, expensive, and invasive. They also require bulky specialist equipment at medical facilities that are not easily accessible to rural populations and therefore not easily adapted for community screening. Simpler and sensitive methods are needed for rapid and reliable detection of lung cancer, ideally in the form of a point-of-care (POC) device. Most importantly it should be non-invasive, and easy to repeat when needed. A potential alternative has emerged through recent knowledge of extracellular vesicles or EV. They are nature’s own nanoparticles with a lipid bilayer membrane and are released by all mammalian cells. Formed by inward budding of a cell’s plasma membrane, they bear cargo molecules such as proteins, lipids and RNAs derived from the cell of origin. This imparts a vast potential to target EVs as diagnostic biomarkers for diseases. A number of such cargo molecules have already been discovered and the list keeps growing. Studies have shown that EV play an important role in intercellular communications and are involved in spread of diseases. The most important group of cargo molecules are the transmembrane proteins which are found elevated when cancer occurs. Their host-specific content offers biochemical information not offered by other analytes. To date, the markers that are most commonly used to assess pulmonary health are volatile organic compounds. A long list of target analytes has been built over several decades. Sadly, the methods for measuring them are also bulky and expensive such as gas chromatography, mass spectrometry and nuclear magnetic resonance. Widespread use is prohibited by the need for specially trained operators. Against the current bioanalytical landscape, EV presents an attractive and novel alternative as a target analyte. Detection via EV would be non-invasive. This is the crucial difference explored by this thesis, a paradigm shift. To capture an EV via targeting a membrane biomarker, a bioreceptor is needed. For this purpose, aptamers are preferred. They are single stranded DNA molecules bearing regions that bind specifically to the target biomarker and are found by a process known as Systematic Evolution of Ligands by Exponential Evolution (SELEX). Under specific conditions of salt, pH and temperature, they fold into secondary structures containing hairpin loops where binding occurs. As they are cheaper, do not require live animals for production, and are easy to modify for specific applications, they were chosen over antibodies. An opportunity thus arises to develop a biosensor platform that can detect EV derived from lung cancer. To detect the target capture, electrochemical measurements are preferred as they are cheaper to operate and simpler to integrate into a portable format. Among the various choices, electrochemical impedance spectroscopy offers a very sensitive technique to detect the formation of a large complex when an aptamer binds with its target. Increase in charge transfer resistance at the electrode interface is easily detected by employing a redox couple such as [Fe(CN)6]3-/4- ions. An interdisciplinary strategy and approach were demanded from the start. Preliminary work focused on surface modifications. Monolayers were optimised to generate sensitive and reliable sensor surfaces. Other EVs not related to lung cancer were detected first, and calibration plots were obtained. A cancer-specific platform was then developed. Using in vitro samples, lung cancer EVs were detected and quantified as particles per unit volume. In the final part of this thesis, the protocol was transferred to gold electrode patterns by photolithography. A prototype of the impedimetric platform was devised. Using this prototype, lung cancer-derived EVs were detected in a much shorter time using a small sample volume. The aptasensor prototype offers an avenue for a cheap, rapid, simple and sensitive test to diagnose and monitor lung cancer. Above all, it proves that EVs can provide a non-invasive route to detect lung cancer.
- Subject
- extracellular vesicle; aptamer; CD44; electrochemical; biosensor; lung cancer; photolithography
- Identifier
- http://hdl.handle.net/1959.13/1514224
- Identifier
- uon:56826
- Rights
- This thesis is currently under embargo and will be available from 06.08.2025, Copyright 2024 Zarinah Mohamed Amin
- Language
- eng
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