Development of a ZnO-based aptamer system for biosensing applications

Abstract

Biosensors are analytical devices that have the capability to convert some information about biological interaction into physical signals. The significance of biosensors in various disciplines, such as medical diagnosis, environmental monitoring, facility security, and food safety, is growing. A significant effort is directed towards developing the recognition element and the transducer component of biosensors to improve their sensitivity and selectivity. One of the elements that has attracted attention in recent years is an aptamer system. Aptamers are single-stranded nucleic acid molecules (DNA or RNA) that bind to specific target molecules, enabling high affinity and selectivity in binding. The transducer is the primary component that transforms and amplifies recognition events. The transducer can be based on a variety of technologies, including optical, electrical, or electrochemical technologies. However, the performance of biosensors can be improved by exploiting specific properties of the transducer’s component material for a better detection technique. In this thesis, it is proposed to utilise zinc oxide (ZnO) as the main material with the aim of developing aptamer-based biosensors. The excellent properties of ZnO including biocompatibility, high electron mobility, ease of fabrication, and labe-free detection, are demonstrated to be an effective approach for biosensing. These features are useful for providing highly selective and sensitive detection, which can accurately and specifically identify a target analyte while maintaining a low detection limit for trace amounts of the substance (down to few femtomolar concentrations). In addition, among semiconductor materials available in the market such as silicon (Si), gallium nitride (GaN), titanium dioxide ( TiO2), ZnO is generally considered a cost-effective regarding synthesis and fabrication. The ZnO-aptamer biosensors system is characterised for its optical, electrical and transducing properties for the detection of the SARS-CoV-19 spike protein and the VEGF-165 protein. Initially, ZnO-aptamer devices have been built using microfabrication and surface functionalisation. ZnO thin films have been deposited using radio-frequency (RF) sputtering, shadow mask evaporation to deposit aluminum (Al) electrodes, and photolithography processes to shape ZnO waveguide configurations. This was followed by functionalisation process of the ZnO surface using different approaches, including amino functional groups of 3-Aminopropyl triethoxysilane (APTES), triethoxysilylpropyl succinic anhydride (TESPSA), a thiol functional group of 3-mercaptopropyl trimethoxysilane (MPTMS), and phosphorothioate oligonucleotide linkage (PS linkage), to facilitate the aptamer bond. Optical and electrical characterisation techniques are utilised to investigate ZnO-aptamer devices responses in various parameters including protein-aptamer dynamic binding, electrical field effect, and transmitted spectra. Spectroscopic ellipsometry (SE) in biosensors is used to measure the thickness and refractive index of adsorbed layers on a sensor surface. SE can provide insights into adsorption kinetics by monitoring these properties over time. The adsorption kinetics of targeted proteins is real-time monitored on the functionalized ZnO surface in an aqueous environment. Current-voltage (I-V) characterisation is used to study the ZnO-FET behaviour upon attaching different protein concentrations. These attached proteins are serving as gate charges. When the target biomolecule binds to the receptor on the functionalised surface, it induces a change in the electrical properties due to charge redistribution. As a result, the conductance of the Bio-FET channel is modulated. A monochromator system is used to optically characterize a ZnO waveguide and its responsiveness to different proteins in concentrations from femtomolar to nanomolar level. SE technique has been employed to examine the thickness and optical characteristics of the ZnO thin film during the chemical reaction of surface functionalisation. This process effectively verifies the stability of ZnO thin film, preventing dissolution and aggregation effects, so ensuring its dependable utilisation. Furthermore, the extraction of optical characteristics before and after functionalization is necessary to verify the stability of ZnO. The validation of the later techniques involved doing I-V characterization to assess the functionalisation roles in achieving ZnO-aptamer specificity to proteins. The APTES coupling agent exhibited strong binding, while the TESPSA, PS linkage, and MPTMS coupling agents showed comparatively weaker binding. In addition, SE in-situ measurements were used to determine the thicknesses and optical characteristics of the adsorbed spike protein and VEGF over time. This helped gain insights into the dynamic binding process to ZnO-aptamers that were modified using optical models based on polarised light reflection over thin layers. The surface mass density (SMD) was determined by considering the thicknesses and refractive indices, yielding a limit of detection (LOD) of around 1 nanomolar (nM) for the spike protein and 100 picomolar (pM) for the VEGF. Moreover, I-V measurements have verified the ZnO-aptamer sensing mechanism through the detection of current responses resulting from the presence of the connected biolayer. The present shift ratio was determined based on concentration, revealing a limit of detection as low as a few picomolars. Furthermore, the same amount of specific proteins exhibited reactions to the ZnO waveguide surface, as evidenced by alterations in the spectral response for various concentrations. These changes were observed at a LOD of 1 nanomolar for spike proteins and 1 pM for VEGF. Ultimately, the obtained optical measurements of the proteins were then employed to construct a model of a micro ring resonator (MRR) for the purpose of creating a small-scale sensing platform at the micro-nano level. The MRR provided extremely sensitive biosensors with a resolution of a few nanometers per refractive index unit (nm/RIU) and a high-quality factor ∼7.66 ×103. This thesis presents a novel approach by conducting experiments to showcase the application of ZnO characteristics in creating a biosensing system. The system is built using aptamer receptors and may be utilised with various detection techniques. Additionally, this study demonstrates several techniques to modify the surface of ZnO with the ability to enhance its sensitivity, while also enhancing the selectivity of ZnO-aptamers towards specific proteins of interest. In addition, a design and model of a ZnO biosensor based on MRR (Micro-Ring Resonator) technology has been introduced to exhibit sensitive biosensor.