Lin, Lih Poh
(2022)
Greenly synthesised biocompatible graphene/zirconia nanocomposite: towards the development of a biosensor for the detection of lung cancer biomarkers.
PhD thesis, University of Nottingham.
Abstract
Early detection improves the survival rate of lung cancer. Routine check-up gives rise to early detection, however, conventional screening methods such as X-ray and computed tomography (CT) are incompetent for routine check-up of lung carcinoma due to health hazards associated with repeated exposure to ionizing radiation. These limitations call for a safer, faster, and cheaper solution - the biosensor. In this study, an electrochemical sensor was fabricated using graphene/zirconia (ZG)-modified screen-printed carbon electrode (SPCE). A green approach using solvent exfoliation alongside hydrothermal treatment was employed to prepare the ZG nanocomposites. Material characterisation suggested that pristine graphene and well-hybridised ZG nanocomposite were synthesised. Alongside material characterisation, a cytotoxicity test was performed on the ZG nanocomposite to address the issue of potential hazards during material synthesis and sensor fabrication. The result showed that the ZG nanocomposite had no adverse effects on human lung cells (MRC5) and skin cells (HaCaT), with a half-maximal inhibitory concentration (IC50) value of >500 µg/ml.
Before the development of an immunosensor and genosensor, the potential of the ZG-based platform as an electrochemical sensor for electroanalytical application was demonstrated via acetaminophen detection. The electrochemical sensor exhibited favourable performances for the detection of acetaminophen ranging from 10 μM to 100 μM. A limit of detection (LOD) of 75.5 nM, repeatability of < 3% relative standard deviation (RSD), reproducibility of 5 % RSD, stability of 95% after 5 days of storage alongside decent selectivity to acetaminophen in the presence of interfering species were observed. This finding subsequently motivated the exploration of the platform to serve as a genosensor enhanced with 1-pyrenebutyric-acid-N-hydroxysuccinimideester (PY) biolinker to differentiate wild-type and mutated exon-19 sequences in epidermal growth factor receptor (EGFR). The exon-19 mutation is common among lung cancers. The fabricated genosensor was able to quantify the concentration of wild-type exon-19 in the range of 5 nM to 1 µM, with a linearity coefficient of R2= 0.99 and a LOD of 1.7 nM. The sensor was observed to produce reproducibility of 8.3% of RSD and stability of 90% after 8 days of storage. Most importantly, the sensor was capable of generating distinctive signals for wild-type and mutated exon-19, making it valuable for the detection of genetic mutation. An immunosensor for the detection of carcinoembryonic antigen (CEA) was proposed. CEA is a glycoprotein involved in cell adhesion and is known to be over-expressed in serum of lung cancer patients. Decent performances were exhibited by the immunosensor for CEA detection with a 4.25 pg/ml LOD and a correlation coefficient of R2= 0.99 within a linear working range from 0.01 ng/ml to 10 ng/ml. The immunosensor exhibited high specificity for CEA against interfering species, decent stability for up to 20 days, and reasonable reproducibility of 4.27% RSD. The immunosensor has also presented a good recovery of CEA in human serum, in which the detected CEA level was in close agreement ( < 7% deviation) with the actual concentration.
The key characteristics of the sensors reported in this work offered appealing merits on their own: the stable PY biolinker immobilisation technique, the biocompatible ZG electrode material, the handiness of disposable design and the potential for miniaturisation; hence, hybridizing all these merits produced a promising electrochemical testing platform that subsequently opened up a new avenue for early detection of malignant conditions.
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