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Fabrication of a gold nanorod metal organic framework sensor for epidermal growth factor ; a biomarker for kidney disease
Gwanzura, Zvikomborero Takunda
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Biosensors have been on the forefront to provide clinical diagnosis tools for various diseases. Proper selection of biomarkers as well as chemical electrode modification is key in the fabrication of electrochemical biosensors. Hence, electrode modified with nanomaterials devices to improve electroanalytical applications. These nanomaterials were functionalized to improve conductivity, accelerate signal transduction and amplify biorecognition events. Thus, resulting in novel sensing platforms that are highly sensitive and selective towards the target analyte. In this study, gold nanorods (Au NRs) capped with CTAB, zeolitic imidazole framework were synthesised using the seed mediated and hydrothermal method respectively. Composites of gold nanorods with cysteine, ZIF-8 or both were also synthesised. All synthesised materials were characterized using ultraviolet–visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-Ray diffraction (XRD) and cyclic voltammetry (CV) techniques. The obtained results confirmed the synthesis of the nanomaterials and composites. Identification of the ideal platform for fabrication of a transducer with the best electrochemical response was determined by studying the combinations of the synthesised nanomaterials and composites. The studied parameters were surface coverage, conductivity, rate of electron transfer constant. Cysteine-Au NRs composites platforms, had exceptional properties hence its synthesis optimisation of was undertaken. The effect of CTAB, reaction time, volume and concentration ratio of Au:Cysteine, temperature and pH on the composite properties were assessed. However, this composite’s electrochemical properties decreased when bioconjugated with the antibodies. Hence, the choice of Au NRs CTAB functionalised ZIF-8 (CTABAu/ZIF-8) as the transducer for biosensor applications due to a more favourable biocompatibility. Biosensor fabrication was done by drop coating glassy carbon electrode with the CTABAu/ZIF-8 forming a transducer followed by immobilisation of the antibody (Ab) using a covalent attachment method with glutaraldehyde (GA) as a cross linker. The target analyte, epidermal growth factor (EGF) was interacted with the Ab binding sites via electrostatic forces. All the fabrication steps were optimized for biosensor components, immobilization technique (drop coating and immersion), concentration and incubation time of linker and bioreceptor, as well as the synthesis of the CTABAu-ZIF-8 composites where in situ and ex situ techniques were compared together with the effect of the concentration ratio of Au: ZIF-8. There was also an analysis of optimum pH. Optimum conditions were found to be immersion in 3 % GA and 2 μg/ml Ab, with incubation times of 8, 10 and 5 minutes for GA, Ab and EGF respectively at a pH of 6. The following electroanalytical techniques: cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV) were utilised for EGF detection. The DPV showed better reproducibility, higher currents and better resolution hence; it was the method of choice. The technique’s optimisation involved assessments of the effect of step potential, starting potential and pulse amplitude. The optimum response for pulse amplitude, step potential and starting potential were 60 mV, 20 mV and 0.5 V respectively. The biosensor analytical parameters were linear towards EGF in the concentration range from 2 to 100 nM with a detection limit of 0.58 nM. Reproducibility and repeatability tests were acceptable, and the biosensor had a stability over 80 % within 15 days. There was no interference observed in the presence of glucose and creatine. The EGF biosensor was successfully applied in urine and saliva analysis, obtaining 67.5 and 3.12 nM respectively. This biosensor’s positive outcome strongly suggests its potential as a diagnosis tool for early detection of kidney disease as it was able to detect EGF concentration within physiological levels of EGF in normal kidney function.