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Fabrication of novel cholesterol nanobiosensors with carbon nanotubes and metallic nanoparticles
thesisposted on 22.02.2017, 03:35 by Abdul Rahim, Mohd Zulkhairi
The determination of cholesterol concentration in blood and other samples is of vital importance in clinical diagnosis. One of the rapid approaches for accomplishing this goal in clinical and domestic settings is by use of biosensors. In general, biosensors are fast, efficient and reproducible devices for measuring cholesterol in biological samples. This thesis presents various strategies for the fabrication of a novel cholesterol nanobiosensor based on carbon nanotubes and metallic nanoparticles. The research has involved the incorporation of carbon nanotubes or/and gold nanoparticles in polypyrrole films by galvanostatic entrapment. The effectiveness of incorporation of multiwall carbon nanotubes (MWCNTs) or/and gold nanoparticles (AuNPs) in polypyrrole (PPy) films for enhancing free and total cholesterol detection were carefully investigated throughout the thesis. Chapter 1 focuses on the review of previous studies conducted on the fabrication of cholesterol biosensors. Specific discussions include importance and health related issues of cholesterol, principles of biosensors, immobilization technique and electrochemical methods of detection. The different strategies for fabrication and characterization of cholesterol biosensor were highlighted. Included also is the scope of the study on fabrication of novel cholesterol nanobiosensors. In Chapter 2, a cholesterol biosensor based on co-immobilization of gold nanoparticles and cholesterol oxidase (COx) in polypyrrole film is described. The optimum condition for the formation of PPy-NO3--Fe(CN)64--AuNP-COx film was an applied current density of 0.5 mA cm−2, polymerization time of 100 s, 0.3 M pyrrole, 15 units/mL COx, 0.1 M KNO3, 0.5 mg/L AuNPs and 1.5 mM Fe(CN)64-. Optimum amperometric response for cholesterol was obtained in 0.05 M phosphate buffer (pH 7.00), achieving a minimum detectable concentration of 10 M, a linear concentration range of 5-25 M and a sensitivity of 1.6 µA cm-2 µM-1. Also, the detection of total cholesterol with the PPy-AuNP based film was accomplished by co-incorporation of cholesterol esterase (CE) with COx and AuNPs. The incorporation of the various components in the PPy film was confirmed by chronopotentiometry, cyclic voltammetry (CV), scanning electron microscopy - energy dispersive x-ray detection (SEM-EDX), Fourier transformed infrared (FTIR) spectroscopy. The optimized additional conditions for film formation and amperometric detection of total cholesterol were 5 units/mL of CE and 15 units/mL COx. Under optimum conditions, a minimum detectable total cholesterol concentration of 25 M was achieved with a linear concentration range of 25-170 M and a sensitivity of 0.1 µA µM-1 cm-2. Furthermore, the presence of common interferants did not affect the total cholesterol measurement with the PPy-NO3--Fe(CN)64--AuNP-COx-CE nanobiosensor. In Chapter 3, an alternative approach is explored for improving the detection of cholesterol by replacing AuNPs with MWCNTs during the fabrication of the PPy based biosensor. The results obtained by chronopotentiometry and cyclic voltammetry indicated that the MWCNTs were successfully incorporated into the PPy film, and their presence improves the performance of the cholesterol biosensor. Optimum free cholesterol response was obtained with 6 mg/L of MWCNTs, a polymerization time of 100 s, 0.3 M pyrrole, 10 units/mL COx, 0.1 M KNO3, current density of 0.5 mAcm-2, and 1.5 mM Fe(CN)64- with an applied potential of -400 mV for the amperometric detection. The optimized pH and buffer were pH 7 and 0.05 M, respectively. A minimum detectable concentration achieved was 5 M free cholesterol with a linear concentration range of 5-25 M and a sensitivity of 3.2 µA cm-2 µM-1. The detection of total cholesterol biosensor was also explored by co-incorporation of cholesterol esterase (CE), cholesterol oxidase (COx) and MWCNTs in the polypyrrole (PPy) film. Chronopotentiometry, cyclic voltammetry (CV), scanning electron microscopy - energy dispersive x-ray detection (SEM-EDX), and fourier transformed infrared (FTIR) spectroscopy were also used to confirm the incorporation of the various components in the PPy film. The additional optimized conditions for fabrication and detection of total cholesterol were 5 units/mL of CE and 10 units/mL COx. A linear range for total cholesterol was obtained from 25 to 170 µM with a minimum detection concentration of 25 µM. The sensitivity of the PPy-NO3--Fe(CN)64--MWCNT-COx-CE electrode was 0.3 µA µM-1 cm-2 and the presence of electroactive substances such as ascorbic acid, uric acid and glucose did not affect the amperometric measurement of total cholesterol. In Chapter 4, further improvement of the performance of the cholesterol biosensor was explored based on co-incorporation of MWCNTs and AuNPs into the PPy film. This involved concurrent galvanostatic entrapment of MWCNTs, AuNPs and COx in PPy film. The optimized conditions for detection of free cholesterol include 6.0 mg/L MWCNTs, 0.25 mg/L AuNPs, applied potential of -400 mv, polymerization time of 100 s, 0.3M pyrrole, 10 units/mL COx, 0.05M KNO3, current density of 0.5 mA cm-2, 1.5 mM of mediator (Fe(CN)64-) and 0.09 M phosphate buffer (pH 7.00). A linear concentration range was obtained from 5 to 25 µM, while achieving a sensitivity of 4.5 µA cm-2 µM-1 and a minimum detectable concentration of 5 µM. The application of the biosensor to the measurement of total cholesterol was also considered by galvanostatic entrapment of MWCNTs, AuNPs, COx and CE in a polypyrrole (PPy) film. The additional optimized conditions for the film growth for total cholesterol detection were 4.0 mg/L of MWCNTs, 0.25 mg/L AuNPs, 10 units/mL of COx and 5 units/mL of CE. The sensitivity for the detection of total cholesterol with this biosensor was 0.5 µA µM-1 cm-2 and a linear concentration range of 25-170 M was achieved. The minimum detectable concentration of total cholesterol was 25 M. Also, no effect was obtained in the presence of interferants such as ascorbic acid, uric acid and glucose. Chapter 5 concludes with the key findings from the study on fabrication of novel cholesterol nanobiosensors with carbon nanotubes and metallic nanoparticles. Comparisons between the biosensors developed in this study and others reported biosensors were made. Also included were some recommendations for future work.