Development of an amperometric alcohol biosensor with modified screen-printed electrodes for the detection of a mixture of ethanol and methanol

The present work mainly aimed the development of a dual enzymes amperometric enzyme biosensor which is modified with various materials, disposable and low-cost, for detection of methanol, ethanol and methanol-ethanol mixtures. Disposable transducers of high electrochemical reactivity were developed by modifying various types of screen-printed electrodes (SPCE) with carbon nanotubes (CNT), gold nanoparticles (AuNP) and poly (neutral) red (PNR). After modifying the developed systems with alcohol oxidase and alcohol dehydrogenase enzymes, the optimum working conditions of these biosensors were investigated for the analysis of ethanol, methanol and ethanol-methanol mixture. The amount of ethanol in real samples and the amounts of ethanol and methanol in ethanol-methanol mixtures were determined with the alcohol biosensors at optimum conditions. Voltammetric techniques such as cyclic voltammetry and chronoamperometry were used in experimental studies. In the work, firstly a new composite electrode of SPCE/MWCNT/AuNP/PNR formulation was developed for the determination of nicotinamide adenine dinucleotide (NADH) at low potentials, without any mediator and without any interference. Optimum pH of this biosensor was determined as 7.5. Reproducibility for 100 µM NADH, sensitivity, limit of detection and limit of quantification, linear range of NADH sensor were found to be 2.55 % (n=10) as relative standart deviation (R.S.D.), 15.82 µA.mM-1, 4.82 µM and 16.09 µM, 16.07 µM – 150 µM respectively, after analytical characterizations. The synergic effect of PNR with nanoparticles in NADH sensors and alcohol dehydrogenase (ADH) based biosensors was investigated and it was observed that PNR increased the sensitivity when it is included in the formulation of sensor or biosensors prepared with only MWCNT or AuNP. Electronic conductivity of sensor and biosensors increased with a synergic effect displayed due to characteristics possessed by PNR and MWCNT. The highest electronic conductivity and electrocatalytic effect were observed in sensors and biosensors prepared with SPCE/MWCNT/AuNP/PNR composite electrode. Quantity optimizations of MWCNT and AuNP were accomplished in SPCE/MWCNT/AuNP/PNR composite electrode. The highest peak currents were obtained when there are 3 layers of MWCNT and 2 layers of AuNP at the SPCE surface. It was found that optimum electrode type and formulation is SPCE/MWCNT/AuNP/PNR, optimum pH is 7.75, working potential is + 0.2 V, the amounts of enzyme and coenzyme are 150 unit and 7 mM, respectively, for the ADH based biosensors. Sensitivity, linear range, limit of detection and limit of quantification were found to be 0.49 µA.mM-1, 320.2 µM – 1000 µM, 96.1 µM and 320.2 µM, respectively, for the ADH based biosensors after analytical characterizations. Reproducibility of ADH based biosensors for 400 µM ethanol relative standard deviation (R.S.D.) was calculated to be 1.57 % (n=10). Alcohol oxidase (AOx) based biosensors were prepared with SPCE/MWCNT/AuNP/PNR composite electrodes which is the optimum electrode type and formulation for ADH based biosensors and NADH sensor. The optimum pH, working potential and enzyme amount were found to be 7.0, - 0.3 V and 1.2 unit, respectively. Reproducibility of AOx based biosensors for 400 µM methanol and 400 µM ethanol relative standard deviation (R.S.D.) were calculated to be 1.12 % (n=5) and 1.65 % (n=5), respectively. It was calculated that the limit of detections were 100.8 µM and 53.5 µM, limit of quantification were 335.9 µM and 178.5 µM, linear ranges 335.9 µM - 1000 µM and 178.5 µM – 1000 µM, sensitivities were 0.509 and 0.482 µA.mM-1 for methanol and ethanol, respectively. Analytical characterization for the determination of ethanol/methanol mixture with AOx based biosensors were also made. The amount of ethanol in ethanol/methanol mixture was determined with ADH biosensors while the amount of methanol was found by substituting the current values obtained by amperometric analysis with AOx based biosensors into the equation of calibration curves. It was concluded that AOx based biosensors were more sensitive for methanol compared to ethanol. The AOx based biosensors were used for ethanol-methanol mixture and the sensitivity for methanol was observed to decrease in the presence of ethanol. A new SPCE/MWCNT/AuNP/PNR composite electrode, NADH sensor and biosensors for ethanol, methanol and ethanol/methanol were developed successfully within the scope of the thesis. KEYWORDS: Alcohol biosensor, screen-printed electrodes, alcohol dehydrogenase alcohol oxidase, poly(neutral) red, carbon nanotubes, gold nanoparticles