Synthesis and optimization of Anodic Aluminium Oxide thin film electrode for DNA sensing

Recent years, anodic aluminum oxide (AAO) has been extensively explored as an inexpensive, portable and sensitive DNA biosensing device. Apart from their ability to self-grow into well-ordered nanoporous with high porosity and huge surface area, AAO has one special feature wherein their nanopores‘ dimension are capable to be altered and engineered by controlling the main anodizing parameters namely voltage, temperature, time and electrolyte concentration. Since sensitivity and limit of detection of AAO-DNA biosensor depends on the nanopores‘ dimension itself, therefore, many researchers have tried to understand the effect of each anodizing parameters to the nanopores‘ dimension while tried to optimize and improve the sensitivity of AAO in detecting DNA. However, those studies were only focused on the trend of single variable parameter at one time by one-factor-at-a-time method (OFAT), rather than investigating all the interactions between the anodizing parameters simultaneously. Hence, they only reported the improvement made to the sensitivity of their AAO-DNA biosensor at the particular parameter range that they have tested without providing the best of optimum combination levels of all anodizing parameters while the most critical and influential anodizing parameter is still remained unknown. As a contribution to solve these problems, Taguchi method has been proposed in this research as an optimization tool to study the existing interaction between the parameters while at the same time providing the best combination of all parameters levels to improve the AAO-DNA biosensor sensitivity at its optimum performance. Meantime, ANOVA analysis has been proposed to obtain the most influential anodizing parameter to the sensitivity of AAO-DNA biosensor. Prior to optimization, correlation study between the pores‘ dimension and resistance charge transfer (Rct) that affects the sensitivity of AAO-DNA biosensor has been conducted. For this research, AAO thin film-DNA biosensor electrode was synthesized using a single step anodization method in oxalic acid. FESEM was used to observe the AAO surface, while EIS was utilised to study the electrochemical system for DNA hybridization detection. From the first finding, sensitivity of AAO-DNA biosensor was found to be influenced by the ratio of AAO thickness to the pore size. Rct remained under 100 kΩ as long as the ratio of AAO thickness to the pore size was maintained in the range between 1:11 to 1:16. Exceeding the ratio of more than 1:25 will result to the sudden increased in Rct and hence affecting the sensitivity to be reduced. From the second finding, optimization through Taguchi method is the main novelty of this research. The optimum combinations of anodizing parameters were found at 40 V, 17 °C, 0.3 M of oxalic acid at 1 hour. A repetition in experiment was conducted to confirm the efficiency of Taguchi where the sensitivity of the optimized AAO-DNA biosensor electrode was successfully improved to 62.57 % at 0.278 kΩ/M (LOD at 6.497 x 10-15 M) compared to the nominal. From the third finding through ANOVA analysis, the sequences of the most influential anodizing parameter to the AAO-DNA biosensor sensitivity are following the order: anodizing voltage (34.13%) > anodizing time (29.85%) > temperature (20.27%) > electrolyte concentration (15.74%). At the end of this research, optimization of the synthesis of AAO thin film electrode by Taguchi method for DNA sensing was successfully achieved. Since this research had used nonspecific type of DNA target analytes for prototyping development purposed, therefore, it is targeted that this optimized and sensitivity improved AAO thin film electrode will have greater chance to be used widely in the future for detecting various types of DNA, such as dengue, E-coli, or salmonella. Besides, it is of great hope that the outcome from this research may help other researchers to synthesize AAO thin film at its optimum anodizing condition as to improve the sensitivity of the AAO-DNA biosensor