Electrochemical DNA biosensor based on nanoporous alumina membrane
Date of Issue2014
School of Physical and Mathematical Sciences
In recent years, due to the high sensitivity and sequence specificity, as well as rapid detection, the electrochemical DNA biosensors are promising for genetic screening and detection of deoxyribonucleic acid. The electrochemical biosensor based on nanoporous structure electrode has attracted considerable attention owing to its unique property of high aspect ratio and high surface area to absorb specific probe molecules which can selectively bind to complementary biomarker molecules of interest. In this thesis, the nanoporous alumina based electrochemical DNA biosensor was investigated thoroughly. Firstly, we proposed an electrochemical DNA biosensor based on homemade alumina-modified electrode which can be obtained through electrochemical anodization method. The anodization technique is relatively easier and simpler than conventional lithographic techniques. The created nanochannel alumina structure with pore size ranging from 10 to 200 nm and pore density of about 1×1010 pores cm-2 is relatively regular. The chemical and thermal stability as well as the high specific surface area of nanoporous alumina suggest that it can be developed as a platform for sensing. Herein the probe DNA molecules were covalently immobilized into the alumina nanochannels. After the complementary target DNA sequences bind to probe DNA sequences inside nanochannels, changes in ionic conductivity of redox species Fe(CN)64- through the nanochannels will be observed due to blocking of the pores. Electrochemical signal can be obtained from the ionic conductivity changes through alumina nanopores by using differential pulse voltammetric (DPV) technique. DPV oxidative peak current of Fe(CN)64- successively decreases as the target complementary DNA concentration increases. Apart from demonstrating extremely low detection limit, the nanoporous alumina-based DNA biosensor can be used to distinguish complementary DNA strand from other mismatched DNA strand. It was further applied to detect the genomic DNA sequence derived from Legionella pneumophila which has been found to contaminate around 4% of drinking water and induces serious form of pneumonia known as Legionellosis or Legionnaires’ disease. Thus, the effective detection technique will be useful in cutting down the risk of transmission of disease. In the following chapter, we demonstrated a novel and integrated membrane sensing platform relying on free-standing anodic aluminum oxide (AAO) for the detection of oligonucleotide sequence using electrochemical impedance spectroscopy. Here, ultra-thin platinum layers were deposited as porous electrodes on both sides of AAO membrane and used as working and counter electrodes, respectively, for the impedance and differential pulse voltammetry measurements. Presently, the approach to DNA membrane sensor is using two external Ag/AgCl electrodes or inert electrodes to measure ionic conductivity across the membrane in a two compartment cell. Our approach used ~50–100 nm thick platinum films that were sputter-coated directly on both sides of the alumina membrane as electrodes to eliminate the solution resistance outside the nanopores. In addition, the target solution can be directly applied in small quantity of only 30 mL in volume onto one side of the membrane without the need of a cell assembly setup, which greatly simplifies the whole sensing procedure and reduces sample volume by a few thousand-fold. Probe DNA sequences were covalently attached within the pores of the nanoporous alumina membrane using glutaraldehyde cross linking. Binding of target DNA to the probe inside nanopores caused impedance changes due to blocking of nanopores, which provided the sensing signals. Pore resistance (Rp) increased nonlinearly in response towards the increasing concentration of the target DNA in the range of 1×10-12 to 1×10-6 M. Moreover, the biosensor selectively differentiated the complementary sequence from single base mismatched (MM-1) strands and non-complementary strands. Additionally, anodic aluminum oxide membrane can be mounted between two compartment cells. The sputter-coated platinum layer on each side of the membrane was still used as working and counter electrode respectively. In this project, an additional Ag/AgCl electrode was introduced as the reference electrode to form a three-electrode system. Electrochemical measurements were carried out using this sensing setup.