Design and Development of a Microfluidic Amperometric Immunosensor for the Quantitative Detection of 2,6-dichlorobenzamide (BAM) Herbicide Residue in Ground Water

Abstract

Access to clean and safe-drinking water is essential to health and it is a basic human right. These days, nearly a billion people of the world‘s population do not have access to this precious commodity. Along with many other causes, pollution of water sources by pesticides poses a real threat to the availability of clean water. Thus, the need of rapid, reliable and on-site early warning systems to monitor the quality of water becomes as important as its preservation. This work describes the design and development of an automated microfluidic biosensor based on immunological methods (immunosensor) to determine quantitatively the presence of pesticides in ground water. A herbicide residue, 2,6- dichlorobenzamide (BAM) is chosen as a model system in this research. The thesis describes how an existing, highly selective and sensitive BAM immunoassay is optimized to transfer it from a lab-based end-point analysis technique (ELISA) to a portable, onsite monitoring system. The optimization of this heterogeneous competitive immunoassay is achieved by a unique approach in which the immunosorbent is engineered by using a newly synthesised BAM hapten library. Additionally, the improvisations made to the existing BAM hapten synthesis route resulted in the isolation of haptens for the parent herbicide (dichlobenil) itself. The affinity constants of new BAM haptens were estimated and compared with the existing BAM hapten. These results were correlated with their regeneration performances. Based on this, one of the newly synthesised BAM haptens (named as hapt D) was chosen for further development of the BAM immunosensor. This immunosensor employs a cost effective, miniaturizable, amperometric detection technique. This thesis details the design and fabrication of a microfluidic platform in order to incorporate the optimized BAM immunoassay and the electrochemical detection method. A modular approach was adopted for the fabrication of the microfluidic platform in order to make the device simple to integrate, automate and maintain. The microfluidic platform has an in-built micro flow-injection analysis (µFIA) system and it is a novel characteristic of the microfluidic device prototype. The microfluidic device was automated using Lego® Mindstorms® servomotors to control its micro pumps and valves. By confirming (amperometrically) the regeneration capability of the optimized immunosurface and generating a standard curve for BAM inhibition assay, the developed Amperometric BAM ImmunoSensor (ABIS) prototype provides the proof of concept. The possibility of interfacing ABIS with the macro world using a nearly developed auto-sampling unit has also been considered in the thesis.