NANOPILLAR BASED ELECTROCHEMICAL BIOSENSOR FOR MONITORING MICROFLUIDIC BASED CELL CULTURE

In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes - Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the `Enzyme electrode\u27 was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the `Working electrode\u27 was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose monitoring devices. Third, the methods used to develop 3D electrodes incorporated with nanopillars can be used for other applications such as neural probes, fuel cells, solar cells etc., and finally, the knowledge obtained from the immobilization of enzymes onto nanostructures sheds some new insight into nanomaterial/biomolecule interactions

Development of a biosensor system to detect bacteria in food systems

The development of biosensors may assist for the on-site detection of foodborne pathogens. The overall goal of this study was to develop a biosensor system for detecting Listeria innocua (non-pathogenic surrogate bacteria used as a model for pathogenic Listeria monocytogenes) in food systems. The study was divided into three main parts: (1) development of a sample collection and interface system for Listeria innocua from food samples, (2) development of a sample concentration system for the collected bacteria prior detection, and (3) development of a detection system based on a carbon nanotube potentiometric biosensor for a quantitative detection of Listeria innocua. In the second chapter, we discussed a sample collection protocol and delivery system developed for bacteria from food surfaces. Listeria innocua was used for testing and illustration. For this purpose, the surface of meat samples was inoculated with Listeria innocua at different concentrations from 10^1-10^5 CFU/mL. Then, cellulose membranes were applied to the surface of products for different times: 5, 10, 15, 20, 25, and 30 min sampling. The cellulose membranes were analyzed for their suitability for bacteria enumeration using a plating method for Listeria innocua. It was observed that sampling times between 5-10 min were the best and collection of \u3e80% of bacteria from the food’s surface was achieved. In the third chapter we discussed a microfluidic device for concentration of biological samples based on removal of liquids by hydrogel films. The performance of the device was demonstrated by concentrating 1-5 µm fluorescent beads followed by concentration of bacteria samples such as Listeria innocua. Results showed that fluorescence intensity of the beads was increased by 10 times at the end of concentration. Recovery efficiencies of 85.60 and 91.75 % were obtained for initial bacteria concentrations of 1x10^1 and 1x10^2 CFU/mL. Moreover, cell counts were observed to increase by up to 10 times at the end of concentration. This study showed that the concentrator device successfully concentrated the samples and no significant loss of living cells was observed for most of the bacteria concentrations. A carbon nanotube potentiometric biosensor for the detection of bacteria from food samples was demonstrated in the fourth chapter. The biosensor was constructed by depositing carboxylic acid (–COOH) functionalized single walled carbon nanotubes (SWCNTs) on a glassy carbon electrode (GCE), followed by the attachment of anti-Listeria antibodies to the SWCNTs between the amine groups and the –COOH by covalent functionalization using EDC/Sulfo-NHS chemistry. The performance of the biosensor was evaluated at various concentrations of L. innocua, for factors such as limit of detection, sensitivity, response time, linearity, and selectivity. In addition, the application of the complete detection system based on sample collection, concentration and detection of bacteria from food samples such as meat and milk was evaluated. Results showed that the system could successfully detect L. innocua with a linear response between electromotive force (EMF/voltage) and bacteria concentrations and a lower limit of detection of 11 CFU/mL. Additionally, similar results were obtained from the biosensor system for L. innocua from food samples

Methods for immobilizing receptors in microfluidic devices: A review

In this review article, we discuss state-of-the-art methods for immobilizing functional receptors in microfluidic devices. Strategies used to immobilize receptors in such devices are essential for the development of specific, sensitive (bio)chemical assays that can be used for a wide range of applications. In the first section, we review the principles and the chemistry of immobilization techniques that are the most commonly used in microfluidics. We afterward describe immobilization methods on static surfaces from microchannel surfaces to electrode surfaces with a particular attention to opportunities offered by hydrogel surfaces. Finally, we discuss immobilization methods on mobile surfaces with an emphasis on both magnetic and non-magnetic microbeads, and finally, we highlight recent developments of new types of mobile supports

Towards a commerical microelectrode array based sensor for improved chlorine detection

The commercial development of a disposable aqueous chlorine sensor based on a novel microelectrode array fabrication process is described. Non-conducting poly(o-phenylenediamine) films are firstly used to passivate conductive surfaces. Ultrasonic ablation of passivated electrode assemblies then results in the formation of a plurality of wells to expose the underlying conductive substrate, thereby forming a microelectrode array. Microelectrode arrays produced in this manner can be exploited within many electrochemical sensing applications; however, portable aqueous chlorine detection has been selected by Microarray Limited (the industrial sponsors of this project) as a primary vehicle for launching its generic production technology. The scale of microelectrode array production has been extended from that of individual gold sputtercoated glass slide electrodes - to the simultaneous production of hundreds of low-cost screen printed carbon-ink based sensors. A focus has been directed at all stages towards permitting the cost-effective large-scale mass production of sensors with a view to challenging existing portable aqueous chlorine measurement technologies both in terms of performance and unit cost. Based on volume batches of 250,000, it has been calculated that Microarray Limited sensors can be manufactured for a unit cost of approximately 2.5 pence, sufficiently low to provide scope for a competitive yet profitable sale price. The Microarray Limited aqueous chlorine detection system has improved the limit of detection from 0.01 ppm to 0.005 ppm total chlorine without sacrificing accuracy. Furthermore, this novel approach to aqueous chlorine detection offers numerous key benefits to the customer including reduced testing time, a more straightforward operation and the elimination of harmful reagents. Product development has been described from an initial concept through to a pre-production phase. The development of an innovative generic sensor packaging technology is also described.EThOS - Electronic Theses Online ServiceGBUnited Kingdo