Electrochemical Biosensors for Real-Time Environmental and Clinical Monitoring

The need for simple, rapid, cost-effective and field-portable screening methods has boosted development in the field of biosensors. Existing biosensors allow detection of various analytes with adequate sensitivity and selectivity. However, their routine use has been hampered by several limitations with respect to operational and storage stability, inefficient transduction between the biological element and the physical transducer, poor reproducibility and interferences from coexisting species in real matrices. Most biosensors that have been reported in the literature have only been tested on standard laboratory samples and not in real matrices. This thesis reports design of new biosensor architectures with improved analytical performance characteristics and describes applications of this technology for the detection of environmental and clinical toxicants with implications in environmental and health monitoring. All biosensors investigated in this work utilize electrochemical detection methods with immobilized enzymes and redox proteins.;The first part of this thesis introduces a new method to site-specifically immobilize proteins onto electrode surfaces as a promising strategy for obtaining bioactive layers of functional enzymes. Specifically, we studied oriented immobilization of an acetylcholinesterase enzyme genetically modified to have a hexa-histidine (6His) residue, onto nickel nanoparticles in a single step procedure. The method is based on the specific affinity binding of the His-tag enzyme to oxidized nickel nanoparticle surfaces in the absence of metal chelators. The advantage of such an approach is the precise control over orientation of the protein on the electrode surface with preservation of bio-functional properties close to the native state. Biosensor fabrication, protein loading, evaluation of non-specific binding and the optimization of the operational variables are discussed. As an example of application, this method was used to design an electrochemical biosensor based on enzyme inhibition for fast and sensitive detection of organophosphorous pesticides in the concentration range from 10-8 to 10-13M. This chapter demonstrates the potential of this method as a promising way to achieve controlled and oriented immobilization of biomolecules for the development of highly sensitive biosensors.;The second part of this thesis explores the use of electrochemical biosensors for real-time monitoring of the evolution and mobility of superoxide anion radicals in a hippocampal brain slice model of ischemia. The monitoring of reactive oxygen (ROS) and nitrogen (RNS) species produced by living organisms in physiological and pathological conditions is of great interest when seeking to understand the role of these species in the onset and evolution of oxidative-stress related diseases. However, real-time in situ monitoring of ROS and RNS in relevant biological matrices is challenging due to their high reactivity, low and variable concentrations and very short half-life. This chapter demonstrates the first use of a cytochrome c biosensor to study and understand the dynamics of superoxide production and release in a mouse hippocampal brain slice model of cerebral ischemia. Using this sensor, we show evidence of superoxide production in the extracellular space in both physiological and pathophysiological conditions of ischemic brain injury. The biosensor allows for real-time continuous measurements and quantitative assessment of superoxide production over a period of 200 minutes of induced ischemia.;The third part of this thesis reports studies of the scavenging activity of inorganic nanoparticles of cerium oxide, or ceria (CeO2) against reactive oxygen and nitrogen species. Ceria nanoparticles are a novel wave of antioxidant agents with potential therapeutic uses in the treatment of oxidative-stress related diseases. In this chapter we studied the scavenging capacity of various types of cerium oxide nanoparticles towards NO and superoxide radicals through fluorescence and electrochemical detection. Using a fluorescence dye binding method, we demonstrate the ability of ceria nanoparticles to inactivate NO radicals in a concentration dependent manner. Using the cytochrome c biosensor, we show that additions of 1 microg/ml ceria reduce superoxide release in the brain slice model of ischemia by approximately 8.1+/-2.2% with each nanoparticle addition. These findings confirm the scavenging activity of cerium oxide nanoparticles against NO and superoxide radicals, and demonstrate the potential success of electrochemical biosensors in the establishment of time-course inactivation profiles and in the analysis of the therapeutic efficiency of pharmaceutical manipulations in real biological matrices.