Development of Measurement Systems for Biosensing Applications

A health condition called “Oxidative Stress” (OS), resulting from an excessive level of Reactive Oxygen Species (ROS) is a “state harmful to the body, which arises when oxidative reactions exceed antioxidant reactions because the balance between them has been lost”[1] OS appears to be associated with and might be a cause of, many serious diseases such as cardio-vascular accidents, cancer, Parkinson’s and Alzheimer’s[2]. This is not surprising as ROS are free oxygen radicals that can attack lipids, proteins, cellular membranes, enzymes and even modify DNA. Extensive correlation studies have shown that the complex impedance spectrum of blood samples from patients diagnosed with an OS syndrome differs significantly from the spectra obtained from the blood of healthy people, which is quite normal as the presence of an excessive amount of ROS should affect the physico-chemical properties of a blood sample. Measuring the complex impedance spectrum of a blood sample can be done quickly by means of low-cost electronic devices, making possible and affordable the early detection of OS among a large population. In order to quantitatively evaluate the OS, the impedance spectra being insufficient, the concentration of oxidative stress markers such as hydrogen peroxyde, malondialdehyde or F2 isoprostanes needs to be measured. Such measurements can, for instance, be used for monitoring the severity of a disease during a treatment. These concentration measurements are traditionally based upon analytical techniques but recently biosensors acting as transducers transforming directly a specific biochemical reaction into a measurable signal have been developed. They are essentially obtained by modifying the surface of metal or carbon electrodes using biomaterials such as enzymes antibodies or DNA that allow bindings or catalytic reactions with other specific biomaterials to occur on the surface of the electrodes. The resulting modifications of the electrical properties of the medium separating the electrodes can be analyzed through ad-hoc electronic and signal processing systems to yield the desired concentration. Biosensors have the advantages of rapid analysis, low-ost and high-precision. They are widely used in various fields, such as medical care, disease diagnosis and food analysis [3]. Hydrogen peroxide (H2O2) generated by cellular processes directly via two-electron reduction of molecular oxygen or indirectly via dismutation of superoxide, is the most widely studied ROS and its overproduction results in OS. Therefore, an ability to quantify the level of hydrogen peroxide and by ricochet the assessment of oxidative stress can be useful in order to assess certain health conditions occurring inside the body and as a result, an integrated electrochemical biosensor coupled with the hydrogen peroxide quantification can become a practical solution as a point of care device at home[4] Most of the time, H2O2 biosensors are based on HRP (Horseradish peroxidase) which is the most commonly used enzyme in the design of biosensors that can supervise the activity of oxidases and determine in terms of concentration, oxidase substrate such as lactate oxidase, cholesterol oxidase, or glucose oxidase, which all induce the production of hydrogen peroxide (HRP’s substrate). In the first part of this research, we explore the development of low-cost and compact measurement systems aiming to determining the impedance of biological samples as they grant access to information from electrical cellular characteristics. It is indeed possible to measure capacitance or conductance that are dependent on the health state of cells. The development of such measurement systems allowing the portability of biological essays requires sensitive electronics. Afterward, in the second part of our work, we explore the design of an electrochemical biosensor by immobilizing an enzyme (HRP) onto the surface of golden electrodes in order to detect and assess the analyte, hydrogen peroxide (H2O2). We also discuss the design of a potentiostat readout circuit to measure and convert the biosensor’s current. The combined results of the two parts of this work can be considered as a first prototype of a low cost and robust instrument easy to use in the field, away from a biological laboratory, with the goal of reaching the so called “point of care diagnostic” [5] The present thesis is organized as follows: Chapter I, introduces the present thesis. In Chapter II, we provide an overview in the field of biosensing technology. Chapter III deals with the design of a portable EIS measurement system to investigate reactive oxygen species in blood. Chapter IV presents an improved version of the previously designed instrument. Moreover, it points out the significance of EIS-based blood analysis through relevant medical diagnosis parameters such as hematocrit and erythrocyte sedimentation rate, extracted from the measured impedance spectra. In Chapter V we discuss on one hand the design of the H2O2 biosensor, and on the other hand the realization of the front-end circuit of the amperometric sensor. Finally, in Chapter VI, a conclusion is drawn..