Development of Capacitive Biosensors for Monitoring of Bacterial Toxins and Other Biomolecules

Detta är en avhandling från Biotechnology, Lund University

Sammanfattning: Sensing and quantification of biomolecules are becoming increasingly important in medical diagnostics, bioprocess technology and recently in homeland security. The majority of techniques currently employed to interrogate biomolecules require some type of radio-, enzymatic- or fluorescent-labeling. However, there is an increasing awareness of novel techniques in which biomolecules are detected in their natural forms and allow the detection of any analyte with minimal assay development. An important step forward in this pursuit is to identify and quantify an analyte using its electrochemical behavior and to convert its presence and concentration into perceivable electrical signals. The widespread exploitation of capacitive biosensors, in which changes in the dielectric properties of an electrode surface are detected, has its origin in the late 1980s. Such changes can be determined by measuring the electrical capacitance by potentistatic methods. In this context, two capacitive biosensors were developed for detection of biological warfare agents namely, staphylococcal enterotoxin B (SEB) and cholera toxin (CT). A linear range of 2.8 pg/ml to 2.8 ng/ml with a detection limit of 0.3 pg/ml was established for the SEB biosensor whereas, a linear range of 8.5 pg/ml to 8.5 ng/ml with a detection limit of 0.85 ng/ml was obtained by the CT biosensor. The developed biosensors were assessed by comparison with the well-established ELISA test and a surface plasmon resonance biosensor. We have explored the possibility of monitoring low molecular weight carbohydrates (e.g. glucose) using a novel competitive capacitive biosensing technique. The developed biosensor responded linearly to glucose in the range from 1.8 µg/ml to 18 mg/ml with a detection limit of 0.18 µg/ml. Such biosensor proved capable of estimation of the molecular weight of polysaccharides (dextran) and evaluation of the binding affinity of small sugars to concanavalin A (Con A). In attempt to improve the sensitivity the glucose sensor, a new sensing platform based on nanogold particles was utilized. Such trend has led to a better sensing range of 0.18 µg/ml to 1.8 mg/ml along with a detection limit as low as 18 ng/ml of glucose. In addition, a multipurpose capacitive biosensor was developed for detection and quality control of human immunoglobulin G (hIgG). The capacitance response depended linearly on hIgG concentration over the range from 5.0 to 100 µg/ml with a detection limit of 1.0 µg/ml. Additionally, the biosensor was able detect hIgG aggregates with concentrations as low as 0.01% of the total hIgG content, providing a potential post size-exclusion chromatography-UV (post SEC-UV) immunoassay for in-process quality control of hIgG, which can not be detected by SEC-UV singly at concentrations below 0.3% of the total hIgG content. Furthermore, optimization of the experimental conditions was addressed in all of the developed sensors. Finally, the developed biosensors are targeting the pharmaceutical research, bioprocess control and biodefense industry sections.