Capacitive Biosensor - A Tool for Ultrasensitive Analysis : Application in Clinical Analysis and Process Monitoring

Sammanfattning: Increasing demands for highly sensitive, accurate, fast and portable analytical assays have led to the development of new analytical tools. The integration of highly specific biological sensing elements and appropriate transducers has enabled design of even more efficient biosensor devices for the detection of target substances. In a clinical setting, early detection of biomarkers is crucial for successful treatment of diseases. Highly sensitive analytical tools are therefore needed to measure these biomarkers which, in several cases, are present at very low levels during the early stages of disease. A specific example is the analysis of trace concentrations of HIV-1 p24 protein in patients infected with HIV. Hence, such an analytical method requires ultrasensitivity in order to facilitate early detection and adequate selection of treatment, ultimately resulting in increased patient survival rates. Recent expansion in the field of biotechnology has led to advanced developments in production of recombinant proteins. Previously, it was acceptable and sufficient to state the percentage of pure product. Now, identification and quantification of impurities are becoming of interest. Host cell proteins, endotoxin, nucleic acids as well as viruses constitute such impurities together with reagents from downstream processing, such as protein A and other affinity ligands. The U.S. Food and Drug Administration has established guidelines requiring all possible removal of these impurities, while remaining levels must be clearly stated, especially for products that will act as injectable biopharmaceuticals. Therefore, bioprocess monitoring of these impurities is essential, and ultrasensitive analytical techniques will undoubtedly be required. This work presents the development of a capacitive biosensor. Certainly, there is an increasing awareness of this novel technique and the work in this thesis was first focused on the development of a thin film electrode, which can save preparation time, reduce cost of analysis and be used as a disposable sensor. Other parts of the work aimed to develop an ultrasensitive capacitive biosensor technique for application in clinical analysis (human serum albumin, HIV-1 p24) and bioprocess monitoring for host cell proteins and protein A. The work describes modifications of the sensor surface that can be used to increase the amount of immobilized antibodies. Self-assembled monolayer, non-conducting polymer, gold nanoparticles, and layer-by layer assembly were utilized. The developed capacitive biosensor was shown to be highly sensitive for detection of target analytes down to sub attomolar levels (1.0×10-18 mole per litre). Another promising result in this thesis was the application for detection of HIV-1 p24 antigen, which is a biomarker for HIV infection. The capacitive p24-sensor developed in this thesis showed a detection limit at least 500 times better than current technologies used in the field. Apart from pursuing ultrasensitivity, the work in this thesis also focused on the development of a new method for measuring capacitance. Compared to an existing method based on potential pulsing, which works very well for many applications, the newly developed system described herein uses a current pulse technique that allows for significantly more stable measurements. Furthermore, the new system is integrated with an automated flow injection system, which facilitates at-line analysis and provides more convenience for a user. Data acquisition is performed by software, which helps to avoid personal bias from data interpretation, ultimately resulting in a more reliable analysis. Finally, some limitations of the capacitive biosensor and future prospects of this technique are discussed.