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

Abstract

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.

Abstract (Swedish)

Popular Abstract in English

A biosensor is an analytical device that combines a transducer with a biological sensing element. This sensor is unique and has played an important role for a broad range of applications including clinical analysis and process monitoring. Between different types of biosensors, the capacitive biosensor has recently gained a lot of interest due to its analytical performances. The sensor can detect the interaction without the need of labelling compound, thus resulting in a cheap and fast analysis. Moreover, the result is obtained in real time and, most importantly, with an ultra-sensitivity characteristic to the technique. It is possible to detect the biomolecule at concentrations far below the detection limit of any other analytical technique or type of biosensor.



This thesis describes the development of a capacitive bio-sensing system. The sensor consists of a gold electrode on which the biological sensing element e.g. antibody was attached. This antibody is specific toward the substance that needs to be detected. The sensor is mounted in a flow injection system, and a continuous stream of liquid containing the target is passed over the sensing surface. The interaction of the target substance with the antibody will cause the capacitance to decrease proportional to the concentration of the target substance.



The development of a capacitive biosensor in this thesis focused on different aspects. The results are summarized in 6 papers. The first study (paper I) described the use of thin gold film electrodes, fabricated by thermal evaporation, as a transducer surface. This thin film electrode can be used without the need of surface pre-treatment e.g. mechanical polishing which is required for solid gold electrode. The great success demonstrated in this study led to the mass production of disposable electrodes. Another thin film deposition called “sputtering” was also studied and the electrodes fabricated from this technique were used in this thesis.





To demonstrate the use of the capacitive system, the sensors were first developed for the analysis of target substances which are important in the diagnosis of diseases. The work in paper I describes the detection of human serum albumin (HSA) from human serum sample. The capacitive biosensor results provide good correspondence with the results obtained in hospital analyses. The second application in clinical analysis is the detection of HIV-1 capsid protein, p24 antigen. The analysis of p24 antigen is of considerable diagnostic interest for monitoring HIV exposure. The analytical challenge for p24 detection is the sensitivity of an analytical device as p24 antigen is only present at very low concentrations when people are initially infected with HIV. The results of this work show the ultra-sensitivity of the capacitive biosensor technique which enables HIV-1 p24 antigen detection at very low concentrations.



The detection of trace impurities in the preparation of biopharmaceutical products is required for process monitoring. Therefore, high sensitivity of analytical techniques is needed as impurities are often present in minute amounts. Paper III described the development of a capacitive biosensor for the detection of host cell protein (HCPs) impurities. Host Cells e.g. E. coli used for recombinant expression are complex systems and contain hundreds, and up to thousands, of host cell proteins. These proteins can contaminate biopharmaceutical products and failure to sufficiently remove contaminants from these products can result in reduced product efficacy or adverse responses in patients. In this study, Lactate dehydrogenase (LDH) is expressed in E. coli and the production of this enzyme was used as a model system for the study of the HCPs impurities profile. The capacitive biosensor was successfully applied in the analysis of HCPs and the sensitivity of the technique is very high. This enables the detection of HCPs even when present at very low amounts.



Another application in bioprocess monitoring is the detection of protein A (paper IV) – a chromatographic column ligand used during the purification of monoclonal antibodies. The leakage of protein A into monoclonal antibody product might cause a serious problem if the antibody is used as therapeutic agent. This paper described the development of the capacitive sensor which can detect protein A down to 1.0×10-17 M.



The next part of this thesis describes the further development of a capacitive transducer using a new concept to measure the capacitance of the biosensor electrode (paper V). The use of the current pulse technique was found to increase the stability of the measurement. Sensor functionalities including data acquisition, automated operating system, and miniaturization are also studied in this work. The results show a good performance of the sensor which could lead to the further development of the sensor into a commercialized product, able to compete with other analytical devices in the market.



Finally, the circumstances promoting the sensitivity of the capacitive biosensor were discussed (paper VI) and some theoretical calculations were provided to support the explanations. This part also comprises examples of some results and limitations of the capacitance measurement technique.

• author: Teeparuksapun, Kosin
• supervisor: Martin Hedström; Bo Mattiasson
• opponent: Professor Ruzgas, Tautgirdas, Faculty of Health and Society, Malmö University, SE-205 06, Malmö, Sweden
• organization: Biotechnology
• publishing date: 2013
• type: Thesis
• publication status: published
• subject: Industrial Biotechnology
• keywords: Capacitive Biosensor, Ultrasensitive Analysis, Clinical Analysis, Bioprocess Monitoring, Human Serum Albumin, HIV-1 p24, Host Cell Proteins, Protein A, Current Pulse
• categories: Higher Education
• pages: 164 pages
• defense location: Lecture hall C at the Centre for Chemistry and Chemical Engineering, Getingevägen 60, Lund
• defense date: 2013-06-12 10:15:00
• ISBN: 978-91-89627-94-9
• language: English
• LU publication?: yes
• id: 10a52abd-308f-4ae2-9df9-9ab2d51c9d4a (old id 3737414)
• date added to LUP: 2016-04-04 11:17:35
• date last changed: 2018-11-21 21:03:53