| Summary: | 博士 === 國立臺灣大學 === 工程科學及海洋工程學研究所 === 104 === Innovations in technological applications not only accelerate the spread of knowledge in Science and Technology (S&T) but it can also increase the value of S&T knowledge. The integration of the fields of Life Science and Engineering has been a major research focus in achieving cross-disciplinary, cross-field knowledge. Since the completion of the Human Genome Project in April 2003, one of the major focus of biotechnology research has been on proteome-related research. New technology or equipment which are real-time, are label-free, and can handle direct detection of biomolecular interactions have been found to be the most advantageous. It is estimated that the global market for immunoassay technologies will grow by US$7 billion from 2015 to 2020 and the biosensor market will increase by US$9.53 billion. Biomolecular interaction detections, especially those between antigens and antibodies, have become more important in Life Science research, new medical development, and clinical studies.
According to the World Health Organization (WHO), tuberculosis (TB) is a top infectious disease killer worldwide. In 2014, it was estimated that 9.6 million people fell ill with TB and 1.5 million died from the disease. Data shows that latent tuberculosis infection (LTBI), affects about one-third of the world’s population and approximately 10% of people with LTBI develop into active TB later on. Methods which facilitate efficient detection and interpretation of biomarkers for screening LTBI have been an important research topic as it attempts to deal with an international public health problem. Interferon-gamma (IFN-γ) as a biomarker indicator for tuberculosis and with antiviral, immunomodulatory and anti-tumor properties can be used as a reference to assess inflammatory status associated with risk of the disease. In addition, since the body immune function abnormalities produce neutralizing anti-IFN-γ antibodies (anti-IFN-γ Ab), it has the potential to be used in the development of drugs for antagonistic auto-antibodies associated with autoimmune diseases. With the limitations of current detection methods and costs, latent tuberculosis infection has not yet undergone thorough, large-scale screening. To this end, the development of a more appropriate and more readily available first-line diagnostic tool is one of the major priorities of the United Nations in combating tuberculosis.
The experimental approach in this investigation was based on creating a bio-sensing platform. This platform, called OBMorph, was developed by the National Taiwan University (NTU), Nano-BioMEMS group. It integrated a surface plasmon resonance (SPR) technology and a phase modulated ellipsometer to improve the performance of biomolecular measurements. Electrochemical impedance spectroscopy (EIS) was also integrated simultaneously onto this measurement platform. Furthermore, a bithiophene-based conductive biolinker was also developed with an attempt to improve the antibody-antigen interactions for biosensors and to verify the performance as well as potential for synchronous measurement of IFN-γ binding interaction by the OBMorph platform and EIS. In addition, the bithiophene biolinker was introduced into our newly developed biochip and adopted into a commercially available SPR instrument (Biacore T200). We wanted to test and verify the suitability of the bithiophene biolinker and to optimize the test procedures for detecting anti-IFN-γ Ab. Our experimental results show excellent measurement performance for the OBMorph as well as for the newly developed biolinker. The interactions of the IFN-γ with immobilized anti-IFN-γ Ab at various concentrations (12.8pM ~ 1000nM) were investigated both optically and electrochemically. It was also verified that the bithiophene biolinker used was an effective biolinker for immobilizing antibody-antigen/antigen-antibody bio-detection. More specifically, we compared the response and concentration of the anti-IFN-γ Ab on a bithiophene-coated and dextran-coated biochip as well as on different thickness-modified surfaces under SPR measurement conditions. Our results indicate that a response to the IFN-γ molecules immobilized on a sensor using a bithiophene biolinker improved more than 8-fold when compared to that of a sensor using a dextran biolinker. A higher sensitivity on the immobilization of the IFN-γ and specific binding of the anti-IFN-γ Abs were both found in the newly developed bithiophene biochips when compared to that of a commercially available dextran biochip SPR measurement tool. Furthermore, the detection range of the anti-IFN-γ Ab obtained was 0.67nM to 83.33nM using a biosensor method without resorting to the use of second molecules for signal amplification. The regeneration ability of the sensor surface showed good repeatability as less than a 1% decrease was found after repeating the experimental work over 6 cycles.
In this dissertation, we found that the OBMorph offered us a good platform for rapid screening, real-time monitoring and the potential for quantitative concentrations of the autoimmune antibody activities. Proper protocols were also developed to demonstrate the detection sensitivity, measurement resolution, dynamic detection range, and chip regeneration capability of this newly developed biochip. Effects associated with using the bithiophene as a biolinker for the anti-IFN-γ Ab interaction with IFN-γ are examined in this dissertation. Moreover, our newly developed integrated bio-sensing system has the potential to provide new insight into various conjugate phenomena and interfacial processes for observing molecular conformation changes. It provides an advantageous platform for proteomic research which is cost effective and cost efficient. In summary, the results obtained from the integrated bio-detection OBMorph platform in this dissertation, can be a good starting point for advancing future technology research. It can be used to further work on disease prevention or extending to other applications which has the potential to boost the local development of in vitro detection devices.
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