1.1 Overview of Biochemical Detection
The biomedical engineering progresses significantly in the past decade. Especially, the disease diagnostics and drug discovery are the two most attributed issues. Useful information is provided to the biomedical fields by biochemical detection technologies with high accuracy and throughput. Because of early detection and diagnostics, advanced pharmaceutical knowledge, early stage disease prevention and appropriate disease treatment are possible. Several biosensors are developed to reach these goals. A biosensor is an analytical device [1, 2] which can identify the presence of specific analytes [3], their concentrations, and kinetics in a sample by transforming the biological signal into an electrical or optical readout. The device combines a biological recognition element [4]
(e.g., antibodies, nucleic acids, enzymes, aptamers) immobilized on a transducer. The ideal properties a biosensor should possesses are high accuracy, high sensitivity, high specificity, ease of operation, cost-effective, high throughput, and mass production easily.
Nowadays, traditional methods for biochemical detection such as tumor marker, blood testing, immunological assays, and urinalysis were widely used. However, it is impossible to apply these methods to real time detection and the detection processes are costly, complicated, and time-consuming. Therefore, a simple, accurate, low-cost, and rapid method for detecting and measuring the biomaterials is highly demanded.
Researchers have proposed many advanced techniques for biochemical detection.
Among them, surface plasma resonance (SPR) [5], enzyme-linked immunosorbent assay (ELISA) [6] (see Figure 1.1), and western blotting [7] are mature and widely available, but these methods require large and expensive instruments in order to integrate many sensing components for high throughput measurement.
A potential method with properties of accurate, sensitive, high specificity, cost-effective, time-saving, high throughput, label-free, simple, and mass production easily is addressed. The candidate is field-effect transistor (FET)-based biosensor. In this thesis, a thin film transistor (TFT)-based biosensor is demonstrated.
Fig. 1.1 Techniques for biochemical detection. (a) Surface plasma resonance (SPR) (b) Enzyme-linked immunosorbent assay (ELISA)
1.2 Introduction of FET-based Biosensors
Field effect transistor (FET)-based biosensors are combined with electrochemical solution, microelectronics transistors, and nano-technology. Because of high sensitivity and selectivity, label-free and real-time measurement, FET based biosensors are getting more and more popular. The structure consists of source, drain, gate and a layer that can sense biomolecules such as protein or nucleic acids. When the target solution is introduced to the system, the analytes will attach to the bio-sensitive layer. The attachment makes an electric field formed by the electrical charges which are carried by biomolecules, which leads to the change of carrier density in FET channel layer.
FET-based biosensors are appropriate to various kinds of detection, such as lung cancer diagnosis by observing threshold voltage change as shown in Fig. 1.2 [8].
Moreover, the semiconductor layers, such as ZnO [9], GaN [9], and IGZO [10], are developed and optimized in order to improve sensitivity. The layers play important roles in their applicability to FET biosensors. To be more specific, here take some FET biosensor research for example: enzyme-coated single ZnO nanowire FET biosensor for detection of uric acid [11], indium-tin-oxide thin-film transistors for detecting AI H5N1 through measuring drain current change as shown in Fig. 1.2 [12], and ZnO-TFT-based biosensor for EGFR detection through measuring drain current change [13], indium gallium zinc oxide (IGZO) thin-film transistors (TFTs) for detecting artificial deoxyribonucleic acid [14].
Fig. 1.2 (a) Optical image of an FET chip and (b) schematic diagram of the multianalyte FET biosensor for detection of multiple tumor markers.
Fig. 1.3 TFT-based biosensor structure immobilized with anti-H5N1 antibodies and attached with negatively charged AI H5N1 virus.
However, the active layer may be damaged by biological solution since oxide semiconductor is sensitive to moisture and oxidant [15]. An extended-gate FET is a robust method to separate electronic devices from the biological solution.
Hence, the IGZO-TFT biosensor with an extended sensing electrode is demonstrated in this research. The extended gold sensing electrode provides an excellent platform for measuring the target biomolecules and also prevents the IGZO-TFT from direct contacting to biological solution effectively.
1.3 Importance of Biochemical Reaction Kinetics
In the biochemical field, analysis of protein-ligand interaction (PLI) raises lots of attentions. Good knowledge of underlying system, i.e., underlying mechanism and kinetic parameters, is indispensable to studies of biological system or applications in drug discovery [16-18]. Because of a drug being effective only when it is bound to its receptor (e.g., proteins) [19], the binding assessment is considered to be important for pharmacological activity. Binding parameters such as the dissociation constant, Kd, were the key to evaluate the drug efficacy in the past decades. Despite many efficacious drug existing on the basis of parameters, recent studies show that the kinetics of drug receptor binding could be more important than affinity in determining drug efficacy. For some drugs, it is not desirable to attain the equilibrium. The dissociation constant measured from experiments no longer well describe the duration of efficacy of a ligand. Instead, the rate of receptor–ligand association and dissociation, generally reflected by association rate constant, ka, and dissociation rate constant, kd, are more appropriate. Also, the clinical differentiation and safety may be influenced by the binding kinetics. The optimization of
binding kinetics helps fulfilling the maximization of a drug's therapeutic index and decrease of drug attrition [20-22].
1.4 Thesis Outline
Chapter 2 “Detection of the mixing reaction situation of the biomaterials”
differentiates biotin, streptavidin, and biotin-streptavidin complex and evaluates the reaction condition of biotin and streptavidin mixing reactions. The fabrication of the TFT biosensor integrated with Y-type microfluidic channel is first introduced. Afterward, diffusion times of both reactant proteins and experiments of the mixing reaction conditions are investigated and discussed. Finally, the non-specific binding situation is verified through the control experiment by applying BSA.
Chapter 3 “Biochemical kinetics investigation” aims at evaluating the binding reaction between the protein and its ligand by extracting the drain current responses.
Lysozyme protein and tri-N-acetylglucosamine (NAG3) ligand are mixed in three ratios for different periods of reaction time. The different mixing conditions make the drain current response vary. By the analysis of drain current responses, the lysozyme and NAG3
binding reaction rate constant and dissociation constant are obtained. The result shows good accuracy of the TFT biosensor setup by comparing with the previously reported values.
Chapter 4 “Conclusions” consists of the summary and the importance of the experiments performed in this thesis.