Fabrication And Characterization Of Novel Biosensors For The Detection Of α-Fetoprotein

The concentration of α-Fetoprotein (AFP) in the mother's blood can be measured for certain fetal defects of the brain and spinal cord. For normal adults, the elevated serum AFP to abnormally high values occurs in several malignant diseases, usually including nonseminomatous testicular cancer and primary hepatocellular carcinoma. Enzyme-linked immunoabsorbent assay (ELISA) is a traditional method for immunoassay of AFP. However, its analytical procedures are complex and time-consuming. Also they are expensive since many bio-reagents are involved. In the recent years, the medical, biological and environmental fields have seen great advances in the development of biosensor due to the unparalleled recognition abilities of biological elements. These biorecognition elements in combination with various transducers have created the rapidly expanding field of biosensors. Electrochemical detection for immunoassay, being safe, economical, simple and easy to handle, and high sensitive, has found wide interest in the recently years.In this thesis, organic compound of Prussian Blue (PB), conductive polymer of polyaniline (PAN), and Silicon-on-insulator (SOI) materials based on Separation by Implantation of Oxygen (SIMOX), have respectively been employed to develop three kinds electrochemical immunosensors for the rapid determination of AFP.Firstly, the PB were employed to develop a rapid method to measure AFP in human serum by use of one-step sandwich ELISA based immunobiosensor with disposable screen-printed carbon electrode. PB modified electrodes display a better electrocatalyst for H₂O₂ reduction than platinum. Since this electrocatalytic process proceeds at a low electrode potential, it is possible to eliminate the effect of interfering species to develop sensors for the determination of H₂O₂ at a low potential. Here, PB was modified on the surface of electrode to catalyze H₂O₂ from the reaction of glucose oxidase (GOD) conjugated with antibody. Compared with typical ELISA, which was high time consuming (2 h), the above methods enabled the detection ofAFP within 30 min through flow injection analysis (FIA). A detection range from 5 ng/ml to 500 ng/ml was obtained. The detection range of the PB modified immunobiosensor is enough for clinical application.Polyaniline and Si material were also used to develop free-label immunosensors for the determination of AFP. Cyclic voltammetry technique was employed to deposited polyaniline on the surface of screen-printed carbon electrode and immobilize antibody (Ab) on the PAN modified electrodes. Scanning electron microscopy (SEM), Fourier infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to characterize the fabrication of biosensors. The fabrication of biosensors and the interaction between antibody and antigen could be studied by impedance spectroscopy. Especially, the thickness of PAN film, the applied potential for immobilization of antibody and that for impedimetric analysis were optimized, which are important for the better sensitivity of impedance monitoring. The resulting immunosensor has a linear dynamic range of 200 to 800 ng/ml of AFP. As free-label immunosensors, the studies are useful for the development of simple, fast and real-time detection methods. Also, the construction of the PAN based immunosensors is simple.Silicon-on-insulator (SOI) wafer produced by SIMOX was employed to develop novel Si ultra-thin-body (UTB) surface sensitive devices for the detection of AFP. An ultra-thin-body (UTB) conductive silicon layer, covered only by the native oxide, was isolated from the bulk wafer through under buried oxide. Devices were built with a combination of five basic processes including photolithography, thin-film growth/deposition, etching, bonding and packaging. Both PPP-type and PNP-type devices were studied by comparison. The results show that The PPP-type Si UTB surface sensitive devices exhibited excellent linear pH-dependent impedimetric response over a large dynamic pH range from 2 to 10, while the PNP-type devices were sensitive to the pH value only at pH of no less than 8.0. The electrolyte concentration didn't interfere with the impedance response of the two kinds of Si UTB devices to pH since the impedance responses to electrolyte concentration and pH were found at different frequency ranges. This property is high appreciated sincethe traditional ISFETs often suffer from the interference of the ionic concentration. Antibody was immobilized onto the sensitive surface with the use of (3-aminopropyl)triethoxysilane (APTES). The response of Si UTB device to the modification and interaction of biomolecules on the surface shows somewhat relationship. Changes of surface potential, the dielectric characters of modified molecules and surface states could influence the space charge region in the sensing layer and influence its conductivity. As a result, the UTB Si based immunosensors could be useful for the development of miniaturized biosensors for the concentration of AFP. Especially, Integration with conventional semiconductor fabrication and integrated-circuit manufacturing technology, many microfabrication processes can be performed as easily on one device as on a thousand. Such batch processing can make thousands of identical devices not subject to the variations present in individually constructed objects. Also as a Si based label-free microdevice, this work would have many advantages with respect to speed, de-skilled analysis and development of multi-analyte sensors and could be useful for the design concept of an integrated chemical microsystem implemented on Si chips for chemical or biomedical application.