| Cancer is one of world’s deadliest diseases, and early diagnosis of cancer is crucial in saving patient’s life. However, there are many limitations on the existing method for cancer diagnosis. Effective approach for early diagnosis of cancer is the advanced research topic in biomedical area. Based on these considerations, a new ferrocenyl-substituted carborane (FcCB) has been explored as bio-molecular probe in potential applica-tion of biomolecular identification of cancer cells. The redox properties of FcCB are studied with a droplet electrochemical technique assisted with spectroscopic methods. Furthermore, the mechanism of intermolec-ular interaction between FcCB and macromolecule is investagted using electrochemical and spectroscopic techniques. To take a step further, FcCB was applied in the anlysis of clinical samples to identify leukemia cells from leukocytes. What’s more, surface plasmon resonance (SPR) with a chip, which is in situ function-alized of aminophenylboroic acid, is used in sensing of cancer cells. The combination of electrochemical and SPR technology is sucssessfully utilized in dynamic monitoring of interaction of bio-molecular probe with cancer cells and quantitative evaluation of live cancer cells.The main contributions of the thesis are as follows:1) Electrochemcal and spectroscopic studies of the redox behavior of FcCB and its interaction with bio-macromoleculesThe redox properties of the FcCB in a wide pH range were investigated by using droplet electrochemical technique and spectroscopic methods. The electrochemical results revealed the reversible and irreversible nature of first and second anodic peaks. The pair of reversible redox peaks and a non-reversible redox peak, which were attributed to the ferrocene group and cyclopentene structure. The oxidationwas found to depend strongly on the pH of the medium. The slopes of fitting lines are near to 59 mV per pH unit at the pH interval 3.0-8.0, indicating the oxidation of FcCB to occur with the transfer of the same number of electron and proton.Electrochemical and spectroscopic results revealed that there was a strong interaction between FcCB and hemoglobin. The magnitude of their binding constant was at 104 M-1. The fluorescence of hemoglobin can be quenched by FcCB through the combined static and dynamic quenching mechanism. When concentration ratio of FcCB and hemoglobin was less than 2, the number of binding sites was 1. However, the number of binding sites was 2 while concentration ratio of FcCB and hemoglobin was greater than 2. It is possible that FcCB can combine to the hydrophobic region of hemoglobin, thereby make the conformation of hemoglobin become loose or even depolymerize the tertiary structure of hemoglobin.2) The application of FcCB in specific recognition of cancer cellsFcCB was explored the potential application in specific recognition of cancer cells for monitoring rel-evant biological process or treatment. The low detection limit and board linearity detection range of the reverse peak for FcCB allowed the potential application in sensitive bioanalysis of specific recognition of cancer cells. The peak potential for normal samples negatively shifted to 0.536 V and for leukemia samples positively shifted to 0.553 V. It is evident that there were significant difference of peak potentials of FcCB after incubated with leukemia cells and leucocytes due to the results of statistical analysis. This raises the pos-sibility for the rapid and highly sensitive analysis of clinical samples by using electrochemistry in a droplet system and thus monitoring the process of cancer diagnosis and therapy.3) In situ modification of SPR chips with p-aminophenylboronic acid and its application in biosens-ing of cancer cellsThe SPR chip was in situ modified with p-aminophenylboronic acid at flow injection-SPR system. The decrease in contact angle indicated the increase of hydrophilicity of the SPR chip. Glucose was taken as an example to investigate the saccharide binding ability of APBA functionalized Au chip. The SPR response to glucose binding was concentration dependent. Furtherly, the modified SPR chip was used in biosensing of cancer cells. As HepG2 cells adherent to the surface of the chip, SPR response gradually increased, and finally stabilized and reached a plateau. The SPR response at sample channel was larger than the reference channel. Meanwhile, the detection sensitivity of the sample channel is much higher than the reference channel. The linear range of sample channel was 5×103～1×106 cells/mL with a detection limit of less than 1,000 cells/mL. This non-labelling, real-time, rapid detection method will provide a new analytical tool for biosensing of cancer cells.4) Real-time evaluation of live cancer cells by an in situ SPR and electrochemical studyA new strategy of the combination of SPR and electrochemical study for real-time evaluation of live cancer cells treated with daunorubicin (DNR) at the interface of the SPR chip and living cancer cells. The observations demonstrate that the SPR signal changes could be closely related to the morphology and mass changes of adsorbed cancer cells and the variation of the refractive index of the medium solution. The results of light microscopy images and MTT studies also illustrate the release or desorption of HepG2 cancer cells, which were due to their apoptosis after treatment with DNR. It is evident that the extracellular concentra-tion of DNR residue can be readily determined through electrochemical measurements. The decreases in the magnitudes of SPR signals were linearly related to cell survival rates, and the combination of SPR with elec-trochemical study could be utilized to evaluate the potential therapeutic efficiency of bioactive agents to cells. Thus, this label-free, real-time SPR-electrochemical detection technique has great promise in bioanalysis or monitoring of relevant treatment processes in clinical applications. |
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