| Summary: | 碩士 === 國立清華大學 === 分子與細胞生物研究所 === 92 === Metallothionein (MT) and serum albumin are two important metal-binding proteins, play roles in metal transport, storage and detoxification in organism. In this study, we attempt to establish protein-based biosensors for the detection of metal ions by surface plasmon resonance. In the first part, rabbit MT was immobilized onto a carboxymethylated dextran matrix (CM5 chip). The optimal pH for the immobilization was determined at 4, and the highest interaction temperature was observed at 30oC. The sensor chip binds cadmium, zinc or nickel, but not magnesium, manganese and calcium. Calibration curves for the quantification of metal ions showed excellent linearity. The sensitivity for metal detection was at the micromolar level. The interaction between the metal ions and the sensor chip was significantly influenced by the presence of NaCl, Tween 20 and the pH of the reaction buffer. In the environment containing 1 mM NaCl, the MT chip effectively differentiated cadmium from zinc and nickel. In displacement analysis, zinc and nickel did not affect cadmium-MT interaction. The binding affinity between the metal ions and the immobilized MT follows the order of cadmium > zinc > nickel.
In the second part, bovine serum albumin was immobilized onto a CM5 chip. The optimal pH for the immobilization was determined at 5, and the highest interaction temperature was found at 25oC. The interaction between the metal ions and the sensor chip was significantly influenced by the reaction buffer. The optimal buffer condition used for the analysis contains 1 mM NaCl, 0.005% Tween-20 and 0.01 M HEPES, pH 7.4. Using this condition, a linear calibration curve can be established within the range of 10-8 to 10-4 M for metals. The sensitivity for cadmium, zinc and nickel detection were at the 0.01~0.1 uM. When measuring the solution containing two species of metal ions by the albumin chip, zinc and nickel were able to reduce cadmium-albumin interaction about 20~30%. However, zinc and nickel show an additive in binding to the chip when both metals are mixed together. The binding affinity between the metal ions and the immobilized albumin follows the order of nickel > zinc > cadmium. A procedure was also developed to analyze cadmium content in the serum using the albumin-based sensor chip. Results from our studies show that the protein-based biosensor can be effectively used for the detection and measurement of metal ions in the solution and in biological sample.
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