| Summary: | 碩士 === 中國文化大學 === 生物科技研究所 === 95 === Staphylococcal enterotoxin B (SEB), is one of many toxins produced by Staphylococcus aureus. Not only does this 28.4 kDa toxin can cause food poisoning, it is also a possible biological weapon. Since the terrorist attack occurred in New York city on September 11, 2001, the threat of Bioterrorism overwhelmed the entire world and thus, the need to detect SEB in food and in human body is inevitably raised. Due to its high disease causing rate and infection ability, SEB in food, water or air would be a great danger to the human health, safety of food supply chain, and economy. Therefore, creating a fast, sensitive, and specific method to detect staphylococcal enterotoxin B in food, water, blood, urine, or tissue is highly required.
The experiments start with cleansing the gold surface with Piranha solution. After the surface being cleaned, self-assembled monolayer (SAM) is adsorbed onto the surface, working as linkers to link the antibodies. When the anti-SEB is linked to the SAM, SEB is ready to be detected. As the immobilization method of SAM has become popular in the recent years, this research will continue to investigate ways of SAM modification in order to increase the sensor’s sensitivity. By altering the physical property of this linker layer using 11-mercapto-1-undecanol (11-MUOH), 6-mercapto-1-hexanol, (6-MCH) and 16-mercapto-1- hexadecanoic acid (16-MHA), steric hindrance which can occur between the adjacent antibodies or between the antibodies and the linker layer itself can be reduced, and the sensitivity of the sensor can then be improved. First of all, SAM with COOH functional group（16-mercapto-1-hexanol, 16-MHA）which can bind the amino group of the antibody was mixed with SAM possessing OH function group（6-mercapto-1-hexanol, 6-MCH, or 11-mercapto-1-undecanol, 11-MUOH）which cannot bind the antibody in different ratios such as 1:20, 1:10, and 1:3 to test the angle shift they each can cause. Later on, after the best ratio is determined, the best spacer length of the OH group was investigated between 6-MCH and 11-MUOH. Secondly, when the most suitable ratio and length of SAM are both determined, unnecessary segments of antibodies were cut off (Fc) before they were put on top of the SAM, leaving only F(ab’)2 and/or F(ab’) fragments in order to further reduce any possible steric hindrance.
The results show that the best mixed SAM for SEB immunosensor is 10 mM 16-MHA/6-MCH in 1:10 ratio. When immobilizing full anti-SEB, the sensor has a good linear relationship（y = 170.3x；R2 = 0.9803）between SEB concentration 0.01 to 1 μg/ml. When immobilizing F(ab’) fragment, the sensor has a higher sensitivity (y = 211.35x), and a linear relationship between SEB concentration 0. 1 to 1 μg/ml (R2 = 0.9877). Detecting SEB in milk has a linear relationship in concentration 0.1 to 1 μg/ml (R2 = 0.9886). Using 0.1 M HCl as the regeneration solution for the first time leads to 75.09 % of the original detection ability; the second time results in 69.77 % of the original detection ability.
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