利用硅烷法將ssDNA 固化T方向切割、3. 58MHz 和10MHz 石英晶體金電極上,構建了壓電DNA傳感器,建立了快速檢測葡萄球菌腸毒素B(SEB) 基因的方法。優化了固定SEB 基因的條件,并用動態法、靜態法和斑點雜交法進行了固化效果觀察。結果表明:該傳感器2 h 后,有較好響應;對不同長度DNA 探針固化效果進行比較,169 bp (即169 個堿基) SEB 探針固定和雜交效果較好;利用0. 5mol/ LNaOH進行電極洗脫,時間為45min ,電極可以再生,且傳感器可以重復使用3 次;利用1. 2mol/ L NaOH和1. 2mol/ L HCl洗滌電極,該電極可多次再利用;固化好的電極于4 ℃條件下可以保存三周之久。
關鍵詞: 壓電式DNA 傳感器; 葡萄球菌腸毒素B ; 3 - 氨基丙基三乙氧基甲硅烷
Abstract : A rapid method has been developed to monitor Staphylococcal enterotoxin B(SEB) gene by using piezoeletric DNA sensor system. AT2cut crystals ,with a based resonant frequency of 3. 58MHz and 10MHz ,were modified by 32 aminopropyltriethoxysilane(32APTES) treatment and then single stranded SEB gene probe was electro2blotted onto the
surface of crystal electrodes. The optimization condition of immobilization for piezoeletric DNA sensor was researched. Im2 mobililzed efficiency was observed by active methods , passive methods and dot hybridization method. There is better re2 sponse of the piezoelectric DNA sensor after DNA hybridization for 2 h. Hybridization effect of 169 bp SEB gene probe was better comparing with the other different length of SEB gene probe. The sensor could be repeatedly used ,the complemen2 tary ssDNA of the SEB DNA probe could be removed from the crystal by washing with 0. 5mol/ L NaOH denaturant. After the hybridized electrode was immersed in 1. 2mol/ L NaOH and then 1. 2 mol/ L HCl ,all substances could be removed
from the crystal surface ,and the quartz crystal could be reused for many times. The 32APTES2coated ssDNA electrode was stable for over three weeks when stored at 4 ℃.
Key words : piezoelectric DNA sensor ; staphylococcal enterotoxin B (SEB) ; 32aminopropyltriethoxysilane (32APTES)