本研究主要為製備金奈米探針，做為免疫沉澱法的工具，針對乳癌
細胞MCF-7 中的雌激素受體阿爾法(estrogen receptor alpha; ERα)進行免疫沉澱抓取實驗。首先在金奈米探針製備方面，先用2-iminothiolane 修飾蛋白質G，使蛋白質G 上的胺官能基硫醇化，再利用硫醇化的蛋白質G 對金奈米粒子表面進行自組裝修飾，使得金奈米粒子表面修飾一層硫醇化蛋白質G。因為蛋白質G 可與抗體的結晶片段（fragment crystallizable;Fc）形成鍵結，因此便可在硫醇化蛋白質G 表面再修飾一層具有方向性的雌激素受體抗體(anti-estrogen receptor alpha; anti-ERα )，製備完成的探針應用於免疫抓取。鍵結在探針上的蛋白質G 與抗體之間需再經過共價交聯，使得抗體不會因為從探針上脫離而造成後續質譜偵測的干擾。
製備條件經系統性最佳化，最後定量出約1.4 μg 蛋白質G 以及4.52 μg 抗體在100 μL 金奈米粒子探針上。所合成好的金奈米探針與傳統免疫沉澱法所用的瓊脂糖凝膠珠體進行專一性抓取能力比較，結果顯示金奈米探針擁有可比凝於瓊脂糖凝膠珠體的專一性與回收率，值得注意的是，金奈米探針在抵抗非專一吸附能力上有更好的效率，這在質譜偵測上尤其重要。最後金奈米探針成功從500 μg 乳癌全細胞中利用蛋白質體結合質譜的方法分離出ERα。

In this study, we fabricated and characterized gold nanoparticle (AuNPs)-based immuno probes and further applied the synthesized probes for imunoprecipitation against estrogen receptor alpha (ERα) in MCF-7 breast
cancer cells. We first utilized 2-iminothiolane to modify the amine groups of protein G in order to form a thiol linker, which could subsequently bind to the surface of AuNPs. Anti-ERα antibody was then bound to the probe
surface via protein G through its Fc domain to form well orientated antibodies on the probe for immuno-capturing. The immobilized antibodies were further cross linked with protein G to minimize the leakage of antibodies that
could interfere the subsequent detection by mass spectrometry (MS). The fabrication conditions were systematically optimized and the final probe was
characterized to have 1.4 μg protein G and 4.52 μg anti-estrogen receptor alpha on 100 μL AuNP. Compared to agarose beads which are conventionally used in immunoprecipitation, our data showed that AuNPs-based probe has comparable specificity and recovery rate but possesses higher resistance against non-specific binding, which, however, is very crucial for MS detection. Finally, we demonstrated that from as little as 500 μ g total proteins of MCF-7 whole cell lysate, ERα could be successfully isolated and identified by proteomics approach using MS
detection.

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