為了在生物感測上發展螢光釕複合物，我們合成一個新穎的硫基反應化合物， 4-bromophenanthroline bis-2,2`-dipyridine Ruthenium bis (hexafluorophosphate) 。將完成的釕複合物接合至硫修飾的蛋白質 G 上形成螢光免疫試驗的通用試劑。並且透過SDS 凝膠電泳確認釕(II)-蛋白質 G 複合體的成功接合。釕(II)-蛋白質 G 複合體在452 奈米激發波長下可以得到602 奈米的放射波峰，表示釕(II)-蛋白質 G 複合體在完成接合後仍保有與釕複合物相似的螢光特性。為了測試接合物在生物感測上的可用性，我們執行免疫球蛋白 G 結合試驗。其結果表現出釕(II)-蛋白質 G 複合體有能力接至免疫球蛋白 G ，並且愈多交聯物修飾就有愈高的接合效率。在測定釕(II)-蛋白質 G 複合體在螢光免疫試驗的可行性中，發展出利用複合體與抗組胺酸抗體去偵測組胺酸標記蛋白的方法。結果顯示組胺酸標記蛋白在不同劑量被下成功的偵測出來，顯示出蛋白質 G 複合體可以當作免疫定量試驗中的通用螢光試劑。To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2`-dipyridineRuthenium bis (hexafluorophosphate). The synthesized Ru(II) complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II)-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The emission peak wavelength of the Ru(II)-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II) complex, indicating that Ru(II)-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG) binding assay was conducted. The result showed that Ru(II)-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II)-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II)-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays.
