Development of peptide-immobilized TiO2 chip for monitoring of intracellular protein phosphorylation signals.

研究了一种利用氧化钛板检测蛋白激酶活性的肽阵列。首先，采用溶胶-凝胶法制备氧化钛板。该板具有相对平坦的表面和较高的光致表面润湿性转化的亲水性。此外，我们还成功地利用低压干燥和紫外线照射对表面润湿性进行了可逆控制。其次，合成了聚丙烯酸-蛋白激酶A （PKA）底物肽配合物（PAA/PEP）。通过偶联酶分析，我们证实了PAA/PEP被PKA磷酸化。最后将荧光聚丙烯酸（F-PAA）固定在氧化钛板上，并在水中进行超声处理。洗涤后，F-PAA仍留在盘子上。这表明F-PAA与氧化钛板之间形成了键合。在此基础上，建立了利用氧化钛板开发肽阵列的基础技术。

A peptide array using a titanium oxide plate for detection of protein kinase activity has been investigated. First, the titanium oxide plate was prepared by sol-gel method. The plate has relatively flat surface and high hydrophilicity rate of photo-induced surface wettability conversion by UV irradiation. Furthermore, we succeeded that the surface wettability was controlled reversibly using hypobaric drying and UV irradiation. Next, complex with poly(acrylic)acid and protein kinase A (PKA) substrate peptide (PAA/PEP) was synthesized. We confirmed that PAA/PEP was phosphorylated by PKA, by coupled enzyme assay. Finally, fluorescenced poly(acrylic)acid (F-PAA) was immobilized on the titanium oxide plate, and the plate was sonicated in water. After washing, F-PAA still remained on the plate. It suggested that a bonding is formed between F-PAA and titanium oxide plate. From above results, we would establish the basis technique to develop the peptide array using the titanium oxide plate.