Application of an enzyme chip to the micro quantification of L-phenylalanine
Application of an enzyme chip to the micro quantification of L-phenylalanine
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DOI：
10.1016/j.ab.2006.09.006
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发表时间：
2006-12-01
影响因子：
2.9
通讯作者：
Asano, Yasuhisa
中科院分区：
文献类型：
--
作者：
Tachibana, Shinjiro;Suzuki, Masayasu;Asano, Yasuhisa
我们在这里描述了一种新的微量定量方法L-苯丙氨酸浓度的提取物从干血斑通过使用心肌黄酶刃天青系统。为了使荧光酶微板检测法小型化以诊断苯丙酮尿症，开发了固定有His标签融合苯丙氨酸脱氢酶（PheDH）的酶芯片。将His-标签融合的PheDH固定在镍涂覆的载玻片的表面上。微阵列片（8 × 30孔）用聚（二甲基硅氧烷）（PDMS）使用双折射技术制造。将不同类型的微阵列PDMS片固定在固定有His标记的PheDH的镍涂层载玻片表面上，构建了双层结构的酶反应室。为了评估对Ni螯合配体的亲和力，构建并表达了8种His标记的PheDH变体。N端的（His）（6-）和（His）（9）-PheDH变体对Ni螯合配体表现出高吸附率。N端的（His）（6）-PheDH变体对L-苯丙氨酸的Vmax和k（cat）值高于（His）9-PheDH变体，并且发现（His）6-PheDH变体最适合于固定到镍包被的载玻片上。通过刃天青偶联的酶促反应（在0.2 μ l反应混合物中）在酶芯片上形成的荧光显示出良好的线性，并且从滤纸上的干血斑提取的含L-苯丙氨酸的样品的相关系数高达12.8 mg/dl。(c)2006年爱思唯尔公司All rights reserved.
We describe here a new microquantification method of L-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase-resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 x 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)(6-) and (His)(9)-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The V-max and k(cat) values of the (His)(6)-PheDH variant at the N terminus for L-phenylalanine were higher than those of the (His)9-PheDH variant, and the (His)6-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-mu l reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the L-phenylalanine-containing sample extracted from a dried blood spot on filter paper. (c) 2006 Elsevier Inc. All rights reserved.