- 學位論文
多反應區域鎳金屬蛋白質晶片之研究
Development of Nickel Coated Protein Chips with Multi-Reaction Wells
摘要
近二十年來在生物領域的學術研究與著作中，生物分子相關的操作技術與方法逐漸備重視。從2000年開始，也陸陸續續有一部份的研究著重在證實這些新穎生物技術的可行性。新穎的分子檢測技術-生物晶片，猶如生物科技領域的一盞名燈日受矚目，更在近幾年，有許的研究方向是利用生物晶片做為主核心技術進而拓展其應用範圍，最終以臨床檢測應用為目標。生物科技的不間斷的突破促使許多新的且誘人的應用於分子生物、遺傳學、免疫學、生物學以及藥物醫學上。 生物晶片技術在蛋白質體研究中扮演著蠻重要的腳色，不論是在科學研究或是醫療檢測上，均能發揮其強大的功能，因此正適合我們深入研究在本研究，我們想製作出一種可以同時處理多個樣品的生物晶片。因此本研究實驗設計分兩個階段完成，並陸續地呈現完整的晶片組織架構。第一個階段為研發出鎳金屬蛋白質晶片表面，藉由鎳離子可以特定的抓取Histidine 蛋白且是具有方向性的理論。再利用田口方法透過直交表探討8種電鍍因子，且每個因子具有2-3個條件進而電鍍沉機製作晶片，之後利用免疫反應來獲得晶片表面固定蛋白之實驗結果，並且由望大法則的田口分析來探討蛋白質晶片表面最佳化的沉積條件。 第二個階段為可剝離式多反應區域薄膜生物晶片的開發。多反應區域薄膜生物晶片是利用聚二甲基矽氧烷(PDMS)作為此晶片的一部份，並且使同一晶片上具有多個生化反應區域。這個多反應區域的結構利用具有良好物理性吸附能力的PDMS所製作，進而貼附於晶片上型成可剝離式的反應空間。此多反應區域薄膜可以同時提供多個樣品在相同的生物晶片上於不同的區域進行生物反應，在實驗之後可以剝離此PDMS薄膜而不會損壞到實驗結果。其中有利用XPS以及洩漏實驗探討其薄膜成型的物理化學特性 最後本實驗的多反應區域鎳金屬蛋白質晶片的組成藉由上敘之兩項研究所完成，未來以蛋白質晶片的研究發展來帶動其在其他疾病診斷跟治療上的應用，藉由蛋白質檢測技術的發展，應用在在臨床醫藥治療跟診斷上更為精確快速，降低疾病所帶來的苦痛與恐懼，追求人類更優質的健康福祉。
並列摘要
In the last two decades an increased attention has been given to molecular biotechnology. The field of biotechnology has undergone many fluctuations and shifts over the years. From the beginning of 2000’s, a number of attempts blossomed to validate the feasibility of novel bio-techniques and the development of biochip is one of the branches. A wealth of researches regarding the characteristics of biochips and their related clinical applications can be found in recent years. The continuous improvements in biotechnology have led to many new and fascinating applications in biomolecule, genetics, immunology, biology, and medicine. In this study, we developed a protein chip whose surface can perform testing of multiple samples in the same chip simultaneously. Therefore, two steps of experimental designs were carried out sequentially. First, the experimental design was aimed to manufacture a protein chip with metallic coated surface. For the traditional ion-metal affinity chromatography, nickel is used to capture the histidines (His) protein to make up a rapid purification system from the organism lysate. Based on this principle, a nickel-coated substrate was manufactured by electroplating technology in order to design a planar protein microarray chip. The Taguchi method was adopted to determine the optimal process of the nickel coated chip using L18 orthogonal array. Eight plating factors with 2~3 levels for each factor were considered. The optimal coating condition was determined by the Taguchi method to get rid of the traditional experiment approach of changing one variable at one time. We utilized the immunoassay to investigate the properties of the protein chip, and the signal was detected by confocal scanner. The result showed the nickel coated slide had better specific binding ability than nitrocellulose coated slide. Afterward a membrane with multi-reaction wells was investigated. The membrane of multi-reaction wells was fabricated from polydimethylsiloxane (PDMS) as a part of biochip so that splitable incubation chambers were provided on a single chip. The conditions of the forming temperature, time and mixing proportion of the materials were investigated in order to acquire optimal physical absorption with the surface of chip substrate. To verify the properties of the multi-reaction wells, immunoassays were performed by the alpha-1-foetoprotein (AFP) antigen sandwich experiment. Attaining excellent physical absorption ability will allow cross contamination or interference between different samples on the same biochip to be avoided. Thus, the merits of nickel protein chip with multi-reaction wells include not only promoting effective surface usage but also providing multiple-sample experiments.