經修飾過竹炭作為多功能免疫分析材料應用於人類血清之研究

Abstract

免疫分析乃是利用抗原與抗體之間具有特異性的親和力，來偵測與定量出檢體中特定物質的濃度。免疫分析已被廣泛地運用在許多醫藥分析領域，如疾病的診斷、血液中藥物濃度的監測及臨床藥動學研究等等，利用放射性、螢光或是酵素顯色來測定抗原或是抗體的濃度。然而，免疫分析不但需要特殊的設備與儀器來測定訊號的強度，因此費用昂貴與耗時，並且容易產生非專一性結合殘留(Non-specific binding)，影響檢驗的準確性。 因此，本研究之目的著眼於降低免疫分析的實驗費用與非專一性結合。利用竹炭表面多孔隙且方便改質的特性，將其修飾成為能夠降低非專一性結合的的免疫分析材料，並且設計一套免疫分析裝置(Immunoassay device)，以減低實驗的繁雜度並降低實驗的費用。 本研究選用竹炭與聚氧乙烯二胺(Polyoxyethylene bis-amine，PEGBA)當做主要材料，乃由於竹炭為多孔性之炭化材料，能夠增加表面積，而且竹炭結構具有豐富的碳雙鍵(C=C)，方便進行表面改質。竹炭利用酸處理使得表面帶有羧基(Carboxyl group)，可與帶有胺基(Amino group)的高分子以穩定的醯胺鍵(Amide bond)接枝。本研究將聚氧乙烯二胺接枝在竹炭的表面後，聚氧乙烯二胺另外一端未鍵結的胺基可以與抗體的C端進行結合，並且不影響抗體與抗原結合的N端，能夠保持抗體與抗原間的專一性與親合力。此外，此高分子的結構更能夠造成極大的空間結構障礙，產生表面泳動效應，使得非專一性的結合或吸附降至最低，增加免疫分析的準確效率。 竹炭經由酸處理並與高分子接枝進行表面修飾後，進一步能夠結合抗體，成為多功能免疫分析的材料。本研究利用設計出的免疫分析裝置，以經過表面修飾的竹炭做為多功能免疫分析的材料，與市售的磁性奈米粒子比較，具有減少非專一性結合並降低實驗費用與時間的功用。

Immunoassays are bioanalytical methods which detect and quantify the concentration of a certain analyte through the specific affinity between an antigen (analyte) and an antibody. Immunoassays have been widely used in many important areas of pharmaceutical analysis, such as diagnosis of diseases, therapeutic drug monitoring, clinical pharmaco¬kinetic, etc. Immunoassays are achieved by measuring the label activity (e.g. radiation, fluorescence, or enzyme) of antigens or antibodies. However, immunoassays often require special apparatus and instruments, which are expensive and time-consuming. Furthermore, signal detection of immunoassays is also easily disturbed by non-specific bindings. Therefore, the purpose of this study focuses on reducing experimental expenses and non-specific bindings of immunoassays. Utilizing its porous structure and the easily modifiable surface, bamboo charcoal can be modified to become a suitable material for immunoassays. In addition, we designed a new immunoassay device in order to reduce the complexity and expenses of the experiment. There are many advantages with using bamboo charcoal and polyoxyethylene bis-amine (PEGBA) as the main experimental materials. First, the porous surface of bamboo charcoal gives rise to a large surface area. Also, the abundant carbon double bonds on surface of bamboo charcoal are easy to be modified. By treating bamboo charcoal with acid, the surface will become full of carboxyl groups, which then can bind with the amino groups of PEGBA through amide bonds. While one end of PEGBA is grafted on the surface of bamboo charcoal, the other end is able to bind with the C-terminus of antibodies, leaving the N-terminus available to antigens. Moreover, the special structure of PEGBA can lead to a waving effect, which can cause steric repulsion and reduce non-specific bindings. This method can improve the accuracy of immunoassays. In conclusion, bamboo charcoal can be modified through carboxylation and PEGBA grafting. Modified bamboo charcoal can then be successfully conjugated with antibodies and become a multiplexed immunoassay material. Comparing with commercial magnetic nanoparticles, our device is more effective in reducing non-specific bindings and saving experimental expenses and time.