本研究之目的在於使用一微流體式光學免疫感測系統(microfluidic optical immunosensor system)，其以表面電漿共振(surface plasmon resonance；SPR)之原理為基礎建構而成並應用於人類生長因子(Human growth factors；HGF)之檢測，如：人類胰島素(human insulin)、人類表皮生長因子(human epidermal growth factor；hEGF)等。
首先，為了使與待檢測抗原具高度專一性的抗人類生長因子單株抗體固定以作為偵測用抗體，對於感測晶片表面的修飾是必要的。於修飾過程中首先是活化感測晶片上已事先修飾好的羧基，採用0.4 M EDC (N-Ethyl-N`-[3-dimethylaminopropyl]cabodiimide) 溶液與0.1 M NHS (N-Hydroxysuccinimide) 溶液以1:1(v/v)比例配製成混合溶液並用以處理晶片表面。由於抗體固定在晶片上的方向性會影響感測器對於待檢測抗原的偵測極限以及偵測濃度範圍，在固定偵測用抗體前先使用一種抗體(peroxidase conjugated goat anti-mouse IgG)作為捕捉用抗體。由於捕捉用抗體對偵測用抗體的恆定區(Fc region)具高度專一性，而使得偵測用抗體的抗原結合部位(Fab region)可在感測晶片表面上容易地與待檢測抗原辨識並結合。因為human insulin的等電點(isoelectric point；pI)大約在pH 5.3而hEGF的pI也約在pH 4.7，因此選用醋酸緩衝溶液(10 mM, pH 4.5)作為進行檢測此兩種生長因子時所用之緩衝液。本研究已成它a建立人類胰島素與人類表皮生長因子之SPR免疫感測系統及其濃度檢量線，分別於 human insulin 濃度0.1至100 ng/mL之間以及 hEGF 濃度1至100 ng/mL具有良好的線性關係。 The aim of this study is to utilize a set of microfluidic optical immunosensor system based on surface plasmon resonance (SPR) for determination of human growth factors (HGF) such as human insulin, human epidermal growth factor (hEGF) etc.
First, the modification for sensing chip surface was necessary in order to immobilize the anti-HGF monoclonal antibodies (MAbs) as detection antibodies which were high specific to the target antigens. The first step of modification procedure was to activate the carboxyl group which had previously modified on the sensing chip surface, a mixed solution with 1:1 (v/v) ratio of 0.4 M EDC and 0.1 M NHS was prepared and used to treat the chip. Since the orientation of the immobilized MAbs would affect the detection limit and concentration range of the sensor to the target molecules, using a kind of antibodies, peroxidase conjugated goat anti-mouse IgG, as capture antibodies which were immobilized prior to the detection antibodies. The capture antibodies were high specific to the Fc region of the detection antibodies and it could facilitate the Fab portion of the immobilized detection antibodies on the sensing chip surface to easily bind the target antigens. Because the isoelectric point (pI) of human insulin is about pH 5.3 and hEGF is also about pH 4.7, an acetate buffer (10 mM, pH 4.5) was chosen as sample buffer for the detection of human insulin and hEGF in the SPR sensor system.
In this study, we had successfully established two kinds of HGF immunosensors, the human insulin and hEGF SPR immunosensor systems. The good linear relationships were obtained between resonant unit (RU) and concentration range from 0.1 to 100 ng/mL for human insulin and 1 to 100 ng/mL for hEGF , respectively.
