外加電場對蛋白質排列方向與之後生物晶片效率之影響

Abstract

本文探討一新式製程，將能增加生物晶片(生物感測器)之效率與敏銳度。本研究嘗試於固定生物分子的過程中外加一8000(V/m)之電場，藉以控制生物分子之方向性，並以不同的電場方向改變生物分子固定於基材時的角度，增加生物分子之活性區暴露於晶片表層的機率，進而提高生物晶片效率，可望發展一套有更高效能之生物晶片。 研究中利用原子力顯微鏡(AFM)測量蛋白質間結合力。此方法以抗癌胜肽藥物CB1a固定於原子力顯微鏡之探針，抗癌胜肽藥物CB1a之免疫球蛋白抗體固定於矽基材，並以兩者間結合力大小差異判斷裸露在晶片表層之活性區比例多寡。 文中詳述測量方式，包括可調式電場之架設，力量加載率，平均結合力大小與晶片上免疫蛋白球之方向預測。在施予固定力量加載率(r = 1.44 x 104 pN/s)的狀況下，使用外加270度電場所固定CB1a探針測量固定CB1a免疫球蛋白之晶片，可發現在製程中外加22.5度電場時所製作之試片分別有最高結合力598.67pN。同樣的，在使用無外加電場所固定CB1a探針測量固定CB1a免疫球蛋白之晶片時，在22.5度電場時所製作之試片亦有最高結合力729.09pN。亦使用無外加電場所固定之Protein A探針測量固定Protein A-Anti免疫球蛋白之晶片時以45度電場所製作之試片有最高結合力6424.19pN。因而推測這兩組製程條件所製作之晶片，其上方的免疫球蛋白的活性區有較多比例裸露於表層，且呈現正確的結合角度，故有較大的結合力量，此方法可提升藥物篩選晶片之敏銳度，使其應用更廣泛。 本研究亦嘗試測量抗過敏藥物Cyn1324對活體T細胞與CD80之影響，以及免疫抑制劑(cynarin1324)阻斷兩者結合的結果，結果顯示Tcell/CD80間的平均分離力由1.537nN降至0.903nN，降低41.25%，此方法亦可直接量測藥物對活體細胞之影響，結果顯示Tcell/Bcell間的平均分離力由0.42nN降至0.34nN，降低19.05%。因此，我們可以利用AFM作為藥物篩選之新方法。

Proteins immobilized on chip (protein bio-chip) have been considered as one of main biotechnologies popularly applied in medical diagnosis and life science. Optimization of the chip efficiency is an expectation. The optimum protein orientation to the best position for undergoing protein-protein interaction may be pursued. In this work, immobilization of protein on chip under the influence of external electric field (EEF) was introduced. A device for producing EEF with adjustable angle from 0o to 360o was built. By using atomic force spectroscopy (AFM) providing the binding force measurement, this EEF device was applied to estimate the efficiency of protein-protein interaction. A custom peptide CB1a immobilized on AFM tip (afm-CB1a) with its antibody antiCB1a immobilized on chip (chip-antiCB1a) was used as an example for their binding tests under EEF. Results showed that the unbinding forces between afm-CB1a and chip-antiCB1a were in average of 337.35 pN (without EEF) and 729.09 pN (with EEF at the best angle 22.5o). Protein A immobilized on AFM tip (afm-Protein A) with its antibody antiProtein A immobilized on chip (chip-antiProtein A) was used as an example for their binding tests under EEF. Results showed that the unbinding forces between afm-Protein A and chip-antiProtein A were in average of 1070.03 pN (without EEF) and 6424.192 pN (with EEF at the best angle 45o). Consequently, the effectiveness of protein-protein interaction was increased to over 200% under EEF as compared with the conventional one (without EEF adjustment). The best angle pursued by using adjustable EEF for improving the efficiency of protein bio-chip was therefore proposed and could be applied to protein industry and related fields in the future. The study also attempts to measure the impact of allergy Cyn 1324 of Tcells and CD80, The results showed that the separation force between Tcell and CD80 by 1.537nN fell to 0.903nN, reduced 41.25%. This method also can measure the impact of drugs on living cells, The results showed that the separation force between Tcell and Bcell by 0.42nN fell to 0.34nN, reduced 19.05%. So we can use the AFM as a new method of drug screening.