利用石英晶體微天平探索人體C反應蛋白與具磷酸膽鹼官能基表面之交互作用

Abstract

心血管疾病是全球的主要死亡原因之一，為了在發病前有更充裕的時間面對心血管疾病，我們可以妥善運用和心血管疾病有關的生物指標，評估人體罹患心血管疾病的風險，並提早投入醫療資源，防患於未然。人體C反應蛋白(C-reactive protein, CRP)就是一種用於評估心血管疾病的生物指標。根據研究，在有鈣離子的環境下，CRP和磷酸膽鹼官能基(phosphorylcholine, PC)會選擇性吸附，此外，PC官能基是極具親水性的兩性離子，可以抵抗非特定性吸附蛋白質的沾粘，以維持生物檢測器的功能。因此，我們將PC官能基和3,4-乙烯二氧基噻吩導電高分子(3,4-ethylenedioxythiophene, EDOT)結合，並用電化學聚合的方式，聚合成具PC官能基的導電高分子薄膜，再藉由調控電聚合溶液的高分子單體比例，來調整高分子薄膜表面的PC官能基密度，而這些帶有不同官能基密度的表面，將會對CRP有不同的偵測極限和範圍。CRP和PC官能基的特定性吸附，將由石英晶體微量天平-耗散偵測系統(quartz crystal microbalance with dissipation, QCM-D)監控，藉由石英震盪頻率的改變，來估量CRP吸附在具PC官能基表面之質量變化。經由QCM的偵測，由單體組成是100% PC官能基的EDOT單體溶液電聚合而成的表面有最好的抗沾黏性質，並可以吸附最多的CRP，偵測範圍落在0.25~5 mg/L，正好符合「高靈敏人體C反應蛋白試驗」(high-sensitive CRP test, hs-CRP teat)的範圍(1~3 mg/L)。霍夫梅斯特序列(Hofmeister series)是探討蛋白質在水中的狀態和水溶液中陰離子的交互關係；根據霍夫梅斯特序列，在我們的系統中導入不同種類和濃度的陰離子，透過改變水溶液中的陰離子種類和陰離子濃度，再藉由從QCM觀察到的蛋白質吸附量，來討論不同的陰離子對高分子表面性質和蛋白質吸附的關聯性。

Cardiovascular disease is the main cause of death in the recent years. Being able to prevent the sudden death caused by the disease, we could get help by using the biomarker to detect the cardiovascular disease and level the risk of cardiovascular disease in advance before the health turning worse. The detection of the human C-reactive protein (CRP) is a potential method to know the risk of cardiovascular disease in advance. It has been found that CRP has strong affinity to phosphorylcholine (PC) in the solutions contained calcium ion. Moreover, phosphorylcholine is one kind of zwitterions which usually provides non-fouling surfaces and prevents the non-specific binding of proteins. To demonstrate this concept, a phosphorylcholine-functionalized 3,4-ethylenedioxythiophene (EDOT-PC) was used to create conducting PEDOT films with PC functional groups on the surfaces. The density of PC groups can be fine-tuned by changing the composition of monomer solutions for the electropolymerization. By changing the surface density of PC groups, the conducting polymer surface can be applied for different linear ranges and detection limits for the CRP detection. The specific interaction of CRP with PC groups was determined by quartz crystal microbalance with dissipation (QCM-D). By monitoring the change of quartz frequency, the change of mass during protein binding can be estimated. The detection range is 0.25~5 mg/L which fit the range of “high sensitive CRP test” (hs-CRP test). Hofmeister series discuss the relation between the conformation of the protein and the kind of the anion. According to the Hofmeister series, the different salt ions can change the water structure and the protein property in water. Therefore, we introduced the different salts into our system to discuss how these anions affected the surface property and protein binding.