物理與化學角度觀察官能化及奈米結構導電高分子界面與水及生物分子之間相互作用

Abstract

生醫傳感的發展高度依賴於生物分子與介面之間複雜的交互關係。這些交互作用將影響傳感器的設計、選擇性和穩定性，進而影響傳感器的效能。而這些作用機制需要考慮到非常表層的水，或是因奈米結構而產生的表面力學性質改變；本研究中，我們將透過物理及化學的方法去剖析材料表面、生物分子與環境之間的關係。 因為具有良好的生物相容性、接近生物組織的機械性質、與在水中穩定的導電等優勢，導電高分子（conducting polymer, CP）中的聚（3,4-乙烯二氧噻吩）（poly(3,4-ethylenedioxythiophene), PEDOT）被廣泛應用在電化學生物傳感當中。PEDOT可以靈活的官能化與控制表面形貌，形成多變的奈米結構來調節生物分子與材料間相互作用。具有磷酸膽鹼基的PEDOT (PEDOT-PC) 是衍生於細胞膜上的親水磷脂質端，賦予導電高分子抗沾黏的特性並防止非特定性生物分子的吸附以維持平台的效率和可靠性。 第一節將探討PEDOT 的奈米結構及官能化與生物分子的交相作用。透過調控EDOT-PC在電化學溶液中的組成比，可以獲得不同抗沾黏程度的表面，而改變電聚合條件也可以得到不同的表面結構。利用石英晶體微天平與耗散量測（quartz crystal microbalance with dissipation, QCM-D）來測量帶有不同等電點蛋白質與平台間的作用力，等電點由低到高為牛血清白蛋白（bovine serum albumin, BSA）、溶菌酶（lysozyme, LYZ）和細胞色素c（cytochrome c, cyt c），而為了連結到生物端的應用，同時也加入了人類纖連蛋白（fibronectin, FN）；此外為了觀測界面上細胞的貼附，我們選擇骨肉瘤細胞（MG-63）、源自子宮頸癌細胞的海拉細胞（HeLa）以及從小鼠胚胎分離的纖維細胞NIH/3T3作為三種細胞源。經由分析細胞數量與細胞核大小可以得知，當表面上有足夠的 PC可以顯著改變細胞貼附的反應，包括附著量的減少、細胞核形態的改變和收縮，最後引起細胞凋亡。這個研究可以了解細胞如何與不同形貌的 PEDOT 相互作用以及抗沾黏表面對細胞型態的影響。 第二部分，我們透過膠體微影技術（colloidal lithography, CL）製備出具有規整奈米結構的導電高分子表面。在此研究中，我們採用原子力顯微鏡（atomic force microscope, AFM）的PeakForce Tapping與力曲線陣列（force−volume）模式來探討這些表面的力學性質，這個技術可以得到精確的力－距離二維陣列並同時提供力的變化和樣品表面形貌，每一點都有獨立的力曲線被記錄下來。接著，我們也利用QCM-D研究材料表面蛋白質吸附行為，除了BSA、LYZ和cyt c作為非特定性吸附蛋白質外，同時還加入了C反應蛋白（C-reactive protein, CRP），該蛋白質表現出對PC特定性結合的特性，以進一步觀察奈米結構對生物分子吸附的效應。AFM 結果顯示出奈米結構的表面會誘發更強的吸附力，提高一般表面的蛋白質吸附；相反的，具有雙親性官能基的PEDOT-PC表面呈現出非常微弱的相互作用。從蛋白質吸附的結果來看，奈米結構削弱了PEDOT-PC的抗沾黏效果，這與水接觸角的結果相符，說明規則的奈米結構可以減弱PC官能基保留住水層的能力。這項研究的結果提供更多物理性的探討，同時評估了奈米結構對抗沾黏的影響。 論文的第三部分專注在探討水分子在導電高分子中的動態行為。材料界面上水分子的表現在各個科學領域中扮演著關鍵角色。為了量測PEDOT及其衍生物中的水分子的狀態，我們採用了原位傅立葉變換紅外光譜（in−situ FT-IR）和差示掃描量熱法（differential scanning calorimetry, DSC）作為分析方法。DSC能夠量化材料中的三種不同狀態的水：非凍結水（non-freezing water, NFW）、中間水（intermediate water, IW）和結晶水（free water, FW）。霍夫梅斯特級數提供了離子與水親和力的排序。過氯酸根的存在會提高水構成四面體結構的能障，屬於會“破壞水結構”的陰離子類別。相反的，“穩定水結構”的陰離子，如硫酸根，表現出較高的水合傾向，並與周圍的水分子形成穩定的結構。通過電位控制結合紅外光譜，我們監測了水在高分子中OH伸展（stretching）的變化與吸附/脫附的現象。對於富含羥基的PEDOT-OH，硫酸根離子明顯影響了吸附水，因為羥基與水有較強的相互作用。然而，過氯酸根離子對PEDOT-OH中的水分子的行為影響不大。對於包含雙親性的導電高分子PEDOT-PC來說，作為有效的抗沾黏表面，非凍結水和中間水普遍存在PC官能基當中，在暴露於鹽類時保護高分子內的水分子而不引起脫水。OH伸展的峰形的變化可以用高斯函數來分析，並可以解析不同的結構的水在光譜顯示的獨特結果。在此章節中，我們闡述了水分子的結構特徵並展示了可藉由電位控制來調節離子、導電高分子和水之間的相互作用。

The development of biomedical sensing highly relies on the complex interactions between biomolecules and interfaces. These interactions affect the design, selectivity, and stability of sensors, which in turn influence the efficiency and sensitivity. These mechanisms need to consider factors such as the interfacial water or changes in surface mechanical properties due to nanostructures. In this study, we used physical and chemical methods to analyze the relationships between the interface of material and biomolecules. Owing to the advantage of biocompatibility and intrinsic conductivity, the poly(3,4-ethylenedioxythiophene) (PEDOT) interface is promising for electrochemical biosensing and modulating biomolecules−interface interaction. As a result, PEDOT has been used as a conductive substrate for organic electrochemical devices. With the flexibility for functionalization and morphology control, derivatives of PEDOT can obtain multipurpose applications. The functionalization of PEDOT with phosphorylcholine (PC) groups (PEDOT-PC) mimics the hydrophilic headgroup found in cell membranes and gives the coating exceptional antifouling ability. Antifouling plays a critical role in maintaining the efficiency and reliability of various biomedical applications to prevent the binding of undesirable biomolecules in recent biotechnology. The first part includes a thorough investigation into how biomolecules interact with different types of PEDOT concerning surface functionalization and structure. We controlled the structure of PEDOT platforms and gradually increased the degrees of antifouling by controlling the electrochemical conditions. The protein binding behavior was measured by quartz crystal microbalance with dissipation (QCM-D) beforehand. We utilized a range of proteins, including bovine serum albumin (BSA), lysozyme (LYZ), and cytochrome c (cyt c), for their distinct isoelectric points to evaluate the binding affinity to PEDOT films. Moreover, a multifunctional adhesive glycoprotein protein, fibronectin (FN), was also included. We also evaluated the cell adhesion behavior on PEDOT interfaces. MG-63 osteosarcoma cell, HeLa derived from the cervical cancer cell, and fibroblast NIH/3T3 were chosen as three cell lines. By assessing the number of cells and measuring the size of their nuclei, sufficient PC contents dramatically changed the adhesive response, including the attached number, morphologies, and cell nuclei shrinkage. Finally, over 70% of the feeding ratio of PEDOT-PC can cause cell apoptosis. In the next part, we created a precisely defined PEDOT nanopattern enriched with antifouling PC moieties (PEDOT-PC) in comparison to PEDOT functionalized with hydroxyl groups (PEDOT-OH) to explore the nanostructure effects at the interfaces. We obtained well-defined nanopatterned PEDOT films using a colloidal lithography (CL) approach. We employed atomic force microscopy (AFM) to investigate the adhesion effect of periodic nanostructures in aqueous solutions. Real-time and quantitative adhesion between the AFM tip and sample was assessed through force-volume mapping. Additionally, we examined the protein adsorption behaviors at these interfaces using QCM-D with BSA, LYZ, and cyt-c, which act as non-specific binding proteins. The C-reactive protein (CRP) was included due to its specific affinity to the PC functional groups. AFM probing nearly the interface revealed that the nanostructured surfaces induced higher adhesion forces than pristine PEDOT-OH films, while the PEDOT-PC coating exhibited minimal interaction during tip scanning. Furthermore, the protein adsorption tests indicated that the nanostructures compromised the antifouling properties of PEDOT-PC films, consistent with water contact angle (WCA) measurements. The periodic structure increased the energy barrier, destroying the retention of a continuous water layer captured by the PC moieties. The results further provided more physical investigation of the nanostructure effect on CP interfaces. Finally, in the third section, we focused on the dynamic water molecule structures of CPs. To assess the hydration state of PEDOT and its derivatives, we employed in-situ Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC) as robust analytical techniques. DSC enabled the quantification of three distinct states of water within the CPs: non-freezing water (NFW), intermediate water (IW), and free water (FW). The Hofmeister series offers a systematic ranking of ion−water affinities. The formation of a hydrogen bonding network within water is hindered in the presence of ClO4−, which belongs to the category of "structure−breaking" anions. Conversely, "structure−making" anions like SO42− exhibit high hydration tendencies and establish stable associations with water molecules. We effectively monitored the adsorption/desorption phenomena through precise potential control and observed changes in the OH stretching bands when in contact with different salts. For CPs enriched with hydroxyl groups, SO42− ions noticeably influenced the adsorbed water due to their strong interaction. In contrast, ClO4− ions didn’t significantly perturb the water structure in PEDOT-OH. In the case of PEDOT-PC, NFW and IW exhibited strong affinities to PC, thereby protecting water molecules within the polymer when exposed to salts. The FT-IR results were analyzed via Gaussian fitting of the sub-bands within the OH stretching region, revealing distinctive changes in spectral shape corresponding to different water states. In this study, the interactions between ions, CPs, and water can be modulated by the applied potential at the CP interfaces, adding a broader dimension to our understanding of these systems.