以功能性聚對二甲苯透過位向選擇鍵結技術於固定生物分子之開發研究

Covalent Immobilization of Biomolecules Based on Functionalized Poly-p-xylenes via Site-Specific Conjugation

蘇巧慈(Chiao-Tzu Su)

指導教授 ： 陳賢燁

國立臺灣大學/工學院/化學工程學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.11151

摘要

在生物界面領域，位向選擇性鍵結(site-specific conjugation)是眾所關注的議題，其中，在材料界面固定生物分子的應用，區域選擇性(regioselective)和正交化學反應 (biorthogonal chemistry)是常見重要的鍵結技術。首先，利用具有光催化性的苯甲醯基聚對二甲苯(poly[(4-benzoyl-p-xylylene)-co-(p-xylylene)]，PPX-benzoyl)，其光反應製程在搭配光罩(photomask)下，可以選擇固定分子於基材特定區域，進而形成幾何形狀(pattern)。在研究中，苯甲醯基聚對二甲苯鍍膜選擇性鍵結一系列抗結垢(anti-fouling)分子:聚乙二醇(poly(ethylene glycol)，PEG，Mn=400)、聚乙二醇甲基醚丙烯酸甲酯(poly (ethylene glycol) methyl ether methacrylate，PEGMA，Mn=475)、葡萄聚醣(dextran)和乙醇胺(ethanolamine)。鍍膜的苯甲醯基(benzoyl group)在波長365 nm紫外光照射下，與碳氫鍵(-CH)或氨基(-NH)進行光交聯反應(photo-crosslinking)，因其廣泛的反應性，不需要額外再替抗垢分子修飾官能基便能直接化學鍵結於表面上。藉由反射吸收式紅外光譜分析技術(infrared reflection absorption spectroscopy，IRRAS)和X射線光電子能譜(X-ray photoelectron spectroscopy，XPS)進行改質後表面的特性分析。利用螢光蛋白質fibrinogen、bovine serum albumin(BSA)的吸附測試可以檢驗其改質過後的抗垢效果，此外，藉由石英晶體微天平(quartz crystal microbalance，QCM)得到改質PEG、PEGMA、dextran和ethanolamine表面吸附fibrinogen的量值分別為: 32.8 ± 4.9ng cm-2、5.5 ± 3.9ng cm-2、21.4 ± 4.5ng cm-2和16.9 ± 3.4ng cm-2，證明其抗垢效果。最後，苯甲醯基聚對二甲苯區域選擇性的改質技術，藉由投影技術(microscopic patterning)延伸應用在立體的人工心臟支架上，其抗蛋白質效果也利用螢光蛋白質吸附測試驗證之。
此外，在化學專一鍵結反應中，利用N-羥基琥珀醯亞胺基聚對二甲苯鍍膜 (poly[(4-N-hydroxysuccinimide ester-p-xylylene)-co-(p-xylylene)] ，PPX-NHS ester)其NHS ester官能基對胺基(amino groups)分子有專屬反應的特性，進行各種胺基分子改質功能之應用: (1)抗垢:表面接上amine-PEG，進行螢光蛋白質吸附測試，並利用QCM得到改質表面吸附FBS (Fetal bovine serum 10%)量值為42.0 ± 6.5ng cm-2。(2)抗菌: 表面改質chlorhexidine gluconate (CHX)，分別培養抗藥性金黃色葡萄球菌(Methicillin-resistant Staphylococcus aureus，MRSA)和陰溝腸桿菌(Enterobacter cloacae)，以驗證其抗菌效果。(3)固定蛋白質:利用螢光微陣列(microarray)掃描和QCM分別計算出PPX-NHS ester表面固定BSA量為557 ng cm-2 、515.3 ng cm-2。另外，也利用QCM監測骨形成蛋白(BMP-2)在其表面的吸附情形。最後，PPX-NHS ester分別接上血管內皮生長因子(VEGF)和QK胜肽，並培養牛主動脈血管內皮細胞(BAEC)觀察其細胞基因表現，基因KDR、FLT1皆有效果，其中FLT1具有顯著效果。
表面官能基密度與反應性是值得深入討論的議題，以客製化雙向進料CVD系統製備不同官能基密度的五氟苯酚酯基聚對二甲苯鍍膜 (poly[(4-carboxylic acid pentafluorophenol ester-p-xylylene)- co-(p-xylylene)]，PPX-PFP)，分別利用帶有螢光短序列核酸適體aptamer和螢光蛋白(Cy5-BSA)與表面pentafluorophenol group (PFP)進行反應，透過微陣列掃描器(microarray scanner)掃描其螢光值訊號，來觀察表面官能基密度與反應性的關係: PFP官能基密度最大的表面螢光未達到最大值，而在比例PFP 1:20達到飽和值，核酸適體與蛋白質都呈現相同的趨勢。推測此現象，是來自於PFP官能基擁有大量的氟，可能在水溶液中帶負電，當PFP官能基密度最高之時，會產生排擠效應，PFP官能基密度低到一定的量之後，也會因可以反應鍵結的PFP官能基太少而固定量下降。最後，此研究所提供的位向選擇性鍵結(site-specific conjugation)技術，預期未來可以應用在醫學檢測儀器(diagnostic devices)、組織工程(tissue engineering)、細胞陣列(cellular arrays)等領域。

關鍵字

生物界面 ； 化學氣相沉積 ； N-羥基琥珀醯亞胺基 ； 苯甲醯基 ； 表面改質

並列摘要

In biointerface science, regioselective and orthogonal fashion of conjugations have played impontant roles. In this study, a vapor-deposited, photodefinable polymer, poly(4-benzoyl-p-xylylene-co-p- xylylene), is introduced as a versatile tool for the regioselective immobilization of antifouling materials, such as poly(ethylene glycol), poly(ethylene glycol) methyl ether methacrylate, dextran, and ethanolamine via photopatterning process. The reported immobilization process relies on the photoactivated carbonyl groups of the polymer, which have the potential to enable light-induced cross-linking of molecules and can rapidly react via insertion into CH- or NH-bonds upon photo-illumination at 365 nm. Characterizations using a combination of X-ray photoelectron spectroscopy (XPS) and infrared reflection absorption spectroscopy (IRRAS) have confirmed the characteristics of the immobilizations of these fouling materials, and the resulting antifouling properties are examined by conducting protein adsorption studies of fibrinogen and bovine serum albumin (BSA) on surfaces that were spatially modified with the aid of a photomask during the photochemical process. In addition, quantitative analysis of adsorbed fibrinogen was performed by using a quartz crystal microbalance (QCM), and the reduced adsorptions were found to be 32.8 ± 4.9ng cm-2, 5.5 ± 3.9ng cm-2, 21.4 ± 4.5ng cm-2 and 16.9 ± 3.4ng cm-2 for poly(ethylene glycol) (PEG), poly(ethylene glycol) methyl ether methacrylate (PEGMA), dextran, and ethanolamine, respectively. Finally, with the microscopic patterning technique, we further applied the antifouling modification technology to the unconventional substrate of a stent wherein PEGMA was immobilized on selected areas during the photoimmobilization procedure. Low levels of fibrinogen and BSA adsorption were also observed at the PEGMA-tethered areas.
In bioorthogonoal chemistry, N-hydroxysuccinimide esters (NHS esters) can site-specifically conjugate with amino molecules. Herein, a facile and versatile approach to prepare NHS ester groups on surfaces based on a NHS ester-functionalized poly-p-xylylene coating, poly[(4-N-hydroxysuccinimide ester-p-xylylene)-co-(p- xylylene)] (PPX-NHS ester), was introduced. This reactive coating is synthesized by chemical vapor deposition (CVD) polymerization and can be prepared on a diversity of substrate materials such as gold, glass, steel, poly(methyl methacrylate) (PMMA) and polystyrene (PS). Herein, we performed a variety of biomolecules containing amino groups were conjugated onto NHS ester modified surfaces and also confirmed by fluorescent proteins, quartz crystal microbalance (QCM), cell culture, antibacterial tests. (1) Antifouling property: Substrates was modified with amine-PEG based on PPX-NHS ester coating and then underwent protein adsorption test and QCM, which performed a FBS adsorption value reduced to 42.0 ± 6.5ng cm-2. (2) Antibacterial property: Chlorhexidine gluconate (CHX), an antibacterial molecule, was immobilized on PPX-NHS ester coatings. Subsequently, the antibacterial assay was conducted to verify the resulting antibacterial property. (3) Proteins immobilization: The density of BSA immobilized on PPX-NHS ester was analyzed by microarray scanning and QCM, 557 ng cm-2 and 515.3 ng cm-2. In addition, the primary antibody and second antibody were used to examine the adsorption of BMP-2 via QCM analysis. Finally, surfaces immobilized VEGF and QK peptide was investigated by using the response of in vitro culture of BAECs and the gene expression data were also collected.
The density of functional groups on functionalized poly-p-xylylene has drawn many interest. Herein, we prepared poly[(4-carboxylic acid pentafluorophenol ester-p-xylylene)-co-(p-xylylene)] in various densities by manipulating the deposition rate during the copolymerization process. Then, Cy3-aptamer and Cy5-BSA were exploited to observe the relation between densities of functional groups and the reactivity. Optimal fluorescent signals were detected through microarray scanning, and this trend both showed in Cy3-aptamer and Cy5-BSA. In brief, above site-specific conjugation techniques can be applied in biomedical devices, tissue engineering and cellular assays.

並列關鍵字

Biointerface ； Chemical vapor deposition ； N-hydroxysuccinimide ester ； Benzoyl ； Surface modification

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