Chemistry Bridging Bio-molecules to Surfaces: Synthesis, Characterization and Applications
由於苯硼酸系統只適用於醣蛋白之修飾上，對於一般原核系統下表現蛋白或無醣基化蛋白無法進行修飾故必須另尋他法進行改善。目前最為廣泛應用之標誌為多重組氨酸 (poly-histidine)序列且可以透過基因工程將其安插於蛋白質兩端(N-, C-)。其後次氮基三乙酸(NTA)、鎳離子(Ni2+)及含組氨酸序列之蛋白質形成一親和性鍵結並得以將其固定於材料或元件表面。因此，在本研究中也合成了一系列NTA衍生物並將具前降鈣素(PCT)辨識能力之單鏈抗體(scFv)成功修飾於晶片表面並進行簡單的示範實驗。
透過上述化學修飾可以針對抗體或人工表現蛋白質做一有效之位向控制。然而目前生醫檢測系統上遇到最大的挫折在於非特異性吸附所產生之干擾進而導致其無法進行有效判定。為了克服非特異性吸附所生成之問題，由兩性離子所修飾而成之介面為目前最具有效之方法：如sulfobetaine, phosphorcholine. 本研究中也成功合成一系列與sulfobetaine相關之衍生物也同時驗證其有效性；而phophocholine-EDOT為尤教授所提供之化合物並透過電化學聚合方式將其修飾於ITO與金表面也成功驗證其抗沾黏之能力。
於另外一部分研究中我們為了探討分子間作用力利用了熱泳法(Thermophoresis)之技術。根據分子間所形成之複合物產生不同程度之變化下可以計算出其解離常數並判斷其作用強度。可以廣泛將此技術用於大分子間作用力分析(抗體之間或抗體抗原間)與小分子間作用力分析(離子與化合物間)。此外，我們也透過不同之修飾方式：(1) 一般修飾，(2) 樹狀苯硼酸修飾成功將其強度訊號提升了5~8倍；同時也比較了抗體於一般修飾與位向修飾所產生之結合力之差別，證實了位向修飾具有較好之鍵結能力。
In this study, phenylboronate is a strategy for overcoming the weakness of low recognition ability of antibody that resulted from conventional modification on biosensors. It is well known that phenylboronate and its derivatives can form cyclic boronate ester with vicinal cis-diol and polyol from carbohydrate or glycerol within pH 7~9. Based on this feature, we designed and sysnthesized a various of multi-functional linkers with one terminal containing multi-phenylboronic acid moieties and the other terminal containing serveral functional groups or even additional group such as photoactive group. The moieties of phenylboronates were employed for specifically forming complex with the glycosylation site at the FC region of glycoproteins such as antibody and consequently to control the orientation. Other side of terminal groups which can be utilized for conjugation of the linker on the solid support (such as gold, ITO, aluminum…etc). The linker density and coverage ratio were characterized and evaluated by cylic voltammetry and electrochemical impedance assessments. The results proved that electrochemical deposition/polymerization is much more efficient than the self-assembly process. Also, the studies proved that electrochemical deposition will not destroy the functional group of either fluorescein or phenylboronate moieties. In the other way, quartz crystal microbalance (QCM) was also employed as the detection platform to analyze the binding capacity of antibodies on the multi-phenylboronate derivatives modified surface. Each probes demonstrated the effective function of antibody immobilization. Especially for the di-phenylboronic derivatives which show the highest binding capacity and rate (~1.1 ng/sec) for antibody. Unfortunately, tri-phenylboeonic acid derivatives did not perfrom well due to the steric hindrance of the adjacent phenylboronate moiety itself. Neverrtheless, through the analysis of antibody desorption from acid environment by QCM system to realize that the binding affinity were determined in the sequence of tri-PBA > di-PBA > mono-PBA derivatives. Also, the demonstration of dengue virus detection can be achieved through phenylboronate fabricated surface with anti-DV1 antibody. To increase the binding capacity of antibody on the solid support, phenylboronate-dendrimer can be a capping reagent for automatically assembled on dopamine-fabricated surface. From the QCM analysis, combination strategy gave the maximum binding capacity for antibody (~390 ng on the electrode) and binding affinity than the previuos works. Nitrilotriacetic acid (NTA) is another well known for its affinity binding with histagged proteins via Co2+ or Ni2+. Through organic synthesis, a series of bi-functional linkers with nitrilotriacetic acid moiety and aniline, isothiocynate, phenylboronate, and maleimide group had been sysnthsized. The moiety of NTA can chelate with metal ions for further immobilization of histagged proteins. Through the electrochemical deposition process, NTA fabricated surface was confirmed by fluorescen image within mCherry and demonstrated that scFv can be immobileized on the QCM chip for procalcitonin detection. In this issue, the construction of antifouling layer is aimed to solve the non-specific binding from serums. Zwitterionic layer has been shown to effectively against the non-speicific binding from previous reports. Based on this feature, we successfully synthesized a series of sulfo-beaine deriveatives and the gift of phophocholine EDOT (EDOT-PC) from Prof.Yu. For the results of sulfo-betine fabricated surface, the antifouling ability can reach to 95 % in the case of electrodeopostion due to high density of sulfo-betaine on the electrode. The eletropoymerization of EDOT-PC also demonstrated the ability against the non-specific interaction from diluted FBS (about 92 %). Furhtermore, under the treatment of 0.1 % N-laurylsarcosine that can fully against the effect from FBS (~100 %). The molecular interaction can be analyzed by microscale thermophoresis (MST). Microscale thermophoresis is the directed movement of particles in a microscopic temperature gradient. Any change of the hydration shell of biomolecules due to changes in their structure/conformation results in a relative change of movement along the temperature gradient and is used to determine binding affinities, binding kinetics and activity kinetics. Base on the concept of MST, we had successfully demonstrated the assay between IgG/anti IgG with two modification strategies: (1) conventional labeling with NHS-Cy5. (2) PBA-dendrimer based with self-assemble of dopamine-cy5. The MST signal can be enhanced by 5-8 times than the traditional method by the dendrimer based strategy. Also, PCT/anti-PCT is another demonstration through above two strategies to show that the randomly labeled antibody with lower dissociation constant with target (29 nM~39 nM) compare with the orientated labeling strategy (6 nM~8 nM). To prove the phenylboronate moieties can maintain the activity of biomolecules. Except for antibody/antigen interactions, MST system can even analyze the interaction between small molucules and macromolecules. In this study, Nitrilotriacetic acid (NTA) as an example to analyze the affinity binding strength between NTA ligand, metal ions, and histagged protein interaction. From the results, Co2+ (KD =12.6±4.45 nM) have higher affinity than the Ni2+ ion (KD =52.2±6.67 nM). By combination of organic synthesis, bio-techniques, and analysis platforms, we now freely to set up for specific targets through different strategies by phenyboronic acid derivateives, nitriletriacetic acid deriveatives, or even the fluorescence image. The chemistries for combining with two or more different molecules or immobilizing on the solid supports now can have more options from our studies.
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