硼酸表面微陣列系統的發展及應用

Development of Boronic Acid Surface Based Microarray System and Its Applications

陳穆麟(Mu-Lin Chen)

指導教授 ： 林俊成

國立清華大學/理學院/化學系/博士(2009年)

https://doi.org/10.6843/NTHU.2009.00315

摘要

近年來，微陣列技術已經被發展成為一種高通量的檢測工具，並且已經廣泛的運用於生物分子交互作用的探討以及生醫技術應用，藉以解決基因質體學、蛋白質體學、以及醣質體學上之問題。除此之外，具有專一性的結合配對更可以以微陣列的模式來進行醫學檢測、環境監控、以及毒性物質偵測等等。為了要建立各種不同的生物分子的微陣列系統，已經有各種不同用於固定的微陣列表面官能基被廣泛的研究，然而目前為止沒有任何一樣表面官能基可以適用於全部的生物分子。因此一個容易建立且容易固定生物分子探針的新式表面工具，其可減少生物探針的處理以及可提供共價鍵固定仍然是必要的目標。
苯基硼酸以及其衍生物已知在水溶液下會與含順式二醇形成可逆環狀酯，而多氫氧基化合物例如甘油及醣體因具有許多如此的結構，因此硼酸長久以來已經被使用來達成醣類追蹤的工具並應用於醣類感測器、多醣體偵測、以及醣胜肽與醣蛋白的濃富化與純化等等。然而就我們所知，目前硼酸並沒有應用於生物晶片的研究。有鑑於此，在此篇論文我們結合了硼酸的醣類追蹤特性及生物晶片的優點首創具有醣類追蹤能力的微陣列晶片表面，並應用於醣蛋白方向性共價鍵固定化來保持蛋白質活性，以及共價鍵固定之醣晶片系統來提升醣體在晶片表面上之保持能力。
為了探討蛋白質的固定化，硼酸被用來追蹤Fc融合凝集素以及抗體上座落於Fc domain的醣鏈，以達到方向性固定的目標。經由比較，我們證明具方向性固定的醣蛋白具有較高的結合訊號。而由硼酸共價鍵且方向性固定的醣蛋白其相較於蛋白質G (protein G)非共價鍵且方向性固定的醣蛋白具有較高的結合訊號。
為了建立簡單實用且具有共價固定能力的醣晶片表面，我們選用已商品化的化合物〝間-氨基苯基硼酸〞做為我們的包覆試劑，利用雙NHS活性酯長鏈與牛血清蛋白包覆的晶片進行連結，建立親水性硼酸晶片表面，再用以製造具有共價鍵固定的醣微陣列。經由共價鍵固定，我們證明醣類小分子可以被固定於晶片表面且只要具有0.5 kDa即具有與凝集素辨認的能力。除此之外，我們也成功的利用三層牛血清蛋白包覆表面來成功的減輕表面物理吸附的能力，使我們可以輕易的觀測到硼酸對醣體共價鍵固定所帶來的影響。我們證明了硼酸表面的共價鍵固定醣微陣列相較於沒有共價鍵固定的醣微陣列可以在強烈清洗下有較好的滯留能力。
另外，由於硼酸對於抗體具有方向性固定的能力，我們也建立了抗體微陣列來進行毒物偵測。我們選用一個低毒性的蓖麻毒素替代物〝RCA120〞做為我們的毒物樣板，並用半乳醣官能基化的金奈米粒子做追蹤，最後用銀染技術做放大以達到裸眼偵測的目標。在此我們為了增進靈敏度，我們更開發了金奈米粒子專一性聚集的訊號放大系統，將靈敏度提升至毒物濃度只需aM，且不需要光學儀器即可偵測。

關鍵字

微陣列 ； 硼酸 ； 具方向性醣蛋白微陣列 ； 醣微陣列 ； 醣金奈米粒子 ； 訊號放大系統

並列摘要

In recent decades, microarray has now been emerged as a high throughput screening tool for expanding biomedical application, such as biomolecular interactions of genomics, proteomics, and more recently in glycomics. The well-known specific binding event can be further developed for clinical diagnostic, environmental monitor, and so on in microarray format. To fabricate various kinds of microarray, there are varieties of surface functional groups available for immobilization of bio-molecules. Even so, none of them could be suitable for immobilization of all kinds of bio-molecules so far. Therefore, a new surface tool, which can be easily constructed and provides a new strategy for probe immobilization that minimizes probe destruction and allows covalent bond formation, are urgently demanded.
It is well known that phenyl boronic acid (BA) and its derivatives can form cyclic boronate esters with vicinal cis-diols and polyols, such as glycerol and carbohydrates, even under aqueous conditions. Accordingly, BA has been employed for achieving glyco-targeting such as carbohydrate biosensors, polysaccharide detection, separation or enrichment of glycoprotein and glycopeptide, and so on. However, to our knowledge, it has not been attempted to use as chips. In this thesis, we take advantage of BA to fabricate oriented protein microarray and covalent polysaccharide microarray to improve accessibility of proteins and retention of polysaccharide on slide surface.
To improve the protein conjugation on solid support, BA was used to site-specifically assemble Fc-fused lectin and antibody from its Fc domain for maintaining the best activity on the solid surface. We demonstrated that the protein activity can be conserved when protein molecule is oriented in an optimal manner on the BA surface. In addition, m-aminophenylboronic acid was used to easily construct covalent microarray of oligosaccharide and polysaccharide of molecular weight at least 0.5 KDa. The triple BSA layers surface was successfully applied to generate hydrophilic BA surfaces to significantly alleviate nonspecific adsorption for elucidation of covalent effect upon the carbohydrate. We demonstrated that BA based oligosaccharide microarrays offer better retention of carbohydrates on supports over non-covalent attachment during more stringent washing conditions.
Furthermore, owing to its potential application in glycoprotein microarrays, we employed antibody microarray to detect RCA120, a low-toxic “ricin” surrogate, by using galactose-gold nanoparticle (g-AuNP) and in combination with silver enhancement by the naked eye. To enhance the detection sensitivity, we developed a method to generate and amplify the signal of a antibody microarray using AuNP-based agglutination to give a new ultrasensitive assay with detection sensitivity at the attomolar (aM) level that doesn’t required any optical instrument for readout.

並列關鍵字

microarray ； boronic acid ； oriented glycoprotein array ； carbohydrate array ； glyco-gold nanoparticle ； signal amplification system

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