蛋白質之毛細管電泳分析與金奈米粒子之生物應用

Capillary Electrophoresis for Protein Analysis and Bioapplications of Gold-based Nanoparticles

黃郁棻(Yu-Fen Huang)

指導教授 ： 張煥宗

國立臺灣大學/理學院/化學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.00713

摘要

近年來人類基因體的成功定序，加速了蛋白質體學的蓬勃發展。毛細管電泳具備快速、高效能及樣品消耗量少等優點，在蛋白質分析上，已成為一項不可或缺的重要工具。另外，當材料縮小至奈米尺度時，由於比表面積的增加，量子化侷限效應的產生，往往會展現出與塊材截然不同的物理、化學性質。以金奈米粒子為例，除了具有極佳的生物相容性，在可見光波段的表面電漿共振吸收，也會隨著粒俓大小、形狀、表面環境介電常數以及聚集程度的不同而改變，呈現出肉眼即可辨識的顏色變化。本論文中，我們以毛細管微胞電層析（micellar electrokinetic capillary chromatography）搭配雷射誘導原生螢光偵側系統，有效的分離牛血清蛋白（bovine serum albumin）之水解產物，藉此可成功地觀察胰蛋白酶（trypsin）與金奈米粒子結合後（AuNP-trypsin）其酵素活性的改變。同時以毛細管區帶電泳（capillary zone electrophoresis）檢視AuNP-trypsin水解牛血清蛋白的動力學過程發現，AuNP-trypsin的酵素活性可能因為蛋白質構型（conformation）的改變，而有下降的趨勢。
此外，我們開發出一種微量蛋白質的線上濃縮和分離之技術，來解決一般毛細管電泳，偵測靈敏度不足的問題；藉由高電滲透流引入黏滯度較高的中性聚合物溶液（聚環氧乙烷，簡稱PEO），使得在Tris-borate （pH 10.0）條件下，帶負電的蛋白質由樣品區帶進入PEO溶液時，因電泳遷移率的下降，在兩溶液的界面形成堆積，達到濃縮大體積（1.0 μL）樣品的目的。實驗中發現，樣品注入前，引入一小段十二烷硫酸納（sodium dodecyl sulfate，簡稱SDS， 0.2%），以及添加少許SDS或PEO（0.01%）於樣品區帶，皆有助於減少蛋白質在毛細管表面的吸附，在分析6種蛋白質之混合樣品時，可解析出12根微異質性（microheterogeneity）蛋白質訊號峰。另外，此方法對蛋白質的濃縮倍數高達84倍，以α-lactalbumin為例，偵測極限可降至0.5 nM。這項技術可直接偵測未經前處理的人體尿液中所含之人血清蛋白（human serum albumin），其含量為0.18（± 0.04）μM，以及50個紅血球萃取液中的血紅素（hemoglobin），其含量為45（± 3）nM，其實際樣品偵測值皆符合文獻報導。
論文中，我們亦提出一套簡單的合成技術。在鹼性條件下（pH > 8.0）合成出形狀多樣且產率、純度（> 90%）相當高的金–銀奈米球、奈米棒和奈米線。不同反應時間、pH、胺基酸溶液的種類、濃度之選擇，會影響維他命C（ascorbic acid）還原金、銀離子以及十六烷基三甲基溴化銨（cetyltrimethylammonium bromide）穩定金–銀奈米粒子不同晶格面結構之能力。在pH = 8.0、9.0和10.0的甘胺酸（glycine，0.1 M）溶液中，分別可合成出棒狀、啞鈴型和球型金–銀奈米結構。在0.05 M和0.1 M離胺酸（Lysine，pH 10.0）溶液中，可成功地合成出項鍊型排列的金–銀奈米粒子和奈米線。其餘在組胺酸（histidine，0.1 M，pH 8.0和9.0）和甲硫胺酸（methionine，0.1 M，pH 10.0）溶液中，亦可巧妙的控制花生米和玉黍蜀形狀金–銀奈米材料之合成。
最後，利用金奈米粒子與硫醇間的強鍵結，以及金奈米粒子能吸收紫外光光子並轉換能量至分析物，使之脫附游離的能力，我們以尼羅紅（Nile Red，簡稱NR）修飾金奈米粒子（NRAuNPs）系統，搭配表面輔助雷射脫附游離化質譜技術，可在不受到生物體樣品中複雜基質的干擾下，成功地選擇性分離並定量出紅血球萃取液和血漿樣品中，具有疾病指標的麩胱甘肽（glutathione）和半胱胺酸（cysteine），其含量分別為0.79（± 0.08）mM和11（± 1）μM並符合文獻報導。另外，利用硫醇分子較容易取代NR產物且中和AuNPs表面電荷使AuNPs沈澱的特性，亦可簡單快速地分離硫醇（沈澱）和非硫醇（上層溶液）分子，並達到濃縮硫醇分子的目的，以偵測麩胱甘肽為例，藉由此離心濃縮步驟，可進一步降低麩胱甘肽的偵測極限至25 nM。

關鍵字

毛細管電泳 ； 金奈米粒子 ； 金-銀奈米粒子 ； 表面輔助雷射脫附游離化質譜

並列摘要

In order to develop novel analytical techniques for the analysis of biomolecules such as proteins and aminothiol compounds, we take the advantages of high efficiency, rapidity of capillary electrophoresis (CE) and unique optical and large surface properties of nanomaterials. CE in conjunction with laser-induced fluorescence has been separately applied to study the differential activity of enzymes in free solution and those are bound to gold nanoparticles (AuNPs) and improved sensitivity and efficiency for protein analysis. Differently shaped and sized Au-based nanoparticles (NPs) have been prepared and some of them have been tested to be efficient selectors and matrices for aminothiols compounds in surface assisted laser desorption ionization-mass spectrometry (SALDI-MS).
The specificity and activity of trypsin that is conjugated to gold NPs (AuNP-trypsin) for proteins have been investigated by micellar electrokinetic chromatography (MEKC) and capillary zone electrophoresis (CZE) with laser-induced-fluorescence (LIF) detection. In the presence of sodium dodecyl sulfate (SDS), adsorption of the tryptic fragments on AuNP-trypsin and on the capillary wall is reduced. Thus, the sensitivity and resolution of the tryptic fragments from bovine serum albumin (BSA) is improved. According to the electropherograms, the activity of AuNP-trypsin is lower than that of free trypsin. It is suggested that changes in the conformations and steric effects contribute to the loss of activity and changes in specificity of trypsin adsorbed on AuNPs.
An on-line concentration and separation method for analyzing large-volume protein samples by CE-LIF is described. After the injection of 1.0-μL samples, proteins migrate against the electroosmotic flow (EOF) and enter the poly(ethylene oxide) (PEO) zone; this process causes them to slow down and stack at the boundary between the PEO. Either 0.01% SDS or 0.01% PEO was used as sample additives to improve the stacking and separation efficiencies. By applying a short plug of 0.2% SDS prior to sample injection, the microheterogeneity of the proteins can be resolved. 12 peaks are detected when injecting 1.0-μL of sample containing six model proteins (0.1 μM). The limit of detection (LOD) for α-lactalbumin is 0.5 nM, which is an 84-fold sensitivity enhancement over the traditional method.
Preparation of Au core–Au–Ag shell NPs in different morphologies can be easily achieved by controlling both the pH and the glycine concentration. Using a seed–growth method, we prepared high-quality Au–Ag NPs from a glycine solution under alkaline conditions (pH > 8.5). Dumbbell- and peanut-shaped Au core–Au–Ag shell NPs were prepared in aqueous solution at the concentrations of glycine greater than 0.5 M and greater than 0.2 M at pH 9.5 and 10.5, respectively. In addition, we were able to affect the shapes and sizes of the Au–Ag NPs by controlling the reaction time. At pH 9.7, we observed the changes in the morphologies of the Au core–Au–Ag shell NPs—from regular (rectangular) to peanut- and dumbbell-shaped, and finally to jewel-, diamond-, and/or sphere-shaped—that occurred during the course of a 60-min reaction.
We further prepared differently shaped and sized Au–Ag nanocomposites from gold nanorod (AuNR) seeds in various amino acid solutions at the pH values ranging over 8.0–11.5. Our study shows that the pH as well as the concentration and species of amino acids have great impacts on the preparation of I-liked, dumbbell-liked, sphere-liked, peanut-liked, and corn-liked NPs as well as nanowires (NWs), mainly through the control of the reducing ability of ascorbate (or amino acids), oxidizing abilities of Au and Ag ions, and/or recognition capability as well as surface charges of the amino acids on the AuNRs. Depending on the value of pH, we were able to prepare I-shaped, dumbbell-shaped, and/or sphere-shaped Au–Ag nanocomposites in 0.1 M solutions of Arg, Gly, Glu, Gln, Lys, and Met. In His solutions at pH 8.0 and 9.0, we obtained peanut-shaped Au–Ag nanocomposites. Corn-shaped Au–Ag nanocomposites were prepared in 0.1 M Met solutions (pH 9.0 and 10.0). By controlling the Lys concentration at pH 10.0, we synthesized pearl-necklace-shaped Au–Ag nanoparticles and Au–Ag wires.
Nile Red-adsorbed AuNPs (NRAuNPs) has been demonstrated as selective probes and matrices for the determination of aminothiols through SALDI-MS. Due to the high specificity of NRAuNPs toward thiol-containing compounds as well as the aggregation induced by the binding of these molecules to the surfaces of NRAuNPs, NRAuNPs are capable to selectively concentrate three aminothiols—glutathione (GSH), cysteine (Cys), and homocysteine (HCys) in the precipitate—from a mixtures containing four amino acids. The preconcentration approach also provides an LOD of 25, 54, and 34 nM, for the determinations of GSH, Cys, and HCys, respectively. This method was validated in the analyses of GSH in red blood cells and of Cys in plasma and has great potential for diagnosis.

並列關鍵字

Capillary electrophoresis ； gold nanoparticles ； gold-silver nanocomposites ； SALDI-MS

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