Development and Applications of Capillary Electrophoresis and Functionalized Gold Nanoparticles

此論文包含毛細管電泳技術開發與功能性金奈米粒子之合成與應用。毛細管電泳部分利用naphthalene-2,3-dicarboxaldehyde (NDA)染劑配合發光二極體誘導螢光偵測系統，同時對胺基酸與胺類分子進行分析，並且使用含有cetyltrimethylammonium bromide (CTAB)與acetonitrile (ACN)的poly(ethylene oxide) (PEO)聚合物溶液來提升分離效率。CTAB可有效降低PEO於毛細管壁之吸附並且提供了穩定又快速的反向電滲流。適當濃度的ACN (25%)使CTAB (20 mM)微胞對分析物有更好的選擇性，有效改善分離的效率。此方法可在4分鐘內完成14種胺基酸與生物胺分子的分析，並提供了良好的分離解析能力(理論板數0.9×105–6.4×105)與靈敏度(LODs~10 nM)，也成功地應用於啤酒樣品的分析。在功能性金奈米粒子部分，開發了一種新型且不需要標記分子的凝血酶檢測法。藉由纖維蛋白原修飾在金奈米粒子表面，當凝血酶與纖維蛋白原作用後，會產生不溶於水、纖維狀的纖維蛋白，使金奈米粒子聚集。透過金奈米粒子吸收變化，可進行高選擇性及高靈敏度的凝血酶偵測，其偵測線性範圍在0.1–10 pM，偵測極限為0.04 pM，並順利應用於血漿中凝血酶之測定，不受生物基質干擾。此外，凝血酶適合體(thrombin binding aptamer; TBA)可有效地與凝血酶接合(Kd~1–100 nM)，常見的凝血酶適合體為TBA15 (15個鹼基)與TBA27 (27個鹼基)。雖然TBA15的結合能力明顯較差(Kd~100 nM)，但TBA15可以直接影響凝血酶活性，而TBA27則不直接影響其活性。故可透過一段互補的DNA將TBA15與TBA27結合成具有較強抑制能力的DNA複合體(TBA15-TBA27)。利用此複合體的抑制能力與先前高靈敏度的凝血酶活性檢測方法，成功開發了新的DNA偵測比色法。此方法不需要繁複的平衡、清洗或溫度變化等處理步驟，其偵測線性範圍為0.050–2.0 nM，偵測極限為25 pM，並且能以肉眼進行single-nucleotide polymorphism (SNP)的觀測。Three different analytical approaches are demonstrated in this thesis. First, naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and -amine derivatives were separated and detected by capillary electrophoresis in conjunction with light-emitting diode-induced fluorescence (LEDIF) detection using poly(ethylene oxide) (PEO) containing cetyltrimethylammonium bromide (CTAB). In the presence of CTAB and acetonitrile (ACN), adsorption of PEO on the capillary wall was suppressed, leading to generation of a fast and reproducible electroosmotic flow (EOF). In order to optimize separation resolution and speed, 100 mM Tris–borate solution (pH 7.0) containing 20 mM CTAB and 25% ACN was used to fill the capillary and to prepare 1.2% PEO that entered the capillary via EOF. The analysis of 14 NDA-amino acid and -amine derivatives by this approach was rapid (< 4 min), efficient [(0.9–6.4) × 105 theoretical plates], and sensitive [the LODs (S/N = 3) range from 9.5 to 50.5 nM]. The RSD values (n = 5) of the migration times and peak heights of the analytes for the intraday analysis were less than 1.5 and 1.2%, respectively. The practicality of this approach was validated by quantitative determination of 10 amino acids and amines in a beer samples within 4 min. Secondly, a novel, label-free, colorimetric assay – using fibrinogen (Fib) and gold nanoparticles (Au NPs) –was developed for the highly selective and sensitive detection of thrombin. Addition of fibrinogen to a solution of Au NPs (average diameter: 56 nm) led to ready conjugation, forming Fib–Au NPs through electrostatic and hydrophobic interactions. Introduction of thrombin (a serine protease) into the Fib–Au NPs solutions in the presence of excess fibrinogen induced the formation of insoluble fibrillar fibrin–Au NPs agglutinates through the polymerization of the unconjugated and conjugated fibrinogen. After centrifugation, the absorbance at 532nm of the supernatants decreased upon increasing the concentration of thrombin. This Fib–Au NP probe provided high sensitivity [limit of detection (LOD): 0.04 pM] for thrombin, with remarkable selectivity over other proteins and proteases. The range of linearity for the absorbance against the thrombin concentration was 0.1–10 pM (R2 = 0.96). This approach provided an LOD for thrombin that is lower than those obtainable using other nanomaterial- and aptamer-based detection methods. The utility of this Fib–Au NP probe was validated through separate analyses of thrombin and Factor Xa at picomolar levels in plasma samples—without the need for sample pretreatment. This technique appears to have practical potential in the diagnosis of diseases associated with coagulation abnormalities and cancers (e.g., pulmonary metastasis). Last, detection of DNA hybridization was demonstrated using a Fib-Au NPs-based assay. Two thrombin binding aptamers (TBAs)－TBA15 (15 bases long) and TBA27 (27 bases long)－that are specific towards thrombin were used to form a TBA15-TBA27 assembly in the presence of a complementary DNA (cDNA). The TBA15-TBA27 assembly relative to TBA15 and TBA27 provided a greater inhibition activity for thrombin, showing bivalent binding capacity. The activity of thrombin decreased upon increasing the concentration of cDNA. This new sensing strategy provides high sensitivity [limit of detection (LOD): 25 pM] and remarkable specificity for cDNA. To test the practicality, another probes [TBA15’ (P-TBA15’) and TBA27’ (P-TBA27’)] were used for the detection of the single-nucleotide polymorphism (SNP) responsible for hepatocellular carcinoma. Unlike conventional approaches, this method requires neither postsynthetic modification of the probe oligonucleotides nor precise temperature control for SNP typing