矽單晶基材上銅矽超微電極陣列之研發及其應用於黏蛋白免疫傳感器之設計與偵測

Development of copper-silica ultramicroelectrode arrays on silicon for use as mucin immunosensors

范原睿(Yuan-Jui Fan)

指導教授 ： 張哲政

國立臺灣大學/理學院/化學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202201026

摘要

唾液黏蛋白可被應用於檢測口腔疾病如囊性纖維化、齲齒與口腔扁平苔癬等。利用黏蛋白的濃度差異，可以診斷不同的口腔疾病。然而，不同的檢測方法在偵測極限上有很大的差異，如ELISA和比色法。因此，為了能靈敏的檢測黏蛋白濃度，本研究發展了一種基於超微電極增強螢光(UMEEF)效應的螢光免疫傳感器，此傳感器是由矽單晶上高密度銅矽奈米陣列結構(CuDS)所製備而得。

在此研究中，通過修改我們實驗室開發的在矽單晶上合成含銅線型結構的製造條件，包含退火速率、停止供氧溫度、氧化持續時間與氧氣壓力的改變，成功得到高密度的CuDS。使用X-ray 電子能譜、原子力顯微鏡、掃描電子顯微鏡分析CuDS的化學成分、表面形貌與導電性。我們發現生成CuDS的機制包含銅模的裂解(dewetting)、銅向二氧化矽的擴散與矽銅的相互作用。

本研究的第二部分是經由化學修飾CuDS以檢測黏蛋白。通過電化學還原重氮鹽，在CuDS的銅基顆粒上形成羧基苯膜後，以EDC/NHS活化羧基，再將5-氨基螢光素(5AF)與抗黏蛋白固定在羧基苯表面。使用二次離子質譜儀、共軛焦雷射掃描顯微鏡等技術分析表面的功能化。CF647綴合的抗黏蛋白會與黏蛋白結合，由5AF與CF647的螢光強度比值量化黏蛋白濃度。CuDS免疫傳感器具有大檢測動態範圍1.2 - 900 ug/mL與良好的偵測極限0.5 ug/mL。由於CuDS超微電極造成的金屬螢光增強效應，使免疫傳感器在較低濃度的黏蛋白(1.2–50 ug/mL)下表現出更高的偵測靈敏度，其靈敏度較高濃度(50–900 ug/mL)高約7倍。

關鍵字

傳感器

並列摘要

Salivary mucin plays an important role for diagnosing oral diseases, such as cystic fibrosis, dental caries and oral lichen planus. There is a significant difference in mucin concentration which may cause different oral diseases. However, different detection methods, such as ELISA and colorimetry, have different limits of detection. Hence, a fluorescent immunosensor based on the ultramicroelectrode enhanced fluorescent (UMEEF) effect was developed in this study for quantitative detection of mucin. The developed UMEEF immunosensor was based on copper-based high-density nano-structure dot arrays (CuDS) fabricated on the silicon chip.
In this study, CuDS was made possible by modifying the fabrication technology developed in our lab for synthesizing copper-based line structure (CuLS) on silicon. The modification included tuning the fabrication parameters of CuLS such as the annealing rate, termination temperature of oxygen supply, duration of oxidation and pressure of oxygen. X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to analyze the chemical composition, the surface morphology and the conductivity of CuDS. The formation mechanism of ultramicroelectrodes in CuDS includes dewetting of the Cu film, the diffusion of Cu to silica and interaction between silicon and copper.
The second part of this study was to chemically modify CuDS for detection of mucin. The modification was conducted via functionalizing CuDS through (i) electrochemically reducing in situ the generated diazonium salts to form carboxyphenyl film on copper-based particles of CuDS, (ii) activating the resulting carboxyphenyl-bonded CuDS surface with EDC/NHS, and then (iii) immobilizing the 5-aminofluorescein (5AF) and anti-mucin on the surface carboxyphenyl to complete the construction of the immunosensor. Secondary ion mass spectrometer (SIMS), confocal laser scanning microscopy (CLSM), XPS and SEM were used to characterize the functionalization. The ratio of fluorescent intensity of 5AF to CF647 conjugated on anti-mucin which, in turn, was bound to mucin was used to quantify mucin concentration. The modified CuDS immunosensor was able to determine the concentration of mucin with a large detection dynamic range 1.2 – 900 ug/ml and a low limit of detection 0.5 ug/ml. Because of metal enhanced fluorescence (MEF) associated with the presence of ultramicroelectrodes on CuDS, the immunosensor exhibited higher sensitivity value at lower concentrations of mucin (1.2 – 50 ug/ml). The sensitivity was about 7 times larger than that at higher concentrations (50 – 900 ug/ml).

並列關鍵字

sensor

參考文獻

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