- 學位論文
應用於阿茲海默症早期診斷之氮摻雜石墨烯磁性複合材料的製備與性質研究
Preparation and Characterization of Magnetic Nitrogen-Doped Graphene Composite for Early Diagnosis of Alzheimer's Disease
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摘要
本研究旨在探討以乙二胺(ethylenediamine, EDA)接枝的四氧化三鐵/氮摻雜石墨烯(magnetic nitrogen-doped graphene)為基礎之磁性奈米複合材料應用於β類澱粉蛋白(Amyloid-Beta, Aβ)生物感測器。Aβ是阿茲海默症病患腦部中斑塊的主要成分，與阿茲海默症的病程有極為密切的關係，故被認為是阿茲海默症的生物指標物(biomarker)。本研究以modified Hummers’ method製備的氧化石墨烯(graphene oxide, GO)可分別經由快速熱膨脹脫層及油浴熱處理製備氮摻雜石墨烯(nitrogen-doped graphene, NG)，經研究後發現以油浴熱處理所製得之NG水溶性較好，擁有較多的-NH2官能基，而且製程簡易。後續再藉由共沉澱法(co-precipitation method)將Fe3O4沉積於NG表面得到Fe3O4-NG (magnetic nitrogen-doped graphene, MNG)，利用EDC/NHS (1-ethyl-1-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide)交聯反應(cross-linking)將 EDA接枝在MNG上得到EDA-MNG，再利用SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane -1-carboxylate)交聯劑將Aβ抗體修飾於EDA-MNG上得到生物感測器工作電極材料，最後利用強力磁鐵將之均勻塗佈於商用金電極(gold screen printed electrode, AuSPE)，便完成了生物感測器的工作電極組裝。此生物感測器工作電極與不同濃度的Aβ抗原反應後，以三電極系統利用電化學儀器測試其電流響應之變化，探討此生物感測器偵測Aβ之靈敏性及選擇性。 本研究第一部分探討實驗所製備之EDA-MNG的材料性質，以TEM和AFM觀察EDA-MNG之表面形貌，得知Fe3O4均勻地沉積於材料表面，且Fe3O4的尺寸介於10~20 nm之間。由XRD圖譜分析EDA-MNG的結果除了可以比對出GO被改質成NG後在(002)位置的特徵峰外，還顯示出具有Fe3O4的特殊結晶面：(220)、(311)、(400)、(422)、(511)及(440)。並可從EDA-MNG的XPS全譜圖觀察到N 1s (400 eV)，Fe 2p1/2 (711 eV)以及Fe 2p3/2 (724 eV)的特徵峰。而TGA試驗的結果可以計算出Fe3O4於MNG中的比例約為58.57 wt%。最後以SQUID分析所製備之MNG材料的飽和磁化強度約31.73 emu g-1，具有超順磁性，無磁滯現象。綜合上述儀器實驗之鑑定，證明已成功合成出乙二胺接枝的四氧化三鐵/氮摻雜石墨烯(EDA-MNG)磁性複合材料，並可利用其超順磁性，將其固定在生物感測器之工作電極上。 本研究第二部分探討EDA-MNG於Aβ生物感測器之應用，首先以循環伏安法(cyclic voltammetry, CV)於工作電位範圍-0.2~0.6 V下測得其氧化還原峰，過程中可以發現EDA-MNG磁性複合材料的導電性能佳。進一步利用微分脈衝伏安法(differential pulse voltammetry, DPV)偵測Aβ濃度。在生物感測器的靈敏度(sensitivity)測試方面，其檢量線(calibration curve)之R-squared值達到0.9977，具有優異的偵測靈敏性，且偵測極限(limit of detection, LOD)為5 pg/mL，偵測線性範圍為5~800 pg/mL；在生物感測器的選擇性(selectivity)方面，加入30 μM 抗壞血酸(ascorbic acid, AA)以及30 μM 尿酸(uric acid, UA)與Aβ做比較，說明生物感測器對於分析物Aβ具有良好選擇性；在可重複性測試方面，工作電極在50次的重複塗佈及洗去電極表面材料的步驟中，其響應電流介於2.5~2.6×10-4 A間，相對標準偏差(relative standard deviation, RSD)為1.40%，具有優異的工作電極重複使用性。在再現性測試方面，生物感測器偵測800 pg/mL的Aβ六次的實驗結果的RSD為2.80%，再現性佳。 本研究建立之Aβ生物感測器具備了可重複使用性、高靈敏度、高選擇性、再現性佳之特性，其偵測範圍涵蓋正常人及阿茲海默症病患體內Aβ濃度之臨界值(cut-off level, 192~603 pg/mL)，可以應用於早期阿茲海默症病患的篩檢。此外，短的偵測響應時間(response time, 30 min)使得此生物感測器具有快速測試的優點。此生物感測器不但可以降低偵測上的成本，亦可以減少偵測時所需人工、時間，非常適合應用於臨床篩檢技術。
並列摘要
This study aims at applying magnetic graphene-based nanocomposite to amyloid-beta (Aβ) biosensor. Aβ is considered as a reliable biomarker for the early diagnosis of Alzheimer’s disease (AD). There is no cure for AD to date, hence, simple and efficient methods for diagnosing AD at its earliest stages is of great importance. Accordingly, a reusable biosensor based on magnetic nitrogen-doped graphene (MNG) modified Au electrode for the detection of Aβ has been developed in this work. To begin with, nitrogen-doped graphene (NG) was synthesized by heat treatment process from graphene oxide (GO) which was synthesized using modified Hummers’ method. In order to obtain magnetic nitrogen-doped graphene (MNG), magnetic nanoparticles (Fe3O4) were introduced onto NG by co-precipitation. To achieve the best use of functional groups on MNG, ethylenediamine (EDA) was utilized to react with carboxyl groups on MNG to form EDA-MNG by EDC/NHS (1-ethyl-1-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) crosslinking method. Then the antibodies of Aβ were immobilized onto EDA-MNG electrode coating material via SMCC crosslinking method to form antibody-EDA-MNG. In addition, the biosensor was fabricated by coating the antibody-EDA-MNG onto the commercial gold electrode by placing an external magnet at the underside of the electrode for the detection of Aβ, and the biosensor can be easily and conveniently regenerated by switching off the magnetic field used to capture the magnetic materials onto the electrode surface. Finally, the sensitivity, selectivity and repeatability of Aβ biosensor were also investigated in this work. The first part of this study was the characterization and the investigation of the morphology of EDA-MNG by XRD, XPS, FTIR and SQUID. Firstly, the XRD pattern of EDA-MNG showed obvious peaks of Fe3O4. The six peaks correspond to (220), (311), (400), (422), (511) and (440) planes of crystalline face-centered cubic Fe3O4 nanoparticles. Secondly, the XPS pattern of NG showed the additional peak at 400 eV, which can be attributed to the nitrogen doping from GO. The peaks at 711 eV and 724 eV of MNG and EDA-MNG were attributed to the Fe 2p1/2 and Fe 2p3/2, repectively. Additionally, the EDA-MNG nanocomposites exhibited the superparamagnetic property as well as Fe3O4. Finally, the morphology of EDA-MNG from AFM and TEM indicates the Fe3O4 nanoparticles were immobilized uniformly with a size range from 10 to 20 nm. These charaterizations suggest that the magnetic graphene-based nanocomposites were synthesized successfully. The second part of this study were the investigations on the EDA-MNG applied in Aβ biosensor using electrochemical method. On one hand, the electrochemical potential window of the biosensor between -0.2 to 0.6 V was obtained by cyclic voltammetry (CV). On the other hand, differential pulse voltammetry (DPV), an electrochemical technique which can reduce the background noise was utilized to detect the Aβ concentration. In terms of sensitivity, the calibration curce was linear within the range from 5 pg mL-1 to 800 pg mL-1 with R2 = 0.9977, covering cut-off level of Aβ and a detection limit of 5 pg mL-1 had been achieved. In terms of selectivity, the influence of detection in the presence of 30 μM asctrbic acid (AA) and 30 μM uric acid (UA) can be neglected. In terms of reusability, the RSD of the current response of reconstruction was 1.40% (n=50) and the RSD of the current reponse of Aβ deteion was 2.80% (n=6), indicating that the biosensor can be reused with good reproducibility and stability. In summary, an Aβ biosensor with high sensitivity, high selectivity and repeatability was fabricated successfully. The fabricated biosensor for Aβ detection not only improves the detection performance but also reduces the cost and shortens the response time. Furthermore, this biosensor could be utilized for early diagnosis of Alzheimer’s disease because the detection range of this biosensor covers the Aβ levels of normal people and patients.
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