標題: 奈米材料結合人工生化材料於生化感測器之開發
Nanomaterials combining with artificial biomaterials for biosensor developments
作者: 李承宇
謝有容
Lee, Cheng-Yu
Hsieh, You-Zung
應用化學系碩博士班
關鍵字: 奈米;生化;電化學;感測器;核酸適體;質譜;nanomaterials;biomaterials;electrochemistry;biosensor;aptamer;LC-MS
公開日期: 2017
摘要: 本論文為利用奈米材料,結合不同的訊號放大機制,輔以自行設計或合成之生化材料,建立平台式的生化感測器。主要之目的在於呈現自行設計訊號放大機制之構想,並透過實驗數據實際展現其訊號放大效益、可行性與應用性。在此論文中包含四個全新開發的生化感測器。在第一個部分中,利用人工設計的互補段核酸序列搭配核酸適體,結合金奈米修飾網版印刷電極,開發出可偵測人體免疫球蛋白E的電化學生化感測器。透過設計於互補段核酸序列之連續鳥嘌呤鹼基,成功將訊號放大。其檢量線之線性範圍為1 – 100,000 pM,所求得之偵測極限為 0.16 pM。此電化學生化感測器同時在100倍以上干擾物存在的環境下依然得到良好的選擇性與回收率。第二部分為結合環滾式擴增法與複合磁性奈米粒子針對攝護腺特異性抗原所設計之電化學生化感測器。透過核酸適體修飾之磁性奈米粒子作為攝護腺特異性抗原之探針,結合互補段引子序列去觸發環滾式擴增法,藉由人工設計之環狀連續胞嘧啶模板,大量複製含有連續鳥嘌呤之核酸序列,再透過其吸引甲基藍產生電化學訊號之放大效果,間接定量攝護腺特異性抗原之濃度。其檢量線之線性範圍為 100 fM 至 10 nM,偵測極限為 22.3 fM。在面對多種人體血液中可能出現之干擾物也呈現良好之選擇性。第三部分為結合複合磁性奈米粒子針對嗜酸性陽離子蛋白開發之電化學生化感測器。利用嗜酸性陽離子蛋白對肝素具有專一性結合之特性,將肝素以化學合成方式透過半胱胺做為橋接分子修飾於金包覆四氧化三鐵之磁性奈米粒子表面,作為偵測嗜酸性陽離子蛋白之探針。當奈米粒子透過肝素抓取嗜酸性陽離子蛋白後,以外加磁場之方式將磁性奈米粒子集中於電極表面。由於肝素本身為帶有大量負電荷之高分子聚醣,當抓取到嗜酸性陽離子蛋白後會因嗜酸性陽離子蛋白所帶之大量正電荷,造成電極表面吸引或排斥所加入之六氨化釕陽離子,令其氧化還原訊號產生變化,藉此定量嗜酸性陽離子蛋白。此電化學生化感測器可於1 至 1000 nM濃度區間內精確定量嗜酸性陽離子蛋白,其偵測極限為 0.30 nM。在經過培養後的培養基 RPMI 1640 中依然不受干擾,具有良好之回收率。最末,為透過金奈米粒子輔助結合條碼核酸序列針對液相層析-質譜偵測所開發之間接偵測技術。乙醇胺為本實驗所選用之範例分子,因其分子量小、高極性、不易於逆向管柱滯留與不具光學活性等特性,並不適於使用液相層析-質譜偵測。透過核酸適體修飾之金奈米粒子結合條碼核酸序列,成功將乙醇胺轉換為容易偵測之腺嘌呤訊號,並透過條碼核酸序列放大訊號,進一步成功以液相層析-質譜偵測並定量。其線性範圍為5 nM 至 5 μM,偵測極限為 1.2 nM。本論文中所設計之生化感測器,均是以平台式開發與訊號放大機制為其主要開發目的。透過替換不同之核酸適體,將可使生化感測器套用並轉換為其他分析物適用之生化感測器。同時,所設計之訊號轉換與放大機制,也可單獨提取並套用於其他分析方法之開發。對相關之研究與分析方法之發展將有所裨益。
In this study, different biosensors combined with unique signal transfer and amplify strategies were presented. Each signal amplification strategy could be used in relative biosensor developments for various applications, which the performance, applicability and feasibility are demonstrated and confirmed in this study. Based on the major purpose to design platform analytical method, four brand new biosensors under the assistance of nanoparticles and biomaterials were developed. In first human IgE electrochemical biosensor, the human IgE is analyzed by aptamer modified gold nanoparticles coated electrode. With the amplification strategy of G-rich cDNA, the extreme low detection limit as 0.16 pM was achieved. The assay also showed good selectivity in the presence of interferences with 100 folds higher concentration than analyte. In the second part, an electrochemical biosensor combined with aptamer and rolling circular amplification for prostate-specific antigen (PSA) detection was developed. With the amplification of the RCA reaction and the isolation of functional magnetic nanoparticles, the biosensor showed good linearity from 100 fM to 10 nM with detection limit as 22.3 fM. Four interferences showed negligible effect with presented biosensor. The third biosensor was discussed about monitoring eosinophil cationic protein (ECP) during cell culture experiments. Heparin modified functional magnetic nanoparticles were used as ECP probe and magnetic drove for analyte gathering. With the pre-concentration and isolation effects provided by functional magnetic nanoparticles, ECP could be analyzed within concentration range from 1 to 1000 nM. The estimated detection limit was 0.30 nM based on the definition of signal to noise ratio. The biosensor also showed good recovery in the cell culture medium RPMI 1640 after incubated with Beas-2B cell line. The last aptamer based biosensor was researched for solving possible issues in LC-MS. Ethanolamine was used as model compound to demonstrate the proposed scheme, which is unsuitable to be direct analyzed by LC-MS. With the transference and amplification strategy by artificial designed barcode DNA, the ethanolamine was indirectly analyzed by LC-MS within linear range from 5 nM – 5 μM. The estimated detection limit was 1.2 nM. The developed biosensor provided a universal platform for other analytes by replacing aptamer and related barcode cDNA sequence.
URI: http://etd.lib.nctu.edu.tw/cdrfb3/record/nctu/#GT079725801
http://hdl.handle.net/11536/140927
顯示於類別:畢業論文