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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48772

Title: 功能性金屬奈米粒子搭配微孔薄膜與雷射脫附游離質譜儀同時檢測多種蛋白質
Simultaneous Detection of Multiple Proteins Using Functionalized Metal Nanoparticles Coupled with Microporous Membrane Substrate and Laser Desorption/ionization Mass Spectrometry
Authors: Chang, Chia-Yin
張家寅
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 奈米粒子;雷射脫附游離法;質譜儀;纖維薄膜;多重檢測;蛋白質
Nanoparticles;Laser desorption/ionization;Mass spectrometry;Cellulose membranes;Multiple detection;Proteins
Date: 2016
Issue Date: 2018-08-22T06:31:10Z
Abstract: 在此研究中,我們使用功能性奈米材料以雷射脫附游離質譜儀(Laser–induced desorption/ionization mass spectrometry, LDI-MS)簡單的同時檢測多種蛋白質。我們使用凝血酶(thrombin)、血管內皮生長因子-A165(vascular endothelial growth factor-A165, VEGF-A165)、血小板生長因子BB(platelet-derived growth factor-BB, PDGF-BB)作為目標蛋白來證實此檢測方法。以三種奈米粒子(金,銀和鉑奈米粒子)修飾上辨識目標蛋白質之適合體或是抗體以達到選擇性分析。修飾後之金屬奈米粒子會專一性地結合目標蛋白,之後將奈米粒子與目標蛋白混合液點樣於乙酸纖維薄膜(cellulose acetate membrane, CAM)以LDI-MS進行分析。多孔洞CAM在此時可以做為一種分離介質,隨著奈米粒子對應之目標蛋白濃度提高而導致奈米粒子質量增加亦或是降低奈米粒子對CAM的親和力,使得奈米粒子更容易滲入薄膜內部或是底部,在質譜中的結果顯示,經雷射照射後產生的金屬團簇離子的訊號([Mn]¬¬+; M = Au和Ag, n = 1-3)以LDI-MS分析CAM表面金屬團簇離子訊號會因此降低。但是鉑團簇訊號卻是相反的,會因為增加目標蛋白質的濃度而提高鉑團簇訊號,這是因為功能化鉑奈米粒子在PDGF-BB的存在下會造成聚集的現象。在LDI-MS的分析過程中,奈米粒子搭配薄膜的系統具有將蛋白質濃度轉變為金屬團簇訊號,同時增幅訊號已達到更好的偵測極限。我們所開發的系統可以選擇性地檢測到不同蛋白質低至pM等級,並且能夠在樣品不需濃縮、分離或純化等預處理情況下,藉由不同金屬團簇的訊號而達到同時檢測的目的。我們所提出的使用奈米材料修飾上辨識分子搭配LDI-MS,應用於未來同時監測不同蛋白、腫瘤標記物、病原體或癌細胞具相當的發展潛力。
In this work we demonstrate a simple assay for the simultaneous detection of multiple proteins by functionalized nanomaterials assisted laser-induced desorption/ionization mass spectrometry (LDI-MS). We chose thrombin, vascular endothelial growth factor-A165 (VEGF-A165) and platelet-derived growth factor-BB (PDGF-BB) as model proteins to demonstrate our sensing method. Selective detection of these analytes was achieved by modifying nanoparticles of three different metals (Au, Ag and Pt) with their corresponding aptamer or antibody. Functionalized metal nanoparticles (NPs) bound to their specific analyte was subjected to LDI-MS on a cellulose acetate membrane (CAM). The microporous CAM served as a medium for an in situ separation of NPs based on the increase in particle weight and/or decreased affinity towards CAM with increasing analyte concentration. As a result, the intensities of the signals of the metal cluster ions ([Mn]¬¬+; M = Au or Ag n = 13) decreased in the mass spectra under pulsed laser irradiation. Platinum cluster ion signal being the only exception, signal increases with analyte concentration, due to aggregation of Pt NPs in the presence of PDGF-BB. The nanoparticles/CAM system can function as a signal transducer and amplifier through transformation of protein concentration to intense metal clusters ions signal during LDI-MS analysis. Our developed system can selectively detect proteins down to picomolar region. Most importantly, the system is able to detect multiple proteins simultaneously without any pre-concentration, separation or purification process since LDI-MS coupled with CAM effectively removes all signals except metal cluster ions. This sensing system shows great potential for the simultaneous detection of different protein, tumor markers, pathogens, and cancer cells by functionalizing with suitable ligands to nanomaterials.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010336007.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48772
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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