發展可應用於偵測類志賀毒素一及金黃色葡萄球菌之官能化磁性奈米粒子分析平台

Abstract

生化樣品通常含有複雜的基質，因此適當的樣品前處理是相當重要的。由於磁性奈米粒子有易合成、易修飾，及具磁性性質等優點，磁性奈米粒子常被應用做為親和探針，用於濃縮複雜樣品中的微量目標物。致病菌和蛋白質毒素會引發感染以及污染食物，因此發展快速的分析方法來確認複雜樣品中的細菌和蛋白質毒素的存在是相當重要的課題。因此在本論文中，我發展了官能化磁性奈米粒子結合基質輔助雷射脫附游離質譜法之分析平台，用於選擇濃縮並偵測複雜樣品中由於受到大腸桿菌O157:H7的污染所產生的的類志賀毒素一及金黃色葡萄球菌。在論文的第一部份，我建立了可偵測類志賀毒素一的官能化磁性奈米粒子分析平台，由氧化鋁包覆的氧化鐵奈米粒子首先在鴿蛋蛋白中抓取鴿卵白蛋白，藉由磷酸蛋白質－鴿卵白蛋白－上的磷酸根與磁性奈米粒子的鋁金屬中心進行螯合作用，製作了表面修飾有鴿卵白蛋白的氧化鋁磁性奈米粒子(POA-Fe3O4@Al2O3 MNPs)。由於類志賀毒素一為一種AB5蛋白質，其B單體能夠與細胞膜上的三醣基Gal-α(14)-Gal-β(14)-GlcNAc結合。因此，此POA-Fe3O4@Al2O3 MNPs具有選擇濃縮存在於小體積(20 L)的細菌分解液和複雜食物樣品中的類志賀毒素一的能力。實驗結果顯示此分析平台對於類志賀毒素一的偵測極限可低至~44 pM左右。在論文的第二部份，則是利用具有聚組胺酸的胜肽(HHHHHHDEEGLFVD)將其修飾在氧化鋁包覆的氧化鐵磁性奈米粒子，合成出表面修飾有DVFLG的官能化磁性奈米探針(DVFLG-Fe3O4@Al2O3 MNPs)，此奈米探針具有辨識抓取金黃色葡萄球菌的能力，並藉由基質輔助雷射脫附游離質譜法確認細菌身份。實驗中也藉由微波輔助加熱的方式加速奈米探針選擇濃縮抓取目標細菌的步驟，可有效將濃縮時間縮短至一分鐘左右，且偵測極限可低達~ 105 cells mL-1。在論文的第三部份，我則是探討如果將磁性奈米探針修飾上不同數目的DVFLG重複胜肽片段，探討這些官能化親和性探針對不同菌屬及不同葡萄球菌種的親和辨識能力，實驗結果顯示表面修飾具有兩重複DVFLG片段的磁性奈米粒子對於金黃色葡萄球菌的辨識選擇能力最佳。總括而言，在本論文所發展的以磁性奈米子為基礎，且以基質輔助雷射脫附游離質譜法為工具之分析平台，可以有效地用於在複雜樣品中濃縮並確認類志賀毒素一及金黃色葡萄球菌的存在，這些方法具有製備簡單、低成本、分析時間短和高靈敏度等優點。因此，本論文中所發展這些分析平台應有潛力應用於實際樣品之分析。

Biochemical analysis is usually used to deal with complex samples. Thus, sample pretreatment is essential in order to reach satisfied analysis results prior to utilizing suitable analytical tools for the analysis of target analytes. Owing to the features of ease of synthesis/modification, and magnetic property, magnetic nanoparticles (MNPs) have been used as affinity probes for concentrating target analytes at trace levels from complex samples. Pathogenic bacteria and protein toxins can cause infections and contaminate food products. Thus, it is significant to have analytical methods that can be used to rapidly enrich and characterize the identity of the bacteria and protein toxins in complex samples. In this study, functional MNP-based platforms combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for selective enrichment and detection of Staphylococcus aureus and Shiga-like toxin 1 (SLT-1) generated from Escherichia coli O157:H7 were developed. In the first part of this thesis, the MNP-based platform for detection of SLT-1 was established. Alumina coated iron oxide (Fe3O4@Al2O3) MNPs were used to trap pigeon ovalbumin (POA) directly from complex pigeon egg white to generate POA-Fe3O4@Al2O3 MNPs through the chelation of aluminum metal center on the surface of the Fe3O4@Al2O3 MNPs and phosphate groups from the phosphorprotein, i.e. POA. POA is also a glycoprotein containing Gal-α(14)-Gal-β(14)-GlcNAc termini. SLT-1 is a AB5 protein, in which B subunits can bind to Gal-α(14)-Gal-β(14)-GlcNAc termini on the cell membrane. Thus, the POA-Fe3O4@Al2O3 MNPs were used to selectively enrich SLT 1B from small volumes (20 L) of complex cell lysates and food samples containing SLT-1. The results show that the limit of the detection of this approach was as low as ~ 44 pM. In the second part of this study, polyhistidine tagged DVFLG (HHHHHHDEEGLFVD) were used to functionalize Fe3O4@Al2O3 MNPs. The generated DVFLG-Fe3O4@Al2O3 MNPs have targeting capacity toward S. aureus. MALDI-MS was used as the detection tool for characterization of the bacteria trapped by the MNPs. Microwave-heating was used to accelerate the selective enrichment, which only took 1 min. The limit of detection toward S. aureus was as low as ~ 105 cells mL-1. In the third part of this study, peptides containing different repeated units of DVFLG were immobilized on the surface of the Fe3O4@Al2O3 MNPs, which were then used as affinity probes to examine the trapping selectivity of the generated MNPs toward different genus bacteria or different Staphylococcus spp. The results show that the peptide containing two repeated units of DVFLG immobilized on the Fe3O4@Al2O3 MNP perform the best targeting capacity toward S. aureus. In summary, these MNP-based platforms combined with MALDI-MS for the analysis of SLT 1B and S. aureus have the advantages including ease-of-generation, cost-effective, short analysis time, and high sensitivity. Thus, these established platforms are potentially to be used in real world applications.