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研究生： 謝易恭
論文名稱： 環境及生物樣品中奈米微粒偵測技術之研究
The determination of nano-particles in environmental and biological samples
指導教授： 王竹方
口試委員: 蔣本基
張怡怡
孫毓璋
李文智
王竹方
學位類別： 博士
Doctor
系所名稱： 原子科學院 - 生醫工程與環境科學系
Department of Biomedical Engineering and Environmental Sciences
論文出版年： 2012
畢業學年度： 100
語文別： 英文
論文頁數： 124
中文關鍵詞： 奈米微粒 、雷射剝蝕感應耦合電漿質譜儀 、電子式低壓衝擊器 、量子點 、半導體排放
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    • 奈米微粒定義為粒徑小於100 奈米的粒子，它具有高氣管沉積性、大的表面積、誘導發炎反應之特性及可能經由血液循環系統累積在多處器官造成健康危害。目前已知大氣中奈米微粒為人類主要的被動暴露於奈米微粒之途徑，因此，發展分析大氣中奈米微粒之技術成為評估奈米微粒健康危害時不可或缺的重要技術。研究中使用電子式低壓衝擊器(ELPI)採集奈米微粒，並嘗試發展以標準溶液滴在濾紙上模擬ELPI收集到之樣品，以此製作實驗室製備之標準濾紙樣品，後續以雷射剝蝕感應耦合電漿質譜儀(LA-ICP-MS)進行元素分析。研究結果顯示實驗室製備之標準濾紙所建立檢量線線性範圍極佳，與傳統之雙粒徑採樣器採樣後續以ICP-MS分析之比對結果證明本研究所發展的方法可靠性甚高，極具實用的價值。實驗中針對新竹科學園區及中部科學園區周界進行採樣，嘗試找出半導體業排放之特性元素。研究結果顯示細微粒之砷及矽可能來自於科學園區內半導體產業的排放，除了砷與矽外，空氣中微量元素如錳、鈷、鎳、銅、硒、銣、銀、鎘及銫亦為可能之半導體工廠排放元素。除分析大氣中奈米微粒外，研究亦將發展以LA-ICP-MS分析生物體內奈米微粒的分布狀況，實驗中用於注入生物體之奈米微粒包括掺雜釓改質之氧化鐵奈米微粒及硒化鎘/硫化鋅無機量子點(CdSe QDs)，用以釐清奈米微粒進入生物體之反應及遷移狀況。研究中以LA-ICP-MS結合MATLAB建立元素分佈的生物影像技術，分析掺雜釓改質之氧化鐵奈米微粒注入小鼠腫瘤之分布及其濃度對於磁共振熱治療(MFH)的影響。研究結果顯示奈米微粒注入腫瘤後仍保持原本結構而未分解，其分布濃度正比於熱治療效果。此外，研究中亦探討CdSe QDs經靜脈注入及氣管灌注後在小鼠器官累積及分布情形，結果顯示靜脈注射後QDs累積在肝、脾及腎上腺，氣管灌注則僅累積於肺臟。由螢光顯微鏡觀察所得到的QDs位置與LA-ICP-MS之Cd-114影像呈現相當高的一致性;而從Cd-114與Se-82樣品中的訊號相關性及比值可做為評估QDs是否有降解現象。

    Table of contents Content Page Publications relevant to the scope of this thesis…………………………………….. i Abstract……………………………………………………………………………… ii Table of contents…………………………………………………………………….. v List of figures………………………………………………………………………... vii List of tables…………………………………………………………………………. xi Chapter 1 Introduction 1 Chapter 2 Elemental analysis of airborne particulate matter using an electrical low pressure impactor and laser ablaion inductively coupled plasma mass spectrometry 9 Chapter 3 The silicon determination in airborne particulate matter using an electrical low pressure impactor and laser ablation inductively coupled plasma mass spectrometry 30 Chapter 4 Determination of high-tech related toxic metals in ultrafine, fine and coarse airborne particles at Hsinchu Science Park 48 Chapter 5 Using LA-ICP-MS to bioimage multiple elements in mouse tumors after hyperthermia 65 Chapter 6 The biodistribution of CdSe quantum dots in mouse tissues: A laser ablation inductively coupled plasma mass spectrometry study 76 Chapter 7 Biointeractions of inhaled CdSe quantum dots in a mouse lung by using laser ablation inductively coupled plasma mass spectrometry 95 Chapter 8 Conclusions and recommendations 107 Reference 110 Appendix 124 List of figures Figure Page Fig. 1-1 Scope of the research………………………………………………………. 7 Fig. 1-2 Flowchart of the research…………………………………………………... 8 Fig. 2-1 Airborne particulates collected on the filters of ELPI stages (a) 1, (b) 3, (c) 8, and (d) 10………………………………………………………………………… 14 Fig. 2-2 Photographs of samples of a standard solution on the filter medium (a) before and (b) after evaporation of the solvent (water)…………………………….. 15 Fig. 2-3 Schematic representation of the laser grid pattern used to ablate a single spot on filter of sample C…………………………………………………………… 17 Fig. 2-4 Qualitative scanning spectrograms of the blank, standard, and real sample filters analyzed through LA-ICP-MS (operating conditions: laser fluence, 16.7 J cm−2; defocused distance, 1.5 mm)…………………………………………………. 19 Fig. 2-5 Responses of 75As ablated using the proposed laser grid patterns from the blank, standard, and real-sample filters……………………………………………... 20 Fig. 2-6 Total analyte signals from the blank filter (a), standard filter (b, 1st scan; c, 2nd scan), and samples A (d, 1st scan; e, 2nd scan), B(f, 1st scan; g, 2nd scan), and C (h, 1st scan; i, 2nd scan)……………………………………………………………… 21 Fig. 2-7 Standard addition calibration curves for As in the sample spots on the standard filter and the real-sample filters A, B, and C……………………………… 24 Fig. 2-8 Correlations between the PM10 and PM2.5 data obtained using dichotomous sampler (Dicho) and ELPI for Zn, Pb, and Cd in an aerosol sample collected in the Hsinchu area (n = 13)………………………………………………. 28 Fig. 3-1 Location of the study area………………………………………………….. 33 Fig. 3-2 Time-resolved signals of blank filter, standard filter (contains 64 ng of silicon), real sample (coarse) and NIST 2783……………………………………….. 37 Fig. 3-3 Calibration graph of 28Si for the laboratory-prepared standard filter……… 38 Fig. 3-4 Annual silicon concentration of ultrafine, fine and coarse airborne particles at HSP and CTSP from 2007 to 2011……………………………………… 41 Fig. 3-5 Characteristics of airborne silicon particle (a) daily concentrations of silicon in ultrafine, fine and coarse airborne particles; (b), (c) size distribution and cumulative mass fraction of silicon in airborne particles…………………………… 44 Fig. 3-5 Size distribution and cumulative mass fraction of silicon in airborne particles collected (a) in the afternoon on August 13 (b) in the evening on August 14 and (c) in the evening on August 15……………………………………………... 46 Fig. 4-1 Location of the study area…………………………………………………. 51 Fig. 4-2 Elemental concentration variations of (a) Ni, (b) Cd, (c) Cu, (d) As, (e) Ga, (f) In, and (g) Tl in different size fractions at HSP from 2007 to 2011 and the annual revenue at HSP…………………………………………………………..…... 58 Fig. 4-3 Mean mass size distributions of the elements collected at HSP. The values and error bars of elements are geometric means and standard derivation calculated from 35 samples except W, Mo, Sn and Se from 6 samples………………………… 60 Fig. 5-1 Photograph of the original tissue slice under an LA camera………………. 68 Fig. 5-2 Photograph of the tissue slice after Prussian blue staining………………… 68 Fig. 5-3 Photograph of the tissue slice after H&E staining…………………………. 69 Fig. 5-4 Mapping and ion intensities of (a) 56Fe and (b) 158Gd atoms. The dashed line indicates the path taken during the time-resolved analyses of Fe and Gd atoms. 71 Fig. 5-5 Mapping and ion intensities of C, P, S, Ni, Cu, and Zn elements. The dashed line indicates the path taken during the time-resolved analyses of C, P, S, Ni, Cu, and Zn atoms………………………………………………………………... 73 Fig. 5-6 Combined mapping of Ni, Cu, and Fe atoms………………………………. 74 Fig. 6-1 (a) the Cd-114 mapping image in spleen from LA-ICP-MS; (b) higher magnification of selected Cd-114 hot zone and its corresponding H&E stained image (c) and fluorescence image (d)………………………………………………. 82 Fig. 6-2 The intensity profiles of each element (from top to bottom, P-31, Cu-63, Zn-66, Fe-56, Cd-114, and Se-82, respectively) in spleen and their corresponding mapping images. Recorded intensity profiles were scanned along the indicated red lines………………………………………………………………………………….. 85 Fig. 6-3 (a) the Cd-114 mapping image in liver from LA-ICP-MS; (b) higher magnification of selected Cd-114 hot zone as shown in (a); and its corresponding H&E stained image (c); fluorescence images of selected area (d-f), and their corresponding H&E stained images (g-i)…………………………………………… 87 Fig. 6-4 The intensity profiles of each element (from top to bottom, P-31, Cu-63, Zn-66, Fe-56, Cd-114, and Se-82, respectively) in liver and their corresponding mapping images. Recorded intensity profiles were scanned along the indicated red lines………………………………………………………………………………….. 88 Fig. 6-5 (a) the Cd-114 mapping image in kidney from LA-ICP-MS; (b) higher magnification of selected Cd-114 hot zone as shown in (a); and its corresponding H&E stained image (c); fluorescence images of selected area (d)………………….. 91 Fig. 6-6 The intensity profiles of each element (from top to bottom, P-31, Cu-63, Zn-66, Fe-56, Cd-114, and Se-82, respectively) in kidney and their corresponding mapping images. Recorded intensity profiles were scanned along the indicated red lines………………………………………………………………………………….. 92 Fig. 7-1 (a) The fluorescence image of lung sample, where the yellow spots are the locations of quantum dots (b) the corresponding LA-ICP-MS Image…………........ 99 Fig. 7-2 The intensity profiles and corresponding LA-ICP-MS mapping image of (a) Cd-114 and (b) Se-82……………………………………………………………. 101 Fig. 7-3 The mapping images of each element observed in this study……………… 102 Fig. 7-4 (top) The H&E stained image pointing out the accumulation of lymphocytes and alveoli surrounded by accumulated macrophages. (bottom) Enlarged Cd-114 hot zones; (a) the distribution of Cd-114 in the lung section; (b) Enlarged view of the rectangular region indicated in panel (a); (c) corresponding H&E stained image of panel (b); (d) corresponding fluorescence image of panel (b); and (e) fluorescent image with the matrix fluorescence filtered out (greens in panel (d))…………………………………………………………………………….. 105 List of tables Table Page Table 2-1 Collecting strategies and size distributions for the ELPI and dichotomous samplers……………………………………………………………..... 13 Table 2-2 Optimized operating conditions for LA-ICP-MS………………………... 16 Table 2-3 Detection limits (3σ) for all analyte elements and correlation coefficients (r2) for the standard curves……………………………………………...................... 22 Table 2-4 RSDs (n = 5) and mean signal counts obtained through LA-ICP-MS for the blank, laboratory-prepared standard, and real sample (sample C) filters……….. 23 Table 2-5 Elemental contents (ng) obtained from samples B and C using external standard calibration (ESC) and standard addition (SA)…………………………….. 26 Table 3-1 Sampling information for sampling sites at the study area………………. 35 Table 3-2 Silicon concentrations (arithmetic mean ± SD; in ng m-3) of ultrafine, fine and coarse particles collected at CTSP and HSP………………………………. 39 Table 4-1 Sampling information for sampling sites at HSP………...………………. 53 Table 4-2 Elemental concentrations (arithmetic mean ± SD, in ng m−3) of ultrafine, fine and coarse particles collected at HSP (n = 117, except marked as *).. 55 Table 4-3 Comparisons of the metal total contents in the airborne particles at HSP with the literature values…………………………………………………………….. 62 Table 4-4 Mean concentrations in the size range of 0.03―0.99 μm of Mn, Ni, As, and Cd collected at HSP and its limit values………………………………………... 63 Table 5-1 Operating conditions for the line scan mode of the LA-ICP-MS system... 70 Table 6-1 The correlation between each monitored elements in spleen……………. 84 Table 6-2 The correlation between each monitored elements in liver……...………. 89 Table 6-3 The correlation between each monitored elements in kidney……………. 93 Table 6-4 The correlation coefficients of Cd-114 and Se-82, S-34 and C-13, S-34 and P-31, respectively……………………………………………………………….. 94 Table 7-1 The correlation between each element observed in this study….…….... 103

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