臺灣博碩士論文加值系統

病毒生物氣膠，如流感病毒 ( Influenza Virus )、嚴重急性呼吸道症候群( severe acute respiratory syndrome, SARS ) 病毒等因其奈米粒徑，可於空氣中快速傳播。而液體式衝擊瓶 ( liquid impingers ) 為廣泛使用的空氣生物氣膠採樣方法之一，其對於生物氣膠的活性保存具有優勢且分析方法多元，但目前市售之液體式衝擊瓶，其最低截取粒徑約為 300 nm，對於 < 100 nm 之生物氣膠收集之效率不佳，僅約 10-20% 左右。因此本研究嘗試以不同實驗條件 (採樣液介質、採樣流速、顆粒過濾床、採樣時間) 提升液體式衝擊瓶對於奈米生物氣膠之採集效率及存活率。
本研究團隊先前利用衝擊瓶 ( All Glass Impinger 30, AGI-30 ) 應用填充床之過濾機制，尋找最佳之實驗參數，結果顯示當採樣流率為 4 lpm、填充玻璃珠粒徑為 3 mm、填充床高度 10 cm、採樣液容積 30 mL 之條件下，對於奈米氣膠的採樣效率可達99％。而本研究利用上述最佳條件，以添加追蹤劑(uranine)之MS2噬菌體作為試驗病毒氣膠，實驗系統結合了零級氣體供應系統、奈米生物氣膠微粒產生系統、生物氣膠採集系統，評估其他不同條件變因，包含以無菌水、peptone、LB Broth作為採樣液介質；採樣流速為 4 lpm、6 lpm、12.5 lpm (使用AGI-30之標準採樣流速) 等，探討隨著採樣時間變化 ( 10min、20min、30min )對於MS2噬菌體氣膠存活率之影響。
研究結果顯示，對於MS2噬菌體氣膠而言，使用Peptone做為採樣液介質，相較於無菌水、LB Broth，採樣液介質Peptone可得到較佳之收集效率。而採樣流速12.5 lpm時，相較於4 lpm、6 lpm，採樣流速12.5 lpm可得到較佳之收集效率。此外，於液體式衝擊瓶中，填充顆粒過濾床，相較於無顆粒過濾床之衝擊瓶，填充顆粒過濾床可增進奈米生物氣膠之收集效率。最後，採樣20分鐘，相較於採樣時間10分鐘、30分鐘，採樣20分鐘之收集效率較佳。
本研究結果，可了解不同實驗變因對於奈米生物氣膠之收集效率影響，有利於監測奈米尺度的病原體，以確實達到公共衛生上預防疾病之目的。

The fact that disease-causing airborne viruses, such as SARS, MERS-coV, and influenza virus, spread fast among human beings is partially due to their nanoscale particle size. The liquid impingers are widely used to sampling air bioaerosols, which have advantages for the preservation of bioaerosols. However, the lowest cutoff diameter of commercially available samplers for bioaerosols is about 0.3 μm and their physical collection efficiency for nanoparticle is only about 10-20%. Therefore, this study aims to improve the collection efficiency and survival rate of the liquid impinge for the collection of nano-bioaerosols with different sampling conditions.
Our previous research has shown that the excellent collection efficiency ( ~99% ) of All Glass Impinger ( AGI-30 ) with packed glass beads for nanoparticles occurred in the condition of 4-lpm, 6-lpm sampling flow rate, grain size of 3 mm, 10-cm depth of packed bed with 30 ml of collection liquid.
In this study, the above conditions and MS2 aerosols mixed with a physical tracer ( uranine ) was used. After periods of 10, 20 and 30 min, sampling liquids were titred using the plaque assay to test biological collection efficiency and survival rate. The experimental system was combined with zero gas supply system, nano-bioaerosols particle production system, bioaersols sampling system. We will try to obtain the optimal efficiency by changing following conditions, including type of sampling liquid ( Sterile water, Peptone, LB Broth ), sampling flow rates ( 4 lpm, 6 lpm, 12.5 lpm ), granular bed and sampling time ( 10 min, 20 min, 30 min ).
An ideal sampler is the one that has steady collection efficiency and preserves bacteriophage viability during sampling. The results showed the highest collection efficiency for airborne nano-bioaerosols occurred in the condition of AGI-30 sampler packing with glass beads, peptone as sampling liquid, 12.5 lpm sampling flow rate, 20 min sampling time.
The study provides a fundamental basis for sampling nano-airborne virus.

目錄
誌謝 I
摘要 II
Abstract IV
目錄 V
圖目錄 VIII
表目錄 X
第一章 前言 1
1-1 研究背景 1
1-2 研究目的 2
第二章 文獻回顧 3
2-1 生物氣膠 3
2-1-1 生物氣膠之來源 3
2-1-2生物氣膠之特性與危害 4
2-1-3 替代性病毒 7
2-1-4 現有之生物氣膠採樣技術優勢與限制 7
2-2 液體衝擊瓶 10
2-2-1 液體衝擊瓶之種類與收集機制 10
2-2-2液體衝擊瓶之收集效率 12
2-3 填充床(顆粒過濾床) 14
2-3-1 填充床之收集效率 14
第三章 材料與方法 16
3-1研究設計 16
3-2實驗系統 18
3-3 實驗設備與方法 20
3-3-1 零級氣體供應系統 20
3-3-2 氣膠生成系統 20
3-3-3 氣膠採樣裝置 21
3-3-4 多功能微量盤式分析儀 (Multimode microplate readers) 21
3-3-5掃描式氣膠粒徑分析儀 22
3-4生物氣膠培養 23
3-4-1 寄主細菌 23
3-4-2 噬菌體之懸浮液 24
3-4-3 採集樣本 26
3-5藥品、材料與其他設備 28
3-5-1實驗藥品 28
3-5-2實驗材料 28
3-6 計算公式與指標參數 29
3-6-1 採樣器品質(sampler quality) 29
3-6-2 追蹤劑採樣之收集效率值 (Etracer) (lower limit on the percentage of tracer collected by the sampler) 29
3-6-3噬菌體採樣之收集效率值 (EPFU) (lower limit on the percentage of viruses collected by the sampler) 30
3-7統計分析 31
第四章 結果與討論 32
4-1 噬菌體氣膠之粒徑分布圖 32
4-2 採樣器品質 ( sampler quality ) 33
4-2-1採樣流速對於採樣器品質之影響 33
4-2-2採樣液介質對於採樣器品質之影響 38
4-3 生物收集效率 42
4-3-1採樣流速對於生物收集效率之影響 42
4-3-2採樣液介質對於生物收集效率之影響 48
4-4 生物收集效率之多變量線性迴歸分析 55
4-5 研究限制 57
第五章 結論與建議 58
5-1 結論 58
5-2 建議 59
參考文獻 60

圖目錄
圖 1、生物氣膠之粒徑分布圖(A)蛋白質(B)病毒(C)細菌(D)真菌孢子(E)花粉[27-33] 5
圖 2、 ICRP 沉積模式預測之沉積曲線[7] 6
圖 3、液體衝擊瓶採樣器 10
圖 4、研究架構流程圖 16
圖 5、實驗系統圖 19
圖 6、Collison Nebulizer 20
圖 7、多功能微量盤式分析儀 21
圖 8、寄主細胞培養流程圖 23
圖 9、噬菌體懸浮液培養流程圖 25
圖 10、實驗培養流程圖 27
圖 11、MS2噬菌體氣膠數量濃度與粒徑關係分布圖 32
圖 12、採樣液介質為無菌水時，不同採樣流速之採樣器品質 ( sampler quality, qS )與時間之關係圖 35
圖 13、採樣液介質為Peptone時，不同採樣流速之採樣器品質 (sampler quality,qS)與時間之關係圖 36
圖 14、採樣液介質為LB broth時，不同採樣流速之採樣器品質 (sampler quality,qS)與時間之關係圖 37
圖 15、採樣流速4 lpm時，不同採樣液介質之採樣器品質 ( sampler quality, qS ) 與時間之關係圖 39
圖 16、採樣流速6 lpm時，不同採樣液介質之採樣器品質 (sampler quality,qS) 與時間之關係圖 40
圖 17、採樣流速12.5 lpm時，不同採樣液介質之採樣器品質 ( sampler quality, qS ) 與時間之關係圖 41
圖 18、採樣液介質為無菌水時，不同採樣流速與病毒採樣之收集效率值( EPFU )、時間之關係圖 45
圖 19、採樣液介質為Peptone時，不同採樣流速與病毒採樣之收集效率值( EPFU )、時間之關係圖 46
圖 20、採樣液介質為LB Broth時，不同採樣流速與病毒採樣之收集效率值( EPFU )、時間之關係圖 47
圖 21、採樣流量4 lpm時，不同採樣液介質與病毒採樣之收集效率值 ( EPFU )、時間之關係圖 52
圖 22、採樣流量6 lpm時，不同採樣液介質與病毒採樣之收集效率值 ( EPFU )、時間之關係圖 53
圖 23、採樣流量12.5 lpm時，不同採樣液介質與病毒採樣之收集效率值( EPFU )、時間之關係圖 54

表目錄
表 1、室內活動佔一日時間之百分比 4
表 2、生物氣膠採樣器之比較 9
表 3、不同液體衝擊瓶採集之粒徑範圍與效率 12
表 4、實驗條件 17
表 5、不同採樣流速之收集效率比較 44
表 6、不同採樣介質與收集效率之比較 50
表 7、不同實驗變因對收集效率影響之比較 51
表 8、收集效率與不同條件之多變量線性迴歸分析參數估計 56

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