單一完整病毒及奈米粒子質譜學
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2007
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摘 要
病毒是地球上最簡單的生命形式,它僅僅由DNA(或RNA)分子和一個蛋白質外殼組成。除了原核生物(細菌和原始細菌)外,病毒是地球上第二類最常見的生物體,它們是海洋中最常見的生命形式。測量這些起源各異、個體微小的生物粒子質量及其在特定群體中的變異情況,對它們結構和特性的瞭解是非常有幫助的。採取一個比較溫和的離子化技術,運用自行研發的微型離子阱質譜儀,準確的測量了單一個完整病毒的重量。
前人測量病毒質量的方法誤差在±15%左右,這無法分辨病毒在質量上微小的差異。為了測定它們的質量,病毒首先必須被轉換成氣相粒子,並獲得電荷。但是,在這項過程中,病毒必須是完好無缺。因此,採用了雷射誘導聲波脫附法。病毒粒子經由雷射激發產生的聲波從樣品中釋出後,被捕獲在一個有特殊幾何結構和交流電場的「離子阱」中。個別的病毒粒子被捕獲後就可以測量其質量了。在離子阱中,病毒粒子受雷射激發後會發出散射光,部分散射光經由數位相機接收,記錄此粒子的運動軌跡,其餘的光進入一個測量裝置來分析此散射光信號的頻率。散射光和入射雷射光的性質不同,這是由於病毒粒子在離子阱中有如彈簧般的振盪,而此振盪頻率反映出該病毒的重量。
測量了直徑在80奈米至300奈米三種不同類型病毒的質量,測得的實驗誤差僅僅在±1%左右。結合其他分析方法的結果後,此病毒的質量就能用來推斷一個病毒蛋白質外殼的組成數目,及其包含遺傳物質的數量。
這項質量測量的準確性相當高,這是由於採用了特殊的離子阱,而不是傳統的四極式離子阱。選擇的是圓柱型離子阱,雖然在這類型的阱中,被捕獲粒子的運動十分複雜;然而,它的優點是幾何結構非常簡單。建造了一個微型的圓柱離子阱,優化其幾何結構,並且用透明的導電板當作圓柱的末端電極。這種特殊的結構使得質譜技術能夠與顯微鏡技術結合,成功的測得單一個完整病毒的重量。
Abstract Measurements of a single whole cell and virus are the technological challenge for mass spectrometry. The sizes of biological particles are from 80nm to 10 μm. It is very difficult to generate the intact and biological particles in the vacuum condition. However, to produce gas-phase ions of viruses and cells that do not have rigid walls, we use the laser-induced acoustic desorption (LIAD) method. We also measure the mass of the National Institute of Standards and Technology (NIST) polystyrene which demonstrates the single particle mass measurement can be applied to determine the diameter of NIST polystyrene particles. In this thesis, the first part measures the macro-cell with the LIAD-QIT. And the second part improves the efficiency of the light scattering with the microscopy-CIT (cylindrical ion trap). In contrast to the QIT, the cylindrical ion trap (CIT) has a simple geometry. It holds the advantage that one of its end-cap electrodes can be replaced by a flat, electrically conductive glass plate that enables collection of more than 10% of the light radiating from the trapped particle. Recent studies show that viruses are the most abundant biological entities in the world’s oceans and are second to prokaryotes in terms of biomass on the planet. It is thus timely and of interest to develop mass spectrometry of whole viruses to expand our understanding of the simplest “life forms”.
Abstract Measurements of a single whole cell and virus are the technological challenge for mass spectrometry. The sizes of biological particles are from 80nm to 10 μm. It is very difficult to generate the intact and biological particles in the vacuum condition. However, to produce gas-phase ions of viruses and cells that do not have rigid walls, we use the laser-induced acoustic desorption (LIAD) method. We also measure the mass of the National Institute of Standards and Technology (NIST) polystyrene which demonstrates the single particle mass measurement can be applied to determine the diameter of NIST polystyrene particles. In this thesis, the first part measures the macro-cell with the LIAD-QIT. And the second part improves the efficiency of the light scattering with the microscopy-CIT (cylindrical ion trap). In contrast to the QIT, the cylindrical ion trap (CIT) has a simple geometry. It holds the advantage that one of its end-cap electrodes can be replaced by a flat, electrically conductive glass plate that enables collection of more than 10% of the light radiating from the trapped particle. Recent studies show that viruses are the most abundant biological entities in the world’s oceans and are second to prokaryotes in terms of biomass on the planet. It is thus timely and of interest to develop mass spectrometry of whole viruses to expand our understanding of the simplest “life forms”.
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Keywords
病毒, virus