以C60+-Ar+共濺射與動態二次離子質譜術平行偵測與定量胜肽分子

Abstract

以簇離子(cluster ions)作為濺射離子源的時間飛行二次離子質譜儀（TOF-SIMS)已被證明是分析生物樣本之頗具發展潛力的一項技術，其中碳簇離子(C60+)已引起廣大的研究興趣。隨著高質量的分子離子飛濺而出，可在不預先分離或同位素標記情況下同時偵測多種分子。目前多數研究是使用靜態二次離子質譜儀作分析，但其在縱深分佈分析上步驟較為複雜且偵測極限較差，因此本研究主要使用動態二次離子質譜儀分析技術。摻雜胜肽的海藻糖基質試片，被用作檢視平行偵測和定量分析生物性樣品時相關參數的模型。海藻糖基質同時混合不同種類與濃度的胜肽，經由實驗發現，於海藻糖分子中胜肽分子的二次離子訊號相對強度是直接正比於其在基質中的濃度。因此，透過繪製各胜肽存在於海藻糖基質的比例對比於其二次離子的相對強度圖，即可得到各種胜肽相對於基質的校正曲線。實驗證實利用這些曲線，即可達成各胜肽在基質中的平行檢測、識別及定量分析。另外，為了抑制高能量C60+離子濺射造成並因而限制後續濺射分析的碳沉積，同時使用低能量Ar+離子共同濺射摻雜胜肽之海藻糖試片。其顯示共同濺射的技術較單獨使用C60+離子濺射可產生較為穩定的分子離子訊號，依此特性，共同濺射技術更適用於分析厚度較大的樣品。此外實驗亦發現，負責產生分子離子的主要仍為C60+離子，輔助濺射的Ar+離子其電流設定並不影響定量分析中校正曲線數值。

Using pulsed primary cluster ions, especially for C60+ cluster ion, time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been shown to be a promising technique for analyzing biological specimens. With molecular secondary ions of high mass, multiple molecules can be identified at the same time without prior separation or isotope labeling. While current reports are based on static-SIMS that makes depth profile more complicated, a dynamic-SIMS based technique is reported in this work. Mixed trehalose and peptides were used as a model for evaluating the parameters that lead to parallel detection and quantification of biomaterials. Trehalose is mixed with different peptides separately with varied concentrations of peptides. It is found that the normalized secondary ion intensity of peptide as respect to trehalose is direct proportional to its concentration in the matrix. Therefore, by plotting the percentages of peptides exist in trehalose versus their normalized SIMS intensities, calibration curves of each peptide are obtained. Using these curves, it is shown that parallel detection, identification, and quantification of multiple peptides in the matrix can be achieved. To suppress the associated carbon deposition with high energy C60+ bombardment that leads to suppressed ion intensity in prolonged profiling, a low energy Ar+ is used to co-sputter the peptide-doped trehalose thin film. It is shown that the co-sputtering technique yields more steady molecular ion intensity than single C60+ beam. In other words, the co-sputtering is more suitable for analyzing thick specimens. Furthermore, because the C60+ is responsible for generating the molecular ions, it is found that the does of the auxiliary Ar+ does not change calibration curve for quantification.