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  • 學位論文

紙片常壓游離質譜法於生物醫學領域之應用

Biomedical Application of Paper-based Ambient Ionization Mass Spectrometry

指導教授 : 徐丞志

摘要


常壓游離質譜法(AIMS)因其樣品前處理簡易、操作方便的特性,已經被廣泛引入生物醫學領域之應用。以紙片為取樣基材的常壓游離法能快速由樣品中游離得到待測物訊號,且具有取樣快速和易於保存樣品的特性。 醫院與醫學檢驗中心常以血液中的代謝物檢測判斷病患的生理狀況。本論文中,我們採用即時直接分析游離法(DART)搭配離子阱質譜儀(ion trap MS),以濾紙作為承載血清樣品的基材,即能從微量的人類血清中偵測到膽固醇(cholesterol)脫水後的[M-H2O+H]+訊號。並加入同位素內標準品輔助,可定量樣品中的膽固醇濃度。以此紙片基質取樣搭配即時直接分析游離質譜法(pDART-MS)得到的膽固醇濃度,與商業化的酵素反應螢光檢測組(fluorometric-enzymatic assay)和傳統定量用之液相層析質譜儀(LC-MS)測量同一樣品所得濃度比較,在統計上並無顯著差異。同時,我們以此法測量二十一位參與超級馬拉松的參賽者於賽前、賽後、賽後一天所得的血清樣品。此常壓游離法僅需簡易的樣品前處理,且只需費時約一分鐘,即可得到一個血清樣品的膽固醇濃度。 此外,我們也嘗試將紙片常壓游離法應用於癌症診斷。以乳癌為例,在醫院的常規檢查中,醫師通常以皮下針取患部組織,並將組織沾黏於濾紙上,作為後續病理學檢驗之樣本。我們以該含有組織的紙片樣品,於紙片電噴灑游離法下進行代謝物質分布的快速檢測,判斷該組織為癌症/非癌症。為了降低來自複雜基質的化學干擾,我們於紙片電噴灑游離源後方裝設場不對稱離子遷移譜(FAIMS-PSI-MS)。我們對磷脂質類代謝物所在的質荷比(m/z)600-900的範圍進行統計分析,結果顯示此法所獲得的組織樣品之非定標性代謝物質掃描,具有區別癌症與健康組織的潛能,能於診療過程中,協助醫師定位腫瘤組織之位置。 常壓游離法因其快速、易於操作的特性,能對複雜的生物樣品進行高效率、直接的定量或定性分析。本論文中,描述兩種紙片為基材的生物樣品檢測法,證明常壓游離法於生物醫學領域、臨床檢驗的適用性。

並列摘要


Ambient ionization mass spectrometry (AIMS) techniques have been widely introduced to the field of biomedical analysis due to their simplicity in sample pretreatment and low cost of operation. The advantages of paper-based ambient ionization contains fast extraction of analytes from the raw sample, featuring in its high sampling efficiency and convenience of preserving sample. Blood test for endogenous small metabolites to determine physiological and biochemical states is routine for laboratory analysis. In this thesis we demonstrate that by combining a commercial direct analysis in real time (DART) ion source with an ion trap mass spectrometer in positive mode, native cholesterol in its dehydration form ([M-H2O+H+], m/z 369) is readily detected from few hundred nanoliters of human serum loaded onto a chromatography paper. Deuterium-labeled cholesterol was used as the internal standard to obtain the absolute quantity of the endogenous cholesterol. The amount of the cholesterol measured by this paper-loaded DART mass spectrometry (pDART-MS) is statistically comparable with that obtained by using commercially available fluorometric-enzymatic assay and LC/MS. Furthermore, serums from twenty-one participants at three different time points in an ultra-marathon were collected to obtain their cholesterol levels. The test requires only minimal sample preparations, and the concentrations of cholesterol in each sample were acquired within a minute. Furthermore, we also explored the possibility of utilizing paper-based AIMS to cancer diagnosis. In the case of breast cancer, breast tissue is extracted with a hypodermic needle, and wiped onto filter papers for the following pathology diagnosis in hospital. We used this routine paper-based tissue sample incorporating with AIMS for a rapid metabolite profiling to discriminate cancerous/noncancerous tissues. To reduce the chemical noise in the complex matrix without additional sample pretreatment, we interfaced field asymmetric waveform ion mobility spectrometry with paper spray ionization mass spectrometry (FAIMS-PSI-MS). Statistical analysis of MS spectral profiles in the mass range of m/z 600-900, in which the phospholipid species were dominant, was performed. Relative signal intensity of 28 species showed significant difference in cancerous and noncancerous breast tissue with p value <0.05 in t-test. These markers were used to discriminate the 43 tissue samples from patients, potentially assist to locate the tumor during clinical treatment. AIMS is characteristic for its speed and ease of use together that allows a rapid, direct, and quantitative analysis of complex biological samples. The two approaches of paper-based analysis of biological samples demonstrate that AIMS has the ability to perform clinical applications.

參考文獻


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