博碩士論文 972203016 詳細資訊




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姓名 周冠廷(Guan-ting Zhou)  查詢紙本館藏   畢業系所 化學學系
論文名稱 利用磁性奈米探針與基質輔助雷射脫附/游離飛行時間質譜儀進行人類血液中前列腺特異抗原的定量
(Quantification of Prostate-Specific Antigen in Human Plasma by Using Magnetic Nanoprobe and MALDI-TOF MS)
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摘要(中) 前列腺(攝護腺)癌是目前醫學上很常見的一種癌症。在世界衛生組織(World Health Organization, WHO)2009年的報導中指出,前列腺癌是全球十大癌症當中排名第六名的癌症。在前列腺癌的臨床診斷上,前列腺特異抗原(prostate-specific antigen, PSA)濃度檢測是目前最廣泛使用的方法。當血清中的前列腺特異抗原濃度超過4毫微克/毫升時,即可視為前列癌的高度危險群。
在本篇論文中,我們應用本實驗室所開發的奈米探針輔助親和性質譜法(nanoprobe-based affinity mass spectrometry, NBAMS)做為前列腺特異抗原的免疫測定方法。利用表面結合有前列腺特異抗原抗體的磁性奈米粒子,藉由抗體對抗原的高專一性作用力,做為濃縮血清中前列腺特異抗原的親合性探針,再結合具有高靈敏度的基質輔助雷射脫附/游離飛行時間質譜儀針對含量極低且不易游離的高度醣化前列腺特異抗原樣品進行定量偵測。實驗結果顯示,此分析方法可以在人體血清中,偵測低至5毫微克/毫升的前列腺特異抗原。另一方面,藉由內標準品的添加以及使用晶種層樣品配製法(seed-layer method) 改善樣品訊號的變動,我們可以於血清環境中,對於5-100毫微克的前列腺特異抗原建立相關系數高於0.99的工作曲線。最後,我們證明了本實驗方法可以應用於血清環境中,並對前列腺癌以及前列腺良性增生的病人樣本進行分析。
摘要(英) Prostate cancer is the NO.6 cancer cause of death among men in global reported by World Health Organization (WHO) in 2009. The serum level of prostate-specific antigen (PSA) has been widely used for the clinical diagnosis of prostate cancer; abundance of PSA above 4 ng/ml indicates the high probability of prostate cancer.
In this study, we implemented our previously developed nanoprobe-based affinity mass spectrometry (NBAMS) as an immunoassay for PSA quantification in human plasma. Based on the specificity of antibody-antigen interaction, anti-PSA antibody-conjugated magnetic nanoparticles (anti-PSA@MNPs) can be used as an affinity probe to extract and enrich PSA from human plasma, followed by quantification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The challenge in this study is the low abundance of PSA in human serum and the low ionization efficiency of the highly glycosylated PSA. Our results showed the limit of detection (LOD) of 5 ng/ml in diluted human plasma. To facilitate quantification of PSA, we integrated the use of internal standard and seed-layer method; the latter improved homogeneous crystallization of sample and matrix to reduce signal fluctuation. The standard working curve constructed by standard PSA spiked in plasma showed dynamic range from 5-100 ng with correlation coefficients better than 0.99. Finally, we demonstrated that this method can be used to analyze plasma sample from patients with prostate cancer and benign prostatic hyperplasia patient.
關鍵字(中) ★ 前列腺特異抗原
★ 奈米探針
關鍵字(英) ★ Magnetic Nanoprobe
★ Prostate-Specific Antigen
論文目次 中文摘要 I
Abstract III
謝誌 IV
Table of Content V
List of Figure VII
List of Table IX
Abbreiations 1
Chapter 1: Introduction 2
1-1. Affinity Mass Spectrometry for Targeted Protein Detection 2
1-2. Nanoscale Affinity Probe 3
1.3. Biomarker and Disease 5
1-3.1. Biomarker 5
1-3.2. Prostate Cancer 5
1-3.3. Prostate Specific Antigen 6
1-4. Objective 7
Chapter 2: Experiment 9
2-1. Materials 9
2-1.1. Chemicals and Materials 9
2-1.2. Synthesis of Antibody-Conjugated Magnetic Nanoparticles 9
2-1.3. Human Plasma Preparation 10
2-2. Instrument 11
2-3. Method 11
2-3.1. Immunoaffinity Extraction 11
2-3.2. Optimization of NBAMS 12
2-3.3. Detection limit of PSA protein 13
2-3.3.2. The capacity of anti-PSA@MNPs 13
2-3.4. Quantification 14
Chapter 3: Results and Discussion 17
3-1. Optimization of NBAMS 17
3-1.1. Matrix Selection 17
3-1.2. Anti-PSA@MNPs background 19
3-2. Detection Specificity and Sensitivity of PSA by NBAMS 20
3-2.1. Enrichment Specificity 20
3-2.2. The capacity of anti-PSA@MNPs 21
3-2.3. Sensitivity in PBS 22
3-2.4. Specificity and Sensitivity in Plasma 23
3-3. Quantification 24
3-3.1. Comparison of Signal Stability Obtained from Seed-layer Method and Dried-droplet Method 25
3-3.2. Determination of Internal Standard 26
3-3.3. The Standard Curve of PSA in PBS 26
3-3.4. The Standard Curve of PSA in Plasma 28
3-3.5. Quantification of PSA in Patient plasma 29
Chapter 4: Conclusion 30
References 61
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指導教授 陳玉如、王家麟
(Yu-ju Chen、Jia-Lin Wang)
審核日期 2010-8-11
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