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

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：14

、訪客IP：3.15.197.123

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)

相關論文

★ 以質譜技術探討非共價鍵結蛋白質聚合物之結構	★ 有機薄膜電晶體材料三併環及四併環噻吩衍生物之開發
★ 以逆吹式氣相層析法分析氣體成份	★ 氣相層析法應用於工業排放連續監測
★ 煙道氣揮發性有機化合物連續監測方法開發	★ 以蛋白質體學探討在大腸桿菌中甲醇利用代謝途徑
★ Data-independent acquisition mass spectrometry analysis for identification of cerebrospinal fluid biomarker of reversible cerebral vasoconstriction syndrome	★ 自製新型除水及熱脫附濃縮裝置用於GC/MS線上分析揮發性有機汙染物
★ 觸媒式非甲烷總碳氫分析儀開發與驗證	★ 自製除水器及熱脫附儀用於線上GC/MS/FID揮發性有機污染物之分析
★ 大氣及水樣中揮發性有機氣體自動化分析技術之建立及應用	★ VOC前濃縮與預警系統之建構
★ 建立自動化甲烷連續量測系統與其在指示大氣輻射冷卻之應用	★ 臭氧前趨物連續監測與臭氧生成之光化學探討
★ 以近連續方式量測空氣中甲烷與異戊二烯及其生成之季節性探討	★ 自行架設光化學測站與商業化儀器平行比對及所得資料初步分析

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

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

參考文獻

[1] Lequin, R. M., Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical Chemistry 2005, 51, 2415-2418.
[2] Loo, J. A.; Udseth, H. R.; and Richard D. Smith, R. D., Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry. Analytical Biochemistry 1989, 179, 404-412.
[3] Karas, M.; Hillenkamp, F., Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Analytical Chemistry 1988, 60, 2299-2301.
[4] Xu, Y.; Bruening, M. L.; Watson, J. T. Non-specific, on-probe cleanup methods for MALDI-MS samples. Mass Spectrometry Reviews 2003, 22, 429– 440.
[5] Gilar, M.; Bouvier, E. S. P., Compton, Bruce J. Advances in sample preparation in electromigration, chromatographic and mass spectrometric separation methods. Journal of Chromatography A, 2001, 909, 111–135.
[6] Tang, N.; Tornatore, P.; Weinberger, S. R. Current developments in SELDI affinity technology. Mass Spectrometry Reviews, 2004, 23, 34–44.
[7] Papac, D. I.; Hoyes, J.; Tomer, K. B. Direct Analysis of Affinity-Bound Analytes by MALDI/TOF MS. Analytical Chemistry 1994, 66, 2609-2613.
[8] Jaina, V.; Deepti Saini, D.; Goswamia, P.; Sinha, S., A phage antibody to the active site of human placental alkaline phosphatase with higher affinity to the enzyme–substrate complex. Molecular Immunology 2007, 44, 369-376.
[9] Bundy, J.; Fenselau, C. Lectin-Based Affinity Capture for MALDI-MS Analysis of Bacteria. Analytical Chemistry 1999, 71, 1460-1463.
[10] Bundy, J. L.; Fenselau, C. Lectin and Carbohydrate Affinity Capture Surfaces for Mass Spectrometric Analysis of Microorganisms. Analytical Chemistry 2001, 73, 751-757.
[11] Olmsted, J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. Journal of Biological Chemistry 1981, 256, 11955-11957.
[12] Brockman, A. H.; Orlando, R. Probe-Immobilized Affinity Chromatography/Mass Spectrometry. Analytical Chemistry 1995, 67, 4581 -4585.
[13] Brockman, A. H.; Orlando, R. New Immobilization Chemistry for Probe Affinity Mass Spectrometry, Rapid Communications In Mass Spectrometry 1996, 10,1688-1692
[14] Liang, X.; Lubman, D. M. On-Probe Immunoaffinity Extraction by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Analytical Chemistry 1998, 70, 498-503
[15] Lopez, M. F.; Rezai, T.; Sarracino, D. A.; Prakash, A.; Krastins, B.; Athanas, M.; Singh, R. J.; Barnidge, D. R.; Oran, P.; Borges, C.; Nelson, R. W.,Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants. Clinical Chemistry 2010, 56, 281–290.
[16] Hutchens, T. W.; Yip, T. T. New desorption strategies for the mass spectrometric analysis of macromolecules. Rapid Communications In Mass Spectrometry, 1993, 7, 576-580
[17] Katz, J. E.; Mallick, P.; Agus, D. B. A perspective on protein profiling of blood. British Journal of Urology International 2005, 96, 477-482.
[18] Rosenblatt, K. P.; Bryant-Greenwood, P.; Killian, J. K.; Mehta, A.; Geho, D.; Espina, V.; III, E. F. P.; Liotta, L. A., Serum proteomics in cancer diagnosis and management. Annual Review of Medicine 2004, 55, 97-112.
[19] Issaq, H. J.; Conrads, T. P.; Prieto, D. A.; Tirumalai, R.; Veenstra, T. D., SELDI-TOF MS for diagnostic proteomics. Analytical Chemistry 2003, 149A-155A.
[20] Diamandis, E. P., Mass spectrometry as a diagnostic and a cancer biomarker discovery tool. Molecular & Cellular Proteomics 2004, 3, 367-378.
[21] Henderson, N. A.; Steele, R. J. C., SELDI-TOF proteomic analysis and cancer detection. Surgeon 2005, 1, 383-390.
[22] Jr, B. C. V.; Bonventre, J. V.; Hsu, S. I.-H., Towards the application of proteomics in real disease diagnosis. Clinical Science 2005, 109, 421-430.
[23] Zhu, W.; Wang, X.; Ma, Y.; Rao, M.; Glimm, J.; Kovach, J. S. Detection of cancer-specific markers amid massive mass spectral data. Proceedings of the National Academy of Sciences 2003, 100, 14666-14671
[24] Johann DJ Jr, McGuigan MD, Patel AR, Tomov S, Ross S, Conrads TP, Veenstra TD, Fishman DA, Whiteley GR, Petricoin EF 3rd, Liotta LA. Clinical proteomics and biomarker discovery. Annals of the New York Academy of Sciences. 2004,1022, 295-305
[25] Guo, J.; Yang, E. C.; Desouza, L.; Diehl, G.; Rodrigues, M.J.; Romaschin, A. D.; Colgan, T. J.; Siu, K. W. A strategy for high-resolution protein identification in
[26] Moore, A., Brave small word. European Molecular Biology Organization 2001, 2, 86-88.
[27] Zhang, C.; Zhang, Z.; Yu, B.; Shi, J.; Zhang, X., Application of the biological conjugate between antibody and colloid Au nanoparticles as analyte to inductively coupled plasma mass spectrometry. Analytical Chemistry 2002, 74, 96-99.
[28] Ho, K.-C.; Tsai, P.-J.; Lin, Y.-S.; Chen, Y.-C., Using biofunctionalized nanoparticles to probe pathogenic bacteria. Analytical Chemistry 2004, 76, 7162-7168.
[29] Hirsch, L. R.; Jackson, J. B.; Lee, A.; Halas, N. J.; West, J. L., A whole blood
immunoassay using gold nanoshells. Analytical Chemistry 2003, 75, 2377-2381.
[30] Bucak, S.; Jones, D. A.; Laibinis, P. E.; Hatton, T. A., Protein separation using colloidal magnetic nanoparticles. Biotechnology Progress 2003, 19, 477-484.
[31] Gu, H.; Ho, P.-L.; Tsang, K. W. T.; Wang, L.; Xu, B., Using Biofunctional Magnetic Nanoparticles to Capture Vancomycin-Resistant Enterococci and Other Gram-Positive Bacteria at Ultralow Concentration. Journal of the American Chemical Society 2003, 125, 15702-15703.
[32] Lin, Y.-S.; Tsai, P.-J.; Weng, M.-F.; Chen, Y.-C., Affinity capture using vancomycin-bound magnetic nanoparticles for the MALDI-MS analysis of bacteria. Analytical Chemistry 2005, 77, 1753-1760.
[33] Kriz, K.; Ibraimi, F.; Lu, M.; Hansson, L.-O.; Kriz, D., Detection of C-reactive protein utilizing magnetic permeability detection based immunoassay. Analytical Chemistry 2005, 77, 5920-5924.
[34] Chou, P.-H.; Chen, S.-H.; Liao, H.-K.; Lin, P.-C.; Her, G.-R.; Lai, A. C.-Y.; Chen, J.-H.; Lin, C.-C.; Chen, Y.-J., Nanoprobe-bsed affinity mass spectrometry for selected protein profiling in human plasma. Analytical Chemistry 2005, 77, 5990-5997.
[35] Lin, P. C.; Chou, P. H.; Chen, S. H.; Liao, H. K.; Wang, K. Y.; Chen, Y. J.; Lin, C. C. Ethylene Glycol-Protected Magnetic Nanoparticles for a Multiplexed Immunoassay in Human Plasma. Small 2006, 2, 485–489.
[36] Chen, S. H.; Liao, H. K.; Chang, C. Y.; Juo, C. G.; Chen, J. H.; Chan, S. I.; Chen, Y. J. Targeted protein quantitation and profiling using PVDF affinity probe and MALDI-TOF MS. Proteomics 2007, 7, 3038–3050.
[37] Atkinson, A. J.; Colburn, W. A.; DeGruttola, V. G.; DeMets, D. L.; Downing, G. J.; Hoth, D. F.; Oates, J. A.; Peck, C. C.; Schooley, R. T.; Spilker, B. A.; Woodcock, J.; Zeger, S. L., Biomarkers and surrogate endpoints: Preferred definitions and conceptual framework. Clinical Pharmacology & Therapeutics 2001, 69, 89–95.
[38] Moertel, C. G.; Fleming, T. R.; Macdonald, J. S.; Haller, D. G.; Laurie, J. A.; Tangen, C., An Evaluation of the Carcinoembryonic Antigen (CEA) Test for Monitoring Patients With Resected Colon Cancer. Journal of The American Medical Association 1993, 270, 943-947.
[39] Goldenberg, D.M.; Kim, E.E.; DeLand, F.H.; Bennett, S.; Primus, F. j. Radioimmunodetection of Cancer with Radioactive Antibodies to Carcinoembryonic Antigen. Cancer Researchearch 1980, 40, 2984-2992
[40] Leonard, D. S.; Hill, A. D. K.; Kelly, L.; Dijkstra, B.; McDermott, E.; O'Higgins, N. J., Anti-human epidermal growth factor receptor 2 monoclonal antibody therapy for breast cancer. British Journal of Surgery 2002, 89, 262-271.
[41] Konety, B. R.; Nguyen, T. T. -S.; Dhir, R.; Day, R. S.; Becich, M. J.; Stadler, W. M.; Getzenberg, R. H., Detection of Bladder Cancer Using a Novel Nuclear Matrix Protein,BLCA-4. Clinical Cancer Researchearch 2000, 6, 2618–2625.
[42] Ludwig, J. A.; Weinstein, J. N., Biomarkers in Cancer Staging, Prognosis And Treatment Selection. Nature Reviews Cancer 2005, 5, 845-856.
surface-enhanced laser desorption/ionization mass spectrometry: calgranulin A and chaperonin 10 as protein markers for endometrial carcinoma. Proteomics 2005, 5, 1953–1966
[43] LaBaer. J., So, You Want to Look for Biomarkers (Introduction to the Special Biomarkers Issue). Journal of Proteome Research 2005, 4, 1053-1059.
[44] Lilja, H. A Kallikrein-like serine protease in prostatic fluid cleaves the
predominant seminal vesicle protein. Journal of Clinical Investigation 1985, 76, 1899–903
[45] Sa¨vblom, C.; Malm, J.; Giwercman, A.; Nilsson, J.; Berglund, G.; Lilja, H. Blood levels of free-PSA but not complex-PSA significantly correlates to prostate release of PSA in semen in young men, while blood levels of complex-PSA, but not free-PSA increase with age. Prostate 2005, 65, 66–72
[46] Stamey, T.A.; Yang, N.; Hay A.R. McNeal, J.E.; Freiha, F.S.; Redwine, E. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. New England Journal of Medicine 1987, 317, 909–16
[47] Gann, P. H.; Hennekens, C. H.; Stampfer, M. J. A prospective evaluation of plasma prostate-specific antigen for detection of prostatic cancer. Journal of The American Medical Association 1995, 273 (4), 289–94
[48] Stenman, U.H.; Leinonen, J.; Alfthan, H.; Ranniko, S.; Tuhkanen, K.; Alfthan, O. A complex between prostate-specific antigen and a1-antichymotrypsin is the major form of prostatic-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Research 1991, 51, 222–226.
[49] Lilja, H.; Christensson, A.; Dahlen, U.; Matikainen, M.T.; Nilsson, O.; Pettersson, K.; Lo¨vgren, T. Prostate-specific antigen in human serum occurs predominantly in complex with a1-antichymotrypsin. Clinical Chemistry 1991, 37, 1618–1627.
[50] Zhang, W. M.; Finne, P.; Leinonen, J.; Vesalainen, S.; Nordling, S.; Rannikko, S.; Stenman, U. H. Characterization and immunological determination of the complex between prostate-specific antigen and a2-macroglobulin. Clinical Chemistry 1998, 44, 2471–2479.
[51] Catalona, W. J.; Partin, A. W.; Slawin, K. M.; Brawer, M. K.; Flanigan, R. C.; Patel, A.; Richie, J. P.; deKernion, J. B.; Walsh, P. C.; Scardino, P. T.; Lange, P. H.; Subong, E. N. P.; Parson, R. E.; Gasior, G. H.; Loveland, K. G.; Southwick, P. C. Use of the Percentage of Free Prostate-Specific Antigen to Enhance Differentiation of Prostate Cancer From Benign Prostatic Disease: A Prospective Multicenter Clinical Trial. Journal of The American Medical Association 1998, 279, 1542-1547.
[52] Ohyama, C.; Hosono, M.; Nitta, K.; Oh-eda, M.; Yoshikawa, K.; Habuchi,T.; Arai,Y.; Fukuda, M., Carbohydrate structure and differential binding of prostate specific antigen to Maackia amurensis lectin between prostate cancer and benign prostate hypertrophy. Glycobiology 2004, 14, 671–679
[53] Mikolajczyk, S. D.; Millar, L. S.; Wang, T. J.; Rittenhouse, H. G.; Marks, L. S.; Song, W.; Wheeler, T. M.; Slawin, K. M. A Precursor Form of Prostate-specific Antigen Is More Highly Elevated in Prostate Cancer Compared with Benign Transition Zone Prostate Tissue. Clinical Chemistry 2004, 50, 1017–1025.
[54] Peter, J.; Unverzagt, C.; Hoesel, W. Release of Free PSA from the PSA-ACT Complex. Clinical Chemistry 2000, 46(4), 474–482.
[55] Jacobs, J. M.; Adkins, J. N.; Qian, W.; Liu, T.; Shen,Y.; Camp, D.G.; and Smith, R.D. Utilizing Human Blood Plasma for Proteomic Biomarker Discovery. Journal of Proteome Research 2005, 4, 1073-1085.
[56] Jin, Y.; Manabe, T. Direct targeting of human plasma for matrix-assisted laser desorption/ionization and analysis of plasma proteins by time of flight-mass spectrometry. Electrophoresis 2005, 26, 2823-2834.
[57] Tirumalai, R. S.; Chan, K. C.; Prieto, D. A.; Issaq, H. J.; Conrads, T. P.; Veenstra, T. D., Characteriz Characterization of the Low Molecular Weight Human Serum Proteome. Molecular & Cellular Proteomics 2003, 2, 1096-1103.
[58] Schweitzer, B.; Kingsmore, S. F. Measuring proteins on microarrays. Current Opinion in Biotechnology 2002, 13, 14–19.
[59] Davies, D. R.; Padlan, E. A. Antibody -antigen complexes. Annual Review of Biochemistry 1990, 59, 439-73.
[60] Onnerfjord, P.; Ekstrom, S.; Bergquist, J.; Nilsson, J.; Laurell, T.; Marko-Varga, G., Homogeneous sample preparation for automated high throughput analysis with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Rapid Commun. Journal of Mass Spectrometry 1999, 13, 315–322.
[61] van Kampen, J. J.; Burgers, P. C.; de Groot, R.; Luider, T. M., Qualitative and Quantitative Analysis of Pharmaceutical Compounds by MALDI-TOF Mass Spectrometry. Analytical Chemistry 2006, 78, 5403–5411.
[62] Phizicky, E.; Bastiaens, P. I. H.; Zhu, H.; Snyder, M.; Fields, S. Protein analysis on a proteomic scale. Nature 2003, 422, 208-215.
[63] Guidance for industry: Q2B Validation of Analytical Procedure, Methodology. http//www.fda.gov

指導教授

陳玉如、王家麟
(Yu-ju Chen、Jia-Lin Wang)

審核日期

2010-8-11

推文