摘要
激酶在细胞增殖、分化和代谢等生理过程的调控中担任着重要的角色。众多的人类疾病与激酶的活性异常有着直接或者间接的关系。因此对激酶活性的分析检测对于生化研究、临床诊断及对重大疾病的药物靶向治疗等都具有重要意义。研究快速、简单、灵敏度高和特异性好的检测激酶活性的新方法具有广泛的应用前景。本论文以蛋白激酶和己糖激酶为研究对象，主要应用荧光共轭聚合物、功能性磁性微球、便携式血糖仪等生物传感器为技术平台，结合分子荧光、荧光成像等检测技术，研究建立了一系列检测激酶活性的新方法，主要内容如下：
1．基于阴离子荧光共轭聚合物的荧光共振能量转移作用，发展了一种均相、通用型的新型生物传感器，用于蛋白激酶活性的检测。水溶性荧光共轭聚合物（PFPaa）带有氨基二乙酸的侧链，它与金属离子（Zr4+）形成配位化合物。荧光标记的肽底物在蛋白激酶的作用下被磷酸化，其磷酸基团与Zr4+特异性结合。以Zr4+为“桥梁”，PFPaa与标记在肽上的荧光基团被拉近，产生了强烈的荧光共振能量转移（FRET），并对荧光信号进行放大，由此可实现对蛋白激酶活性的高灵敏度检测。该方法操作简便，可检测0.0005U·μL-1~1U·μL-1的蛋白激酶，并可用于抑制剂的筛选。
2．将TiO2修饰在Fe3O4/SiO2磁性微球表面。荧光标记的底物肽在蛋白激酶存在时被磷酸化，其磷酸基团与Ti4+特异性结合，从而被富集在磁性微球表面。通过对富集的磷酸化肽上标记的荧光进行检测或对磁性微球表面荧光成像进行分析，可以实现对蛋白激酶活性的高灵敏度检测。该方法对蛋白激酶活性的测定范围为0.0005U·μL-1~0.5U·μL-1，检出限为0.0001U·μL-1。该方法成功的应用于细胞裂解液中蛋白激酶活性检测及蛋白激酶活性抑制剂的筛选。此外对不同的蛋白激酶底物肽标记不同的荧光基团，还可以实现对不同蛋白激酶的同时检测。
3．基于便携式血糖仪，建立了一种简单、快速、均相测定已糖激酶活性的方法。己糖激酶催化葡萄糖的磷酸化，磷酸化的葡萄糖在血糖仪上不产生响应信号，以商品化的便携式血糖仪作为实验信号测定设备，根据体系中剩余葡萄糖的含量，间接测得己糖激酶的活性。本方法的检测范围为10-6U·μL-1~10-2U·μL-1，这种新的分析方法也可应用于己糖激酶抑制剂的高通量筛选。
Kinases play an important role in regulation of fundamental biological processes,including cell proliferation, differentiation and metabolism. Aberrations in kinase activitiescan result in a number of diseases. Therefore, sensitive and widely applicable assays formonitoring kinase activity are of great signifcance for further understanding the molecularmechanism in biochemical research, clinical diagnosis, and development of targeted therapy.In this study, we focus on the development of sensitive methods for detection of proteinkinase activity and hexokinase activity. This dissertation presents a series of new methods fordetection of kinase activity by using conjugated polymers, functionalized magneticmicrospheres and glucose meter technology integrated with molecular fluorescence andfluorescence images detection. The main contents are as follows:
1. Anionic fluorescent conjugated polymer (PFPaa) coupled with the metal ion-mediatedFRET, can be used to design a versatile, homogeneous, and simple platform for detection ofprotein kinase activities. In conclusion, we have demonstrated that PFPaa can complex withZr4+and the complexed Zr4+can selectively recognize the phosphate group on the peptidesubstrates, resulting in e cient FRET from PFPaa to the fluorophores labeled on thephosphorylated peptides. Based on the light-harvesting property and amplification offluorescence signals, PFPaa can be used to design a versatile, homogeneous, robust platform todetect protein kinase activity with high sensitivity. Protein kinase A (PKA) activity can bequantitatively detected over a wide concentration range from0.0005U·μL-1~1U·μL-1. Thenew strategy provides a simple detection procedure, easy readout and cost e ective manner forprotein kinase assay, which shows great potential for high-throughput assay in clinicaldiagnostics and drug discovery applications.
2. A simple, highly sensitive, and dual-readout fuorescent assay is developed for thedetection of protein kinase activity based on the specifc recognition utility of TiO2-coatedFe3O4/SiO2magnetic microspheres (TMSPs) for kinase-induced phosphopeptides. When thefuorophore-labeled substrate peptides are phosphorylated by the kinase reaction, they canbind specifcally to the TiO2layer of TMSPs by means of phosphate groups, resulting infuorophore enrichment on the TMSP surfaces. The accumulated fuorophores on the TMSPsare proportional to the kinase activity, and the fuorescence signal readout could be run through either direct fuorescent imaging of the TMSPs or measurement of the fuorescenceintensity by simply detaching the fuorescent phosphopeptides into the solution. The TMSPsexhibit extremely high selectivity for capturing phosphorylated peptides over thenonphosphorylated ones, resulting in an ultrahigh fuorescence signal-to-background ratio of42, which is the highest fuorescence change thus far in fuorescent assays for detection ofprotein kinase activities. Therefore, the proposed fuorescent assay presents high sensitivity,low detection limit of0.0001U·μL-1, and wide dynamic range from0.0005U·μL-1to0.5U·μL-1with protein kinase A (PKA) as a model target. Moreover, the TMSP-based fuorescentassay can simultaneously quantify multiple kinase activities with their specifc peptideslabeled with di erent dyes. This new strategy is also successfully applied to monitoringdrug-triggered PKA activation in cell lysates. Therefore, the TMSP-based fuorescent assay isvery promising in high-throughput screening of kinase inhibitors and in highly sensitivedetection of kinase activity, and thus it is a valuable tool for development of targeted therapy,clinical diagnosis, and studies of fundamental life science.
3. We provide a simple, rapid, homogeneous method for measuring hexokinase activiteby quantitating the amount of glucose remaining in solution following a kinase reaction,which is monitored by the personal glucometer. The glucose level is correlated with theamount of glucose present and is inversely correlated with the amount of kinase activity. Thewide dynamic range was detected from10-6U·μL-1to10-2U·μL-1by using HK as a modeltarget. Moreover, this new method is also successfully applied to high-throughput screeningof HK inhibitors. Thus is a valuable tool for development of targeted therapy as well as thestudies of fundamental life science.
引文
[1] J. E. Hutti, E. T. Jarrell, J. D. Chang, et al. A rapid method for determining protein kinasephosphorylation specificity[J]. Nat. Methods,2004,1:27-29.
[2] C. W. Lewis, R. G. Taylor, P. M. Kubara, et al. A western blot assay to measure cyclin dependent kinaseactivity in cells or in vitro without the use of radioisotopes[J]. FEBS Lett.,2013,587(18):3089-3095.
[3] S. Gupta, H. Andresen, J. E. Ghadiali et al. Kinase-Actuated Immunoaggregation ofPeptide-Conjugated Gold Nanoparticles[J]. Small,2010,6(14):1509-1513.
[4] S. Martic, M. Gabriel, J. P. Turowec, et al. Versatile Strategy for Biochemical, Electrochemical andImmunoarray Detection of Protein Phosphorylations[J]. J. Am. Chem. Soc.,2012,134(41):17036-17045.
[5] K. Kerman, H. Song, J. S. Duncan, et al. Peptide Biosensors for the Electrochemical Measurement ofProtein Kinase Activity[J]. Anal. Chem.,2008,80(24):9395-9401.
[6] J. Ji, H. Yang, Y. Liu, et al. TiO2-assisted silver enhanced biosensor for kinase activity profling[J].Chem. Comm.,2009,12:1508-1510.
[7] P. Miao, L. Ning, X. Li, et al. Electrochemical Strategy for Sensing Protein Phosphorylation[J].Bioconjugate Chem.,2012,23(1):141-145.
[8] A. Wieckowska, D. Li, R. Gill, et al. Following protein kinase acivity by electrochemical means andcontact angle measurements[J]. Chem. Comm.,2008,20:2376-2378.
[9] O. I. Wilner, C. Guidotti, A. Wieckowska, et al. Probing Kinase Activities by Electrochemistry,Contact-Angle Measurements, and Molecular-Force Interactions[J]. Chem.-Eur. J.,2008,14(26):7774-7781.
[10] B. T. Houseman, J. H. Huh, S. J. Kron, et al. Peptide chips for the quantitative evaluation of proteinkinase activity[J]. Nat. Biotechnol.,2002,20:270-274.
[11] K. Inamori, M. Kyo, Y. Nishiya, et al. Detection and Quantification of On-Chip PhosphorylatedPeptides by Surface Plasmon Resonance Imaging Techniques Using a Phosphate Capture Molecule[J].Anal. Chem.,2005,77(13):3979-3985.
[12] R. Freeman, T. Finder, R. Gill, et al. Probing Protein Kinase (CK2) and Alkaline Phosphatase withCdSe/ZnS Quantum Dots[J]. Nano Lett.,2010,10(6):2192-2196.
[13] H. W. Rhee, S. H. Lee, I. S. Shin, et al. Detection of Kinase Activity Using Versatile FluorescenceQuencher Probes[J]. Angew. Chem. Int. Ed.,2010,122(29):5039-5043.
[14] A. Y. Ting, K. H. Kain, R. L. Klemke, et al. Genetically encoded fluorescent reporters of proteintyrosine kinase activities in living cells[J]. Proc. Natl. Acad. Sci. U. S. A.,2001,98(26):15003-15008.
[15] J. Zhang, Y. Ma, S. S. Taylor, et al. Genetically encoded reporters of protein kinase A activity revealimpact of substrate tethering[J]. Proc. Natl. Acad. Sci. U. S. A.,2001,98(26):14997-15002.
[16] E. A. Gaudet, K. Huang, Y. Zhang, et al. A Homogeneous Fluorescence Polarization Assay Adaptablefor a Range of Protein Serine/Threonine and Tyrosine Kinases[J]. J. Biomol. Screening,2003,8(2):164-175.
[17] R. A. Nakashima, L. J. Scott, P. L, Pedersen. The role of mitochondrial hexokinase binding in theabnormal energy metabolish of tumor cell lines[J]. Ann. Ny Acad. Sci.,1986,488:438-450.
[18] K. Garber. Energy boost: The Warburg effect returns in a new theroy of cancer[J]. J. Natl. Cancer Inst.,2004,96(24):1805-1806.
[19] C. L. Tiago, G. C. Raquel, D. S. Daniel, et al. Lactate downregulates the glycolytic enzymeshexokinase and phosphofructokinase in diverse tissues from mice[J]. FEBS Lett.,2011,585(1):92-98.
[20] L. Pan, Y. Yu, L. Sun. An LC-MS Method for a Hexokinase Inhibitor Study Based on Adenosine5-Triphosphate Determination and Application to the Anticancer Mechanism of Momordicacochinchinensis[J]. Chromatographia,2010,72(5-6):425-430.
[21] T. Wang, J. Kang. Hexokinase inhibitor screening based on adenosine5-diphosphate determinationby electrophoretically mediated microanalysis[J]. Electrophoresis,2009,30(8):1349-1354.
[22] Y. Tang, F. Feng, M. Yu, et al. Direct Visualization of Glucose Phosphorylation with a CationicPolythiophene[J]. Adv. Mater.,2008,20(4):703-705.
[23] S. Ren, B. Li, L. Zhang. Visual detection of hexokinase activity and inhibition with positively-chargedgold nanoparticles as colorimetric probes[J]. Analyst,2013,138(11):3142-3145.
[24]张先恩.生物传感器[M].北京：化学工业出版社，2006.1-382.
[25]王镜岩，朱圣庚，徐长法.生物化学[M].北京：高等教育出版社，第三版（上册），2005.424-425.
[26] R. H. Stevan, H.T. Jeffrey. Protein Tyrosine Kinase Structure and Function[J]. Annu. Rev. Biochem.,2000,69:373-398.
[27] S. K. Binz, A. M. Sheehan, M. S. Wold. Replication protein A phosphorylation and the cellularresponse to DNA damage[J]. DNA Repair (Amst),2004,3(8-9):1015-1024.
[28] M. S. Kobor, J. Greenblatt. Regulation of transcription elonga-tion by phosphorylation[J]. Biochim.Biophys. Acta,2002,1577(2):261-275.
[29] P. P. Ruvolo, X. Deng, W. S. May. Phosphorylation of Bcl2and regulation of apoptosis[J]. Leukemia,2001,15(4):515-522.
[30]黄文林，朱孝峰.信号转导[M].北京：人民卫生出版社，第一版，2005.33-114.
[31]敖世洲.蛋白质可逆磷酸化对细胞活动的调节[M].上海：上海科学技术出版社，1994.1-253.
[32]翟中和，王喜忠，丁明孝.细胞生物学[M].北京：高等教育出版社，第四版，2011.156-192.
[33] X. Zhao, I.R. León, S. Bak, et al. Phosphoproteome Analysis of Functional Mitochondria Isolatedfrom Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane ProteinComplexes and Enzymes[J]. Mol. Cell. Proteomics,2011,10: M110.000299.
[34] A. Salminen, K. Kaarniranta, A. Haapasalo, et al. AMP-activated protein kinase: a potential player inAlzheimer’s disease[J]. J. Neurochem.,2011,118(4):460-474.
[35] J. Brognard, T. Hunter. Protein Kinase Signalling Networks in Cancer[J]. Curr. Opin. Genet. Dev.,2011,21(1):4-11.
[36] S. B. Lee, S. H. Kim, D. W. Bell, et al. Destabilization of CHK2by a missense mutation associatedwith Li-Fraumeni Syndrome[J]. Cancer Res.,2001,61:8062-8067.
[37]金海晓.蛋白激酶A结构、功能及抑制剂的分子模拟研究[D].浙江大学博士学位论文，2006.
[38] S. K. Hallks, A. M. Quiim. Protein kinase catalytic domain sequence database: identification ofconserved features of primary structures and classification of family members[J]. Meth. Enzymol,1991,200:38-62.
[39] J. L. Meinkoth, Y. Ji, S. S. Taylor, et al. Dynamics of the distribution of cyclic AMP-dependentProtein kinase in living cells[J]. Proc. Natl. Acad. Sci, U.S.A.,1990,87(24):9595-9599.
[40] G. Manning, D. B. Whyte, R. Martinez, et al. The Protein Kinase Complement of the HumanGenome[J]. Science,2002,298(5600):1912-1934.
[41]孙大业，郭艳林，马力耕，等.细胞信号转导[M].北京：科学出版社，第三版.2001.136-156.
[42] B. E. Turk, J. E. Hutti, L. C. Cantley. Determining protein kinase substrate specificity by parallelsolution-phase assay of large numbers of peptide substrates[J]. Nature Protocols,2006,1(1):375-379.
[43] C. J. Hastie, H. J. Mclauchlan, P. Cohen. Assay of protein kinases using radiolabeled ATP: aprotocol[J]. Nature Protocols,2006,1(2):968-971.
[44] K. Kerman, M. D. Vestergaard, E. Tamiya. Label-free electrical sensing of small-molecule inhibitionon tyrosine phosphorylation[J]. Anal. Chem.,2007,79(17):6881-6885.
[45] K. Viht, S. Schweinsberg, M. Lust, et al. Surface-plasmon-resonance-based biosensor withimmobilized bisubstrate analog inhibitor for the determination of affinities of ATP-andprotein-competitive ligands of cAMP-dependent protein kinase[J]. Anal. Biochem.,2007,362(2):268-277.
[46] M. Mann, S. Ong, M. Gr nborg, et al. Analysis of protein phosphorylation using mass spectrometry:deciphering the phosphoproteome[J]. Trends Biotechnol.,2002,20(6):261-268.
[47] J. D. Watts, M. Affolter, D. L. Krebs, et al. Identification by electrospray ionization mass spectrometryof the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosinekinase ZAP-70[J]. J. Biol. Chem.,1994,269(47):29520-29529.
[48] Z. Wang, R. Lévy, D. G. Fernig, et al. Kinase-Catalyzed Modification of Gold Nanoparticles: A NewApproach to Colorimetric Kinase Activity Screening[J]. J. Am. Chem. Soc.,2006,128(7):2214-2215.
[49] H. Song, K. Kerman, H. B. Kraatz. Electrochemical detection of kinase-catalyzed phosphorylationusing ferrocene-conjugated ATP[J]. Chem. Comm.,2008,4:502-504.
[50] M. Gr nborg, T. Z. Kristiansen, A. Stensballe, et al. A mass spectrometry-based proteomic approachfor identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specificantibodies identification of a novel protein, frigg, as a protein kinase a substrate[J]. Mol. Cell.Proteomics,2002,1(7):517-527.
[51] J. Oishi, Y. Asami, T. Mori, et al. Measurement of Homogeneous Kinase Activityfor Cell LysatesBased on the Aggregation of Gold Nanoparticles[J]. ChemBiochem.,2007,8(8):875-879.
[52] X. Xu, X. Liu, Z. Nie, et al. Label-Free Fluorescent Detection of Protein Kinase Activity Based on theAggregation Behavior of Unmodifed Quantum Dots[J].Anal. Chem.,2011:83(1),52-59.
[53] Y. J. Li, W. H. Xie, G. J. Fang. Fluorescence detection techniques for protein kinase assay[J]. Anal.Biochem.,2008,390(8):2049-2057.
[54] C. L. Neary, J. G. Pastorino. Nucleocytoplasmic shuttling of hexokinase Ⅱ in a cancer cell[J].Biochem. Biophys. Res. Commum.,2010,394(4):1075-1081.
[55] V. Adams, W. Kempf, S. Hassam, et al. Determination of hexokinase isoenzyme Ⅰ and Ⅱcomposition by RT-PCR: increased hexokinase isoenzyme Ⅱ in human renal cell carcinoma[J].Biochem. Mol. Med.,1995,54(1):53-58.
[56] D. Palmieri, D. Fitzgerald, S. M. Shreeve, et al. Analyses of resected human brain metastases of breastcancer reveal the association between up-regulation of hexokinase2and poor prognosis[J]. MolCancer Res.,2009,7(9):1438-1445.
[57] B. Altenberg, K. O. Greulich. Genes of glycolysis are ubiquitously overexpressed in24cancerclasses[J]. Genomics,2004,84(6):1014-1020.
[58] Y. Guan, J. Wang, J. Sun. A Method for Determination of Hexokinase Activity by RP-HPLC[J].Wuhan Univ. J. Nat. Sci.,2011,16(6):535-540.
[59] X. R. Duan, Z. P. Li, F. He, et al. A sensitive and homogeneous SNP detection using cationicconjugated polymers[J]. J. Am. Chem. Soc.,2007,129(14),4154-4155.
[60] F. Rininsland, W. Xia, S. Wittenburg, et al. Metal ion-mediated polymer superquenching for highlysensitive detection of kinase and phosphatase activities[J]. Proc. Natl. Acad. Sci. U. S. A.,2004,101(43):15295-15300.
[61] A. Nomura, S. Shin, O. Oulad Mehdi, et al. Preparation, Characterization, and Application of anEnzyme-Immobilized Magnetic Microreactor for Flow Injection Analysis[J]. Anal. Chem.2004,76(18):5498–5502.
[62] C. T. Chen, Y. C. Chen. Fe3O4/TiO2core/Shell Nanoparticles as Affinity Probes for the Analysis ofPhosphopeptides Using TiO2Surface-Assisted Laser Desorption/Ionization Mass Spectrometry[J].Anal. Chem,2005,77(18):5912-5919.
[63] K. Sparbier, T. Wenzel, M. Kostrzewa. Exploring the binding profiles of ConA, boronic acid andWGA by MALDI-TOF/TOF MS and magnetic particles[J]. J. Chromatogr. B,2006,840(1):29-36.
[64] J. Wang, G. Liu, A. Merko i. Particle-based detection of DNA hybridization using electrochemicalstripping measurements of an iron tracer[J]. Anal. Chim. Acta,2003,482(2):149-155.
[65] I. afa ík, M. afa íková. Magnetic nanoparticles and biosciences[J]. Mon. Chem.,2002,133:737-759.
[66] X. Xu, C. Deng, M. Gao, et al. Synthesis of Magnetic Microspheres with Immobilized Metal Ions forEnrichment and Direct Determination of Phosphopeptides by Matrix-Assisted Laser DesorptionIonization Mass Spectrometry[J]. Adv. Mater.,2006,18(24):3289-3293.
[67] Y. Li, D. W. Qi, C. H. Deng, et al. Cerium Ion-Chelated Magnetic Silica Microspheres for Enrichmentand Direct Determination of Phosphopeptides by Matrix-Assisted Laser Desorption Ionization MassSpectrometry[J]. J. Proteome Res.,2008,7(4):1767-1777.
[68] C. Y. Lo, W. Y. Chen, C. T. Chen, et al. Rapid Enrichment of Phosphopeptides from Tryptic Digests ofProteins Using Iron Oxide Nanocomposites of Magnetic Particles Coated with Zirconia as theConcentrating Probes[J]. J. Proteome Res.,2007,6(2):887-893.
[69] C. T. Chen, W. Y. Chen, P. J. Tsai, et al. Rapid Enrichment of Phosphopeptides and Phosphoproteinsfrom Complex Samples Using Magnetic Particles Coated with Alumina as the Concentrating Probesfor MALDI MS Analysis[J]. J. Proteome Res.,2007,6(1):316-325.
[70] J. H. Lee, Y. Kim, M. Y. Ha, et al. Immobilization of Aminophenylboronic Acid on Magnetic Beadsfor the Direct Determination of Glycoproteins by Matrix Assisted Laser Desorption Ionization MassSpectrometry[J]. J. Am. Soc. Mass Spectrom.,2005,16(9):1456-1460.
[71] A. Ela s sari, M. Rodrigue, F. Meunier, et al. Hydrophilic magnetic latex for nucleic acid extraction,purification and concentration[J]. J. Magn. Magn. Mater.,2001,255(1-2):127-133.
[72] J. Su, J. Xu, Y. Chen, et al. Personal glucose sensor for point-of-care early cancer diagnosis[J]. Chem.Comm.,2012,48:6909-6911.
[73] J. Xu, B. Jiang, J. Xie, et al. Sensitive point-of-care monitoring of HIV related DNA sequences with apersonal glucometer[J]. Chem. Comm.,2012,48:10733-10735.
[74] Y. Xiang, Y. Lu. Using Commercially Available Personal Glucose Meters for Portable Quantificationof DNA[J]. Anal. Chem.,2012,84(4):1975-1980.
[75] P. Blume-Jensen, T. Hunter. Oncogenic kinase signalling[J]. Nature,2001,411:355-365.
[76] J. Schlessinger. Cell Signaling by Receptor Tyrosine Kinases[J]. Cell,2000,103:211-225.
[77] A. Arora, E. M. Scholar. Role of Tyrosine Kinase Inhibitors in Cancer Therapy[J]. J. Pharmacol. Exp.Ther.,2005,315(3):971-979.
[78] X. Xu, J. Zhou, X. Liu, et al. Aptameric Peptide for One-Step Detection of Protein Kinase[J]. Anal.Chem.,2012,84(11):4746-4753.
[79] S. Xu, Y. Liu, T. Wang, et al. Highly Sensitive Electrogenerated Chemiluminescence Biosensor inProfling Protein Kinase Activity and Inhibition Using Gold Nanoparticle as Signal TransductionProbes[J]. Anal. Chem.,2010,82(22):9566-9572.
[80] Y. P. Kim, E. Oh, Y. H. Oh, et al. Protein Kinase Assay on Peptide-Conjugated Gold Nanoparticles byUsing Secondary-Ion Mass Spectrometric Imaging[J]. Angew. Chem. Int. Ed.,2007,119(36):6940-6943.
[81] Y. Yu, R. Anjum, K. Kubota, et al. A site-specific, multiplexed kinase activity assay usingstable-isotope dilution and high-resolution mass spectrometry[J]. Proc. Natl. Acad. Sci. U. S. A.,2009,106(28):11606-11611.
[82] G. D. Meredith, C. E. Sims, J. S. Soughayer, et al. Measurement of kinase activation in singlemammalian cells[J]. Nat. Biotechnol.,2000,18:309-312.
[83] A. Zarrine-Afsar, S. N. Krylov. Use of Capillary Electrophoresis and Endogenous FluorescentSubstrate To Monitor Intracellular Activation of Protein Kinase A[J]. Anal. Chem.,2003,75(15):3720-3724.
[84] K. Martin, T. H. Steinberg, L. A. Cooley, et al. Quantitative analysis of protein phosphorylation statusand protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye[J].Proteomics,2003,3(7):1244-1255.
[85] R. Seethala, R. Menzel. A Homogeneous, Fluorescence Polarization Assay for Src-Family TyrosineKinases[J]. Anal. Biochem.,1997,253(2):210-218.
[86] X. Feng, L. Liu, S. Wang, et al. Water-soluble fuorescent conjugated polymers and their interactionswith biomacromolecules for sensitive biosensors[J]. Chem. Soc. Rev.,2010,39:2411-2419.
[87] X. Duan, L. Liu, F. Feng, et al. Cationic Conjugated Polymers for Optical Detection of DNAMethylation, Lesions, and Single Nucleotide Polymorphisms[J]. Acc. Chem. Res.,2010,43(2):260-270.
[88] Y. Zhang, Y. Wang, B. Liu. Peptide-Mediated Energy Transfer between an Anionic Water-SolubleConjugated Polymer and Texas Red Labeled DNA for Protease and Nuclease Activity Study[J]. Anal.Chem.,2009,81(10):3731-3737.
[89] X. Feng, X. Duan, L. Liu, et al. Fluorescence Logic-Signal-Based Multiplex Detection of Nucleaseswith the Assembly of a Cationic Conjugated Polymer and Branched DNA[J]. Angew. Chem. Int. Ed.,2009,121(29):5316-5321.
[90] Y. Wang, Y. Zhang, B. Liu. Conjugated Polyelectrolyte Based Fluorescence Turn-On Assay forReal-Time Monitoring of Protease Activity[J]. Anal. Chem.,2010,82(20):8604-8610.
[91] X. Duan, L. Liu, X. Feng, et al. Assemblies of Conjugated Polyelectrolytes with Proteins forControlled Protein Photoinactivation[J]. Adv. Mater.,2010,22(14):1602-1606.
[92] L. Zhao, R. Wu, G. Han, et al. The Highly Selective Capture of Phosphopeptides by ZirconiumPhosphonate-Modified Magnetic Nanoparticles for Phosphoproteome Analysis[J]. J. Am. Soc. MassSpectrom.,2008,19(8):1176-1186.
[93] C. A. Chen, R. H. Yeh, D. S. Lawrence. Design and Synthesis of a Fluorescent Reporter of ProteinKinase Activity[J]. J. Am. Chem. Soc.,2002,124(15):3840-3841.
[94] M. D. Shults, B. Imperiali. Versatile Fluorescence Probes of Protein Kinase Activity[J]. J. Am. Chem.Soc.,2003,125(47):14248-14249.
[95] M. D. Shults, K. A. Janes, D. A. Lau enburger, et al. A multiplexed homogeneous fuorescence-basedassay for protein kinase activity in cell lysates[J]. Nat. Methods,2005,2:277-284.
[96] J. J. Witt, R. Roskoski. Rapid Protein Kinase Assay Using Phosphocellulose-Paper Absorption[J].Anal. Biochem.,1975,66(1):253-258.
[97] D. M. Olive. Quantitative methods for analysis of protein phosphorylation in drug development[J].Expert Rev. Proteomics,2004,1(3):327-341.
[98] R. Seethala, R. Menzel. A Fluorescence Polarization Competition Immunoassay for TyrosineKinases[J]. Anal. Chem.,1998,255(2):257-262.
[99] M. G. Shapiro, J. O. Szablowski, R. Langer, et al. Protein Nanoparticles Engineered to Sense KinaseActivity in MRI[J]. J. Am. Chem. Soc.,2009,131(7),2484-2486.
[100] C. L. Wang, L. Y. Wei, C. J. Yuan, et al. Reusable Amperometric Biosensor for Measuring ProteinTyrosine Kinase Activity[J]. Anal. Chem.,2012,84(2):971-977.
[101] H. Wei, C. Chen, B. Han, et al. Enzyme Colorimetric Assay Using Unmodifed SilverNanoparticles[J]. Anal. Chem.,2008,80(18):7051-7055.
[102] S. A. Beausoleil, J. Villén, S. A. Gerber, et al. A probability-based approach for high-throughputprotein phosphorylation analysis and site localization[J]. Nat. Biotechnol.,2006,24:1285-1292.
[103] T. Yoshida, M. Sato, T. Ozawa, et al. An SPR-Based Screening Method for Agonist Selectivity forInsulin Signaling Pathways Based on the Binding of Phosphotyrosine to Its Specific BindingProtein[J]. Anal. Chem.,2000,72(1):6-11.
[104] L. Qiao, C. Roussel, J. J. Wan, et al. Specifc On-Plate Enrichment of Phosphorylated Peptides forDirect MALDI-TOF MS Analysis[J]. J. Proteome Res.,2007,6(12):4763-4769.
[105] W. Li, J. Yang, Z. Wu, et al. A Versatile Kinetics-Controlled Coating Method To Construct UniformPorous TiO2Shells for Multifunctional Core-Shell Structures[J]. J. Am. Chem. Soc.,2012,134(29):11864-11867.
[106] H. Zhou, S. Xu, M. Ye, et al. Zirconium Phosphonate-Modified Porous Silicon for Highly SpecificCapture of Phosphopeptides and MALDI-TOF MS Analysis[J]. J. Proteome Res.,2006,5(9):2431-2437.
[107] M. Kokubu, Y. Ishihama, T. Sato, et al. Specificity of Immobilized Metal Affinity-Based IMAC/C18Tip Enrichment of Phosphopeptides for Protein Phosphorylation Analysis[J]. Anal. Chem.,2005,77(16):5144-5154.
[108] A. M. Lipchik, L. L. Parker. Time-Resolved Luminescence Detection of Spleen Tyrosine KinaseActivity through Terbium Sensitization[J]. Anal. Chem.,2013,85(5):2582-2588.
[109] X. Xu, Z. Nie, J. Chen, et al. A DNA-based electrochemical strategy for label-free monitoring theactivity and inhibition of protein kinase[J]. Chem. Comm.,2009,45:6946-6948.
[110] Y. Shinohara, T. Ishida, M. Hino, et al. Characterization of porin isoforms expressed in tumor cells[J].Eur. J. Biochem.,2000,267(19):6067-6073.
[111] Y. Xiang, Y. Lu. Portable and quantitative detection of protein biomarkers and small molecular toxinsusing antibodies and ubiquitous personal glucose meters[J]. Anal. Chem.,2012,84(9):4174-4178.