基于磁性纳米微粒与微流控芯片的血栓形成及药物溶栓机理研究
|
摘要
血栓栓塞性疾病是威胁人类健康的一大原因,且其发病率不断升高,对该疾病的研究也就刻不容缓。为了增加现有溶栓药物的溶栓作用及靶向性,本研究主要将溶解血栓的两种天然酶类蛋白(纳豆激酶和蚓激酶)连接于磁性纳米颗粒上,对其连接条件进行优化,产物性质进行表征,并进一步检测了其溶栓活性。此外为了实现体外条件下血栓形成及溶解的微型化、高效化模型检测系统,为血栓栓塞性疾病及治疗药物的研究提供依据和方法,本实验以微流控芯片技术为平台在体外逼真模拟体内血管血栓的形成,并用上述两种溶栓蛋白酶(Nattokinase and Lumbrukinase)进行溶栓行为研究。
用EDC将两种重要的溶栓酶(NK和LK)固定于Fe3O4磁性纳米颗粒上,并研究了它们的溶栓活性。用透射电镜,傅立叶变换红外分光镜,振动探针式磁强计,X射线衍射和紫外可见分光光度计对Fe3O4磁性纳米粒子及NK和LK连接的磁性纳米粒子进行性能分析。在405nm和630nm处进行双波长吸光值测定,研究其溶栓活性。通过载药率分析,NK连接的最佳条件为pH6.00,MNPs : protein : EDC的值为2 : 1 : 1;LK连接的最佳条件为pH6.00,MNPs : protein : EDC的值为2 : 1 : 2。溶栓活性试验表明NK连接的磁性粒子溶栓活性可达91.89%,LK可达207.74%,甚至比纯的NK(82.86%)和LK (106.57%)还高。
用PDMS制备不同管径的芯片,用注射泵以一定流速注入血小板丰富的血浆和凝血酶,在倒置荧光显微镜下实时观察血栓生成情况。芯片管径越小生成血栓越大且容易堵塞管道,溶解需较长时间;芯片管径越大则生成血栓较细小,溶解时所需时间也较少。在已生成血栓的管径注入不同浓度NK和LK使其溶解,在显微镜下观察其溶解过程,并用CCD照相机取图。通过溶解时间及血栓面积变化情况分析,管径内凝块在高浓度药物作用下溶解较快,2.5 mg/mL NK和LK溶解时间分别为21分钟和12分钟,且同等浓度下,LK的溶解效率比NK高(平均速率分别为23409.5 a.u./min和14208.4 a.u./min)。
Thromboembolic diseases is the major cause of threatening human health, it’s of great urgency to study it as the morbidity is increasing every year. In order to strengthen the function and drug targeting of thrombolytic reagents, we conjugated two thrombolytic emzyms (nattokinase and lumbrukinase) to magnetic nanopartiles, optimized the conjugation conditions, characteristiced the products properties and tested the thrombolytic activity. In addition, a microfluidic chip was used to mimic the in vivo conditions of thrombosis in vessel, also the thrombolysis process was analyzed at the function of NK and LK, thus realized the foundation of micro and efficient detection system in vitro and provided groundwork and method for studying thromboembolic diseases and drugs.
Two important thrombolytic enzymes, nattokinase (NK) and lumbrukinase (LK), were immobilized onto fine magnetic Fe3O4 nanoparticles using 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide (EDC) as the coupling reagent, and their thrombolytic activities were studied. The Fe3O4 nanoparticles and NK- and LK-conjugated magnetic nanoparticles were characterized by transmission electron microscopy, Fourier transform infrared spectrophotometry, vibrating sample magnetometry, X-ray diffraction, and UV–vis absorption spectroscopy. Dual kinetic absorbance measurements at 405nm and 630 nm were employed to measure their thrombolytic activity. Analysis of protein amount showed that the optimum conditions for NK and LK binding to nanoparticles were respectively at a mass ratio of 2 : 1 : 1, 2 : 1 : 2 (magnetic nanoparticles : protein : EDC), and pH6.00. Thrombolytic activity assay showed that the best thrombolytic activity could reach 91.89% for NK–nanoparticle conjugates and 207.74% for LK–nanoparticle conjugates, which are much higher than the pure enzymes (NK, 82.86%; LK, 106.57%).
PDMS was used as the material of making this microfluidic chip with different size, pletelet rich plasma and thrombin were injected into the channel by syringe pump under a certain flow rate, then observed the thrombosis with converted fluorescence microscope. With smaller channel the thrombus formed was big and easy to block, also needed longer time to lysis, whereas the thrombus was small and thin in the bigger channel and the thrombolysis time was shorter. Different concentrations of NK and LK were infused into the channel with formed thrombi, watched the thrombolytic process by microscope and got the pictures in real time by CCD camera. Analysis of thrombolysis time and thrombus area showed that the clot lysised faster in high concentration of the thrombolytic agents, the thrombolysis time were 21min and 12min respectively when the concentration was 2.5 mg/mL of NK and LK, the anverage thrombolysis rate of LK was higher than NK (23409.5 a.u./min and 14208.4 a.u./min respectively) at the same conditions.
用EDC将两种重要的溶栓酶(NK和LK)固定于Fe3O4磁性纳米颗粒上,并研究了它们的溶栓活性。用透射电镜,傅立叶变换红外分光镜,振动探针式磁强计,X射线衍射和紫外可见分光光度计对Fe3O4磁性纳米粒子及NK和LK连接的磁性纳米粒子进行性能分析。在405nm和630nm处进行双波长吸光值测定,研究其溶栓活性。通过载药率分析,NK连接的最佳条件为pH6.00,MNPs : protein : EDC的值为2 : 1 : 1;LK连接的最佳条件为pH6.00,MNPs : protein : EDC的值为2 : 1 : 2。溶栓活性试验表明NK连接的磁性粒子溶栓活性可达91.89%,LK可达207.74%,甚至比纯的NK(82.86%)和LK (106.57%)还高。
用PDMS制备不同管径的芯片,用注射泵以一定流速注入血小板丰富的血浆和凝血酶,在倒置荧光显微镜下实时观察血栓生成情况。芯片管径越小生成血栓越大且容易堵塞管道,溶解需较长时间;芯片管径越大则生成血栓较细小,溶解时所需时间也较少。在已生成血栓的管径注入不同浓度NK和LK使其溶解,在显微镜下观察其溶解过程,并用CCD照相机取图。通过溶解时间及血栓面积变化情况分析,管径内凝块在高浓度药物作用下溶解较快,2.5 mg/mL NK和LK溶解时间分别为21分钟和12分钟,且同等浓度下,LK的溶解效率比NK高(平均速率分别为23409.5 a.u./min和14208.4 a.u./min)。
Thromboembolic diseases is the major cause of threatening human health, it’s of great urgency to study it as the morbidity is increasing every year. In order to strengthen the function and drug targeting of thrombolytic reagents, we conjugated two thrombolytic emzyms (nattokinase and lumbrukinase) to magnetic nanopartiles, optimized the conjugation conditions, characteristiced the products properties and tested the thrombolytic activity. In addition, a microfluidic chip was used to mimic the in vivo conditions of thrombosis in vessel, also the thrombolysis process was analyzed at the function of NK and LK, thus realized the foundation of micro and efficient detection system in vitro and provided groundwork and method for studying thromboembolic diseases and drugs.
Two important thrombolytic enzymes, nattokinase (NK) and lumbrukinase (LK), were immobilized onto fine magnetic Fe3O4 nanoparticles using 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide (EDC) as the coupling reagent, and their thrombolytic activities were studied. The Fe3O4 nanoparticles and NK- and LK-conjugated magnetic nanoparticles were characterized by transmission electron microscopy, Fourier transform infrared spectrophotometry, vibrating sample magnetometry, X-ray diffraction, and UV–vis absorption spectroscopy. Dual kinetic absorbance measurements at 405nm and 630 nm were employed to measure their thrombolytic activity. Analysis of protein amount showed that the optimum conditions for NK and LK binding to nanoparticles were respectively at a mass ratio of 2 : 1 : 1, 2 : 1 : 2 (magnetic nanoparticles : protein : EDC), and pH6.00. Thrombolytic activity assay showed that the best thrombolytic activity could reach 91.89% for NK–nanoparticle conjugates and 207.74% for LK–nanoparticle conjugates, which are much higher than the pure enzymes (NK, 82.86%; LK, 106.57%).
PDMS was used as the material of making this microfluidic chip with different size, pletelet rich plasma and thrombin were injected into the channel by syringe pump under a certain flow rate, then observed the thrombosis with converted fluorescence microscope. With smaller channel the thrombus formed was big and easy to block, also needed longer time to lysis, whereas the thrombus was small and thin in the bigger channel and the thrombolysis time was shorter. Different concentrations of NK and LK were infused into the channel with formed thrombi, watched the thrombolytic process by microscope and got the pictures in real time by CCD camera. Analysis of thrombolysis time and thrombus area showed that the clot lysised faster in high concentration of the thrombolytic agents, the thrombolysis time were 21min and 12min respectively when the concentration was 2.5 mg/mL of NK and LK, the anverage thrombolysis rate of LK was higher than NK (23409.5 a.u./min and 14208.4 a.u./min respectively) at the same conditions.
引文
程敬泉,高政,马惠卿,段晓旭,张美月. 2008.纳米氧化铁的制备及其应用.衡水学院学报, 2(10): 41~43
方肇伦,方群. 2001.微流控芯片发展与展望.现代科学仪器, 4: 3~6
黄辉. 2006.基于磁性微球的PMMA微流控免疫分析芯片系统的研究. [博士学位论文].重庆:重庆大学
何运兵. 2006.磁性靶向药物载体Fe3O4的制备及性能研究. [硕士学位论文].江西:南昌大学
刘国卿. 2003.药理学.北京:中国医药科技出版社: 273~277
李清. 2008.大众公共科技. http://heal.cpst.net.cn/jknw/2008_03/204531778.html [2009-04-23]
李小花. 2006.螺旋藻激酶抗凝和溶栓作用的实验研究. [硕士学位论文].广西:广西医科大学
莫锦秋,梁庆华,汪国. 2004.微机电系统设计与制造.北京:化学工业出版社:1~30
秦润华,姜炜,刘宏英,李凤生. 2003.纳米磁性四氧化三铁的制备和表征.材料导报, 17:66
秦国宏,熊小超,张菊花,邢建民,刘会洲. 2008.枯草芽孢杆菌联产纳豆激酶和γ-聚谷氨酸.过程工程学报, 8(1):120~123
史旭波,胡大一. 2007.血栓形成与凝血机制及调节.临床荟萃, 22(14): 989~991
宋宏新,李敏康. 2002.现代生物化学实验技术教程.西安:陕西人民出版社: 66~68, 240~243
孙兆军,袁桂枝,梁国栋. 2001.蚯蚓纤溶酶分子生物学进展.生物工程进展, 21(6): 15~18
汪世华,胡开辉,沙莉,杨燕凌,林尾珠. 2006.溶栓酶的研究与开发,现代食品科技, 1: 174~176
薛巍,姜雯,苏荣国. 2007.微流控芯片检测技术进展.化学分析计量, 16(3): 77~79
许芳,杨艳燕,阎达中. 2003.溶栓药物的研究进展及发展方向.现代商贸工业, 7: 45~47
赵洪,何执中. 2003.溶栓药物研究进展.中国生化药物杂志, 24(1):15, 25, 35
郑虎. 2005.药物化学.北京:人民卫生出版社: 151
Adams T E, Huntington J A. 2006. Thrombin–Cofactor Interactions: Structural Insights Into Regulatory Mechanisms. Arterioscler. Thromb. Vasc. Biol. 26:1738~1745
Aizawa H, Kurosawa S, Tozuka M, Park J W, Kobayashi K, Tanaka H. 2003. Conventional detection method of fibrinogen and fibrin degradation products using latex piezoelectric immunoassay. Biosensors and Bioelectronics, 18: 765~771
Al Shwafi K, Meester A, Pirenne B, Renkin J, Col J. 2000. Rapid detection of streptokinase resistance using a bedside lytic assay of dry reagent technology. Fibrinolysis & Proteolysis, 14 (6): 351~357
Blinc A, Magdi? J, Fric J, Mu?evi? I. 2000. Atomic force microscopy of fibrin networks and plasma clots during fibrinolysis. Fibrinolysis & Proteolysis, 14 (5): 288~299
Bacri J C, Perzynski R, Salin D, Cabuil V and Massart R. 1990. Ionic ferrofluids: A crossing of chemistry and physics. J. Magn. Magn. Mater, 85: 27~32
Collen D, Lijnen H R. 2000. Recent developments in thrombolytic therapy. Fibrinolysis & Proteolysis, 14 (2/3): 66~72
Darton N J, Hallmark B, Han X, Palit S, Slater N K H, Mackley M R. 2008. The in-flow capture of superparamagnetic nanoparticles for targeting therapeutics. Nanomedicine: Nanotechnology, Biology and Medicine, 4: 19~29
Dunn E J, Philippou H, Ari?ns R A S, Grant P J. 2006. Molecular mechanisms involved in the resistance of fibrin to clot lysis by plasmin in subjects with type 2 diabetes mellitus. Diabetologia, 49: 1071~1080
Dziubla T D, Shuvaev V V, Hong N K, Hawkins B J, Madesh M, Takano H, Simone E, Nakada M T, Fisher A, Albelda S M, Muzykantov V R. 2008. Endothelial targeting of semi-permeable polymer nanocarriers for enzyme therapies. Biomaterials, 29: 215~227
Eteshola E, Leckband D. 2001. Development and characterization of an ELISA assay in PDMS microfluidic channels. Sensors and Actuators B, 72: 129~133
Erdo?an S, Yekta?zer A, Bilgili H. 2005. In vivo behaviour of vesicular urokinase. International Journal of Pharmaceutics, 295: 1~6
Furie B and Furie B C. 2007. In vivo thrombus formation. Journal of Thrombosis and Haemostasis, 5 (Suppl. 1): 12~17
Falati S, Gross P, Merrill-skoloff G, Furie B C, Furie B. 2002. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. NATURE MEDICINE , 8(10): 1175~1180
Fujita M, Ito Y, Hong K, Nishimuro S. 1995. Characterization of Nattokinase-degraded Products from Human Fibrinogen or Cross-linked Fibrin. Fibrinolysis, 9:157~164
Falati S, Liu Q, Gross P, Merrill-Skoloff G, Chou J, Vandendries E, Celi A, Croce K, Furie B C, and Furie B. 2003. Accumulation of Tissue Factor into Developing Thrombi In Vivo Is Dependent upon Microparticle P-Selectin Glycoprotein Ligand 1 and Platelet P-Selectin. J. Exp. Med.197(11): 1585~1598
Fernández -Pacheco R, Marquina C, Gabriel Valdivia J, Gutiérrez M, Soledad Romero M, Cornudella R, Laborda A, Viloria A, Higuera T, García A, Antonio García de Jalón J, Ricardo Ibarra M. 2007.
Magnetic nanoparticles for local drug delivery using magnetic implants. Journal of Magnetism and Magnetic Materials, 311: 318~322
Fermandes E G R, Queiroz A A A D, Abraham G A, Román J S. 2006. Antithrombogenic properties of bioconjugate streptokinase-polyglycerol dendrimers. Journal of Materials Science:Materials in Medicine, 17: 105~111
Fan R, Vermesh O, Srivastava A, Yen B K H, Qin L, Ahmad H, Kwong G A, Liu Ch Ch, Gould J, Hood L, Heath J R. 2008. Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood. Nature Biotechnology, 26(12):1373~1378
Gu F X, Karnik R, Wang A Z, Alexis F, Levy-Nissenbaum E, Hong S, Langer R S, and Farokhzad O C. 2007.Targeted nanoparticles for cancer therapy. Nanotoday, 3:14~21
Goldenberg N A, Hathaway W E, Jacobson L, Manco-Johnson M J. 2005. A new global assay of coagulation and fibrinolysis. Thrombosis Research, 116: 345~356
Graham D A, Huang T C, Keyt B A, and Rita Alevriadou B. 1998. Real-Time Measurement of Lysis of Mural Platelet Deposits by Fibrinolytic Agents under Arterial Flow. Annals of Biomedical Engineering, 26: 712~724
Giesen P L A, Rauch U, Bohrmann B, Kling D, RoquéM, Fallon J T, Badimon J J, Himber J, Riedrer M A, Nemerson Y. 1999. Blood-borne tissue factor: Another view of thrombosis. Proc. Natl. Acad. Sci. 96: 2311~2315
Gilbert C, White II. 2008. Current status of thrombolytics—the need for better, especially safer, agents. Thrombosis Research, 122: S1~S2
He S, Antovic A and Blomb?ck M. 2001. A Simple and Rapid Laboratory Method for Determination of Haemostasis Potential in Plasma II. Modifications for Use in Routine Laboratories and Research Work. Thrombosis Research 103:355~361
He S, Bremme K and Blomb?ck M. 1999. A Laboratory Method for Determination of Overall Haemostatic Potential in Plasma. I. Method Design and Preliminary Results. Thrombosis Research, 96: 145~156
Hong J, Edel J B and deMello A J. 2009. Micro- and nanofluidic systems for high-throughput biological screening. Drug discovery today, 14:134~146
Hsu Y C, Lin S J, Hou C C. 2007. Development of peristaltic antithrombogenic micropumps for in vitro and ex vivo blood transportation tests. Microsyst Technol, 14:31~41
Hamano A, Tanaka S, Takeda Y, Umeda M, Sakata Y. 2002. A novel monoclonal antibody to fibrin monomer and soluble fibrin for the detection of soluble fibrin in plasma. Clinica Chimica Acta, 318: 25~32
Ionov L, Houbenov N, Sidorenko A, Stamm M, and Minko S. 2006. Smart Microfluidic Channels. Adv. Funct. Mater., 16: 1153~1160
Jonathan M, Gibbins Martyn P, Mahaut-Smith. 2008. Platelets and Megakaryocytes. Humana press: vol. 272:165~186
Jonathan M, Gibbins Martyn P, Mahaut-Smith. 2008. Platelets and Megakaryocytes . Humana press. 272 (1): 187~197
Ji H M, Samper V, Chen Y, Heng C K, Lim T M, Yobas L. 2008. Silicon-based microfilters for whole blood cell separation. Biomed Microdevices, 10:251~257
Johnson A K, Zawadzka A M, Deobald L A, Crawford R L, Paszczynski A J. 2008. Novel method for immobilization of enzymes to magnetic nanoparticles. J Nanopart Res, 10:1009~1025
Kling J. 2006. Moving diagnostics from the bench to the bedside. Nature Biotechnology, 24(8): 891~893
KonerackáM, Kop?ansky P, Timko M, Ramchand C N, Sequeira A, Trevan M. 2002. Direct binding procedure of proteins and enzymes to fine magnetic particles. Journal of Molecular Catalysis B: Enzymatic, 18: 13~18
Ku C J, Teresa D’Amico Oblak, and Spence D M. 2008. Interactions between Multiple Cell Types in Parallel Microfluidic Channels: Monitoring Platelet Adhesion to an Endothelium in the Presence of an Anti-Adhesion Drug. Anal. Chem, 80: 7543~7548
Kurotobi K, Yamamoto A, Kikuta A, Hanawa T. 2007. Short term evaluation of material blood compatibility using a microchannel array. J Mater Sci: Mater Med, 18: 1175~1184
Ko J H, Yan J P, Zhu L, Qi Y P. 2004. Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02. Comparative Biochemistry and Physiology Part C, 137: 65~74
Lu A S, Bergemann C, Brock J, McClure D G. 1999. Physiological aspects in magnetic drug-targeting. Journal of Magnetism and Magnetic Materials 194: 149~155
Li G Y, Huang K L, Jiang Y R, Yang D L, Ding P. 2008. Preparation and characterization of Saccharomyces cerevisiae alcohol dehydrogenase immobilized on magnetic nanoparticles. International Journal of Biological Macromolecules 42: 405~412
Li H P, Hu Z, Yuan J L, Fan H D, Chen W, Wang S J, Zheng S S, Zheng Z L and Zou G L. 2007. A Novel Extracellular Protease with Fibrinolytic Activity from the Culture Supernatant of Cordyceps sinensis: Purification and Characterization. Phytother. Res. 21: 1234~1241
Linder V, Verpoorte E, Thormann W, Rooij N F, Sigrist H. 2001. Surface biopassivation of replicated poly(dimethylsiloxane) microfluidic channels and application to heterogeneous immunoreaction with on-chip fluorescence detection. Analytical Chemistry, 73(17): 4181~4189
Lim C T, Zhang Y. 2007. Bead-based microfluidic immunoassays: The next generation. Biosensors and Bioelectronics, 22: 1197~1204
Mackman N. 2008. Triggers, targets and treatments for thrombosis. NATURE, 451: 914~918
Matsuda M. 2000. Structure and function of fibrinogen inferred from hereditary dysfibrinogens. Fibrinolysis & Proteolysis,14(2/3): 187~197
Mosesson M W. 2000. Fibrinogen functions and fibrin assembly. Fibrinolysis & Proteolysis, 14(2/3): 182~186
Martinoia S, Bove M, Tedesco M, Margesin B, Grattarola M. 1999. A simple microfluidic system for patterning populations of neurons on silicon micromachined substrates. J-Neurosci-Methods. 87(l): 35~44
Mutch N J, Moore N R, Wang E and Booth N A. 2003. Thrombus lysis by uPA, scuPA and tPA is regulated by plasma TAFI. Journal of Thrombosis and Haemestasis, 1: 2000~2007
Mizuno T, Sugimoto M, Matsui H, Hamada M, Shida Y, Yoshioka A. 2008. Visual evaluation of blood coagulation during mural thrombogenesis under high shear blood flow. Thrombosis Research, 121: 855~864
Matsui H, Sugimoto M, Mizuno T, Tsuji S, Miyata S, Matsuda M, and Yoshioka A. 2002. Distinct and concerted functions of vonWillebrand factor and fibrinogen in mural thrombus growth under high shear flow. BLOOD, 100(10): 3604~3610
Moresco R N, Vargas L C R, Silla L. 2007. Estimation of the levels of D-dimer by use of an alternative method based in the reaction time of fibrinogen/fibrin degradation products assay. J Thromb Thrombolysis, 24: 73~76
Mine Y, Wong A H K and Jiang B. 2005. Fibrinolytic enzyme in Asian traditional fermented foods. Food Res.Int., 38:243~250
Novokhatny V. 2008. Structure and activity of plasmin and other direct thrombolytic agents. Thrombosis Research, 122: S3~S8
Ngaboni Okassa L, Marchais H, Douziech-Eyrolles L, Cohen-Jonathan S, SoucéM, Dubois P, Chourpa I. 2005. Development and characterization of sub-micron poly particles loaded with magnetite/ maghemite nanoparticles. International Journal of Pharmaceutics, 302: 187~196
Olkhov R V, Shaw A M. 2008. Label-free antibody–antigen binding detection by optical sensor array based on surface-synthesized gold nanoparticles. Biosensors and Bioelectronics, 23: 1298~1302
Pihl J, Karlsson M and Chiu D T. 2005. Microfluidic technologies in drug discovery. DDT, 10: 1377~1383
Park Y D, Kim J W, Min B G, Seo J W, and Jeong J M. 1998. Rapid purification and biochemical characteristics of lumbrokinase III from earthworm for use as a fibrinolytic agent. Biotechnology Letters, 20(2): 169~172
Peng Y, Yang X J, Zhang Y Z. 2005. Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo. Appl Microbiol Biotechnol, 69: 126~132
Packer L, Tristram S, Herz J M, Russell C and Boraers C L. 1979. Chemical modification of purple membranes: role of arginine and carboxylic acid residues in bacteriorhodopsin. FEBS Letters, 108:243~248
Razzacki S Z, Thwar P K, Yang M, Ugaz V M, Burns M A. 2004. Integrated microsystems for controlled drug delivery. Advanced Drug Delivery Reviews, 56:185~198
Sarkar N K. 1960. Mechanism of Clot Lysis. Nature 185:624~625
Sorger P K. 2008. Microfluidics closes in on point-of-care assays. Nature Biotechnology, 26(12): 1345~1346
Smith A A, Jacobson L J, Miller B I, Hathaway W E, Manco-Johnson M J. 2003. A new euglobulin clot lysis assay for global fibrinolysis.Thrombosis Research, 112: 329~337
Song H, Li H W, Munson M S, Van Ha T G, and Ismagilov R F. 2006. On-Chip Titration of an Anticoagulant Argatroban and Determination of the Clotting Time within Whole Blood or Plasma Using a Plug-Based Microfluidic System. Anal. Chem., 78: 4839~4849
Sugimoto M, Matsui H, Mizuno T, Tsuji S, Miyata S, Matsumoto M, Matsuda M, Fujimura Y, and Yoshioka A. 2003. Mural thrombus generation in type 2Aand 2B vonWillebrand disease under flow conditions. BLOOD, 101(3): 915~920
Sun Y K, Ma M, Zhang Y, Gu N. 2004. Synthesis of nanometer-size maghemite particles from magnetite. Colloids and Surfaces A: Physicochem. Eng. Aspects, 245: 15~19
Torno M D, Kaminski M D, Xie Y M, Meyers R E, Mertz C J, Liu X Q, O'Brien Jr W D, Rosengart A J 2008. Improvement of in vitro thrombolysis employing magnetically-guided microspheres. Thrombosis Research, 121: 799~811
Tovar-Lopez F J, Rosengarten G, Westein E, Khoshmanesh K, Jackson S P, Mitchell A and Nesbitt W S . 2010. A microfluidics device to monitor platelet aggregation dynamics in response to strain rate micro-gradients in flowing blood. Lab Chip, DOI: 10.1039/b916757a
Tsuji S, Sugimoto M, Miyata S, Kuwahara M, Kinoshita S, and Yoshioka A.1999. Real-Time Analysis of Mural Thrombus Formation in Various Platelet Aggregation Disorders: Distinct Shear-Dependent Roles of Platelet Receptors and Adhesive Proteins under Flow. Blood, 94(3): 968~975
Ueda M, Kubo T, Miyatake K, Nakamura T. 2007. Purification and characterization of fibrinolytic alkaline protease from Fusarium sp. BLBAppl Microbiol Biotechnol, 74: 331~338
Whitesides G M. 2006. The origins and the future of microfluidics. NATURE, 442(27): 368~373
Wang C T, Ji B P, Li B, Nout R, Li P L, Ji H, Chen L F. 2006. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. J Ind Microbiol Biotechnol, 33: 750~758
Wang Y J, Wang X H, Luo G S, Dai Y Y. 2008. Adsorption of bovin serum albumin (BSA) onto the magnetic chitosan nanoparticles prepared by a microemulsion system. Bioresource Technology, 99: 3881~3884
Wang F, Wang C, Li M, Gui L L, Zhang J P, Chang W R. 2003. Purification, characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia fetida. Biotechnology Letters, 25: 1105~1109
Walker J M, Rapley R. 2008. Second edition. Molecular biomethods handbook. Humana press: Chapter 48: 851~859
Xu Z R, Fang Z L. 2004. Composite poly(dimethylsiloxane)/glass microfluidic system with an immobilized enzymatic particle-bed reactor and sequential sample injection for chemiluminescencedeterminations. Analytica Chimica Acta, 507: 129~135
Yoshiok K, Mizuno S, Miyata H, and Maki S. 1982. Distinction Between Fibrinogen and Fibrin Degradation Products Produced During Disseminated Intravascular Coagulation in Childhood. Eur. Pediatr, 138: 46~48
Zacharowski K, Zacharowski P, Reingruber S, Petzelbauer P. 2006. Fibrin(ogen) and its fragments in the pathophysiology and treatment of myocardial infarction. J Mol Med, 84: 469~477
Zhao X M, Wu Y P, Cai H X, Wei R, Lisman T, Han J J, Xia Z L, Groot P G. 2008. The influence of the pulsatility of the blood flow on the extent of platelet adhesion. Thrombosis Research 121: 821~825
方肇伦,方群. 2001.微流控芯片发展与展望.现代科学仪器, 4: 3~6
黄辉. 2006.基于磁性微球的PMMA微流控免疫分析芯片系统的研究. [博士学位论文].重庆:重庆大学
何运兵. 2006.磁性靶向药物载体Fe3O4的制备及性能研究. [硕士学位论文].江西:南昌大学
刘国卿. 2003.药理学.北京:中国医药科技出版社: 273~277
李清. 2008.大众公共科技. http://heal.cpst.net.cn/jknw/2008_03/204531778.html [2009-04-23]
李小花. 2006.螺旋藻激酶抗凝和溶栓作用的实验研究. [硕士学位论文].广西:广西医科大学
莫锦秋,梁庆华,汪国. 2004.微机电系统设计与制造.北京:化学工业出版社:1~30
秦润华,姜炜,刘宏英,李凤生. 2003.纳米磁性四氧化三铁的制备和表征.材料导报, 17:66
秦国宏,熊小超,张菊花,邢建民,刘会洲. 2008.枯草芽孢杆菌联产纳豆激酶和γ-聚谷氨酸.过程工程学报, 8(1):120~123
史旭波,胡大一. 2007.血栓形成与凝血机制及调节.临床荟萃, 22(14): 989~991
宋宏新,李敏康. 2002.现代生物化学实验技术教程.西安:陕西人民出版社: 66~68, 240~243
孙兆军,袁桂枝,梁国栋. 2001.蚯蚓纤溶酶分子生物学进展.生物工程进展, 21(6): 15~18
汪世华,胡开辉,沙莉,杨燕凌,林尾珠. 2006.溶栓酶的研究与开发,现代食品科技, 1: 174~176
薛巍,姜雯,苏荣国. 2007.微流控芯片检测技术进展.化学分析计量, 16(3): 77~79
许芳,杨艳燕,阎达中. 2003.溶栓药物的研究进展及发展方向.现代商贸工业, 7: 45~47
赵洪,何执中. 2003.溶栓药物研究进展.中国生化药物杂志, 24(1):15, 25, 35
郑虎. 2005.药物化学.北京:人民卫生出版社: 151
Adams T E, Huntington J A. 2006. Thrombin–Cofactor Interactions: Structural Insights Into Regulatory Mechanisms. Arterioscler. Thromb. Vasc. Biol. 26:1738~1745
Aizawa H, Kurosawa S, Tozuka M, Park J W, Kobayashi K, Tanaka H. 2003. Conventional detection method of fibrinogen and fibrin degradation products using latex piezoelectric immunoassay. Biosensors and Bioelectronics, 18: 765~771
Al Shwafi K, Meester A, Pirenne B, Renkin J, Col J. 2000. Rapid detection of streptokinase resistance using a bedside lytic assay of dry reagent technology. Fibrinolysis & Proteolysis, 14 (6): 351~357
Blinc A, Magdi? J, Fric J, Mu?evi? I. 2000. Atomic force microscopy of fibrin networks and plasma clots during fibrinolysis. Fibrinolysis & Proteolysis, 14 (5): 288~299
Bacri J C, Perzynski R, Salin D, Cabuil V and Massart R. 1990. Ionic ferrofluids: A crossing of chemistry and physics. J. Magn. Magn. Mater, 85: 27~32
Collen D, Lijnen H R. 2000. Recent developments in thrombolytic therapy. Fibrinolysis & Proteolysis, 14 (2/3): 66~72
Darton N J, Hallmark B, Han X, Palit S, Slater N K H, Mackley M R. 2008. The in-flow capture of superparamagnetic nanoparticles for targeting therapeutics. Nanomedicine: Nanotechnology, Biology and Medicine, 4: 19~29
Dunn E J, Philippou H, Ari?ns R A S, Grant P J. 2006. Molecular mechanisms involved in the resistance of fibrin to clot lysis by plasmin in subjects with type 2 diabetes mellitus. Diabetologia, 49: 1071~1080
Dziubla T D, Shuvaev V V, Hong N K, Hawkins B J, Madesh M, Takano H, Simone E, Nakada M T, Fisher A, Albelda S M, Muzykantov V R. 2008. Endothelial targeting of semi-permeable polymer nanocarriers for enzyme therapies. Biomaterials, 29: 215~227
Eteshola E, Leckband D. 2001. Development and characterization of an ELISA assay in PDMS microfluidic channels. Sensors and Actuators B, 72: 129~133
Erdo?an S, Yekta?zer A, Bilgili H. 2005. In vivo behaviour of vesicular urokinase. International Journal of Pharmaceutics, 295: 1~6
Furie B and Furie B C. 2007. In vivo thrombus formation. Journal of Thrombosis and Haemostasis, 5 (Suppl. 1): 12~17
Falati S, Gross P, Merrill-skoloff G, Furie B C, Furie B. 2002. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. NATURE MEDICINE , 8(10): 1175~1180
Fujita M, Ito Y, Hong K, Nishimuro S. 1995. Characterization of Nattokinase-degraded Products from Human Fibrinogen or Cross-linked Fibrin. Fibrinolysis, 9:157~164
Falati S, Liu Q, Gross P, Merrill-Skoloff G, Chou J, Vandendries E, Celi A, Croce K, Furie B C, and Furie B. 2003. Accumulation of Tissue Factor into Developing Thrombi In Vivo Is Dependent upon Microparticle P-Selectin Glycoprotein Ligand 1 and Platelet P-Selectin. J. Exp. Med.197(11): 1585~1598
Fernández -Pacheco R, Marquina C, Gabriel Valdivia J, Gutiérrez M, Soledad Romero M, Cornudella R, Laborda A, Viloria A, Higuera T, García A, Antonio García de Jalón J, Ricardo Ibarra M. 2007.
Magnetic nanoparticles for local drug delivery using magnetic implants. Journal of Magnetism and Magnetic Materials, 311: 318~322
Fermandes E G R, Queiroz A A A D, Abraham G A, Román J S. 2006. Antithrombogenic properties of bioconjugate streptokinase-polyglycerol dendrimers. Journal of Materials Science:Materials in Medicine, 17: 105~111
Fan R, Vermesh O, Srivastava A, Yen B K H, Qin L, Ahmad H, Kwong G A, Liu Ch Ch, Gould J, Hood L, Heath J R. 2008. Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood. Nature Biotechnology, 26(12):1373~1378
Gu F X, Karnik R, Wang A Z, Alexis F, Levy-Nissenbaum E, Hong S, Langer R S, and Farokhzad O C. 2007.Targeted nanoparticles for cancer therapy. Nanotoday, 3:14~21
Goldenberg N A, Hathaway W E, Jacobson L, Manco-Johnson M J. 2005. A new global assay of coagulation and fibrinolysis. Thrombosis Research, 116: 345~356
Graham D A, Huang T C, Keyt B A, and Rita Alevriadou B. 1998. Real-Time Measurement of Lysis of Mural Platelet Deposits by Fibrinolytic Agents under Arterial Flow. Annals of Biomedical Engineering, 26: 712~724
Giesen P L A, Rauch U, Bohrmann B, Kling D, RoquéM, Fallon J T, Badimon J J, Himber J, Riedrer M A, Nemerson Y. 1999. Blood-borne tissue factor: Another view of thrombosis. Proc. Natl. Acad. Sci. 96: 2311~2315
Gilbert C, White II. 2008. Current status of thrombolytics—the need for better, especially safer, agents. Thrombosis Research, 122: S1~S2
He S, Antovic A and Blomb?ck M. 2001. A Simple and Rapid Laboratory Method for Determination of Haemostasis Potential in Plasma II. Modifications for Use in Routine Laboratories and Research Work. Thrombosis Research 103:355~361
He S, Bremme K and Blomb?ck M. 1999. A Laboratory Method for Determination of Overall Haemostatic Potential in Plasma. I. Method Design and Preliminary Results. Thrombosis Research, 96: 145~156
Hong J, Edel J B and deMello A J. 2009. Micro- and nanofluidic systems for high-throughput biological screening. Drug discovery today, 14:134~146
Hsu Y C, Lin S J, Hou C C. 2007. Development of peristaltic antithrombogenic micropumps for in vitro and ex vivo blood transportation tests. Microsyst Technol, 14:31~41
Hamano A, Tanaka S, Takeda Y, Umeda M, Sakata Y. 2002. A novel monoclonal antibody to fibrin monomer and soluble fibrin for the detection of soluble fibrin in plasma. Clinica Chimica Acta, 318: 25~32
Ionov L, Houbenov N, Sidorenko A, Stamm M, and Minko S. 2006. Smart Microfluidic Channels. Adv. Funct. Mater., 16: 1153~1160
Jonathan M, Gibbins Martyn P, Mahaut-Smith. 2008. Platelets and Megakaryocytes. Humana press: vol. 272:165~186
Jonathan M, Gibbins Martyn P, Mahaut-Smith. 2008. Platelets and Megakaryocytes . Humana press. 272 (1): 187~197
Ji H M, Samper V, Chen Y, Heng C K, Lim T M, Yobas L. 2008. Silicon-based microfilters for whole blood cell separation. Biomed Microdevices, 10:251~257
Johnson A K, Zawadzka A M, Deobald L A, Crawford R L, Paszczynski A J. 2008. Novel method for immobilization of enzymes to magnetic nanoparticles. J Nanopart Res, 10:1009~1025
Kling J. 2006. Moving diagnostics from the bench to the bedside. Nature Biotechnology, 24(8): 891~893
KonerackáM, Kop?ansky P, Timko M, Ramchand C N, Sequeira A, Trevan M. 2002. Direct binding procedure of proteins and enzymes to fine magnetic particles. Journal of Molecular Catalysis B: Enzymatic, 18: 13~18
Ku C J, Teresa D’Amico Oblak, and Spence D M. 2008. Interactions between Multiple Cell Types in Parallel Microfluidic Channels: Monitoring Platelet Adhesion to an Endothelium in the Presence of an Anti-Adhesion Drug. Anal. Chem, 80: 7543~7548
Kurotobi K, Yamamoto A, Kikuta A, Hanawa T. 2007. Short term evaluation of material blood compatibility using a microchannel array. J Mater Sci: Mater Med, 18: 1175~1184
Ko J H, Yan J P, Zhu L, Qi Y P. 2004. Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02. Comparative Biochemistry and Physiology Part C, 137: 65~74
Lu A S, Bergemann C, Brock J, McClure D G. 1999. Physiological aspects in magnetic drug-targeting. Journal of Magnetism and Magnetic Materials 194: 149~155
Li G Y, Huang K L, Jiang Y R, Yang D L, Ding P. 2008. Preparation and characterization of Saccharomyces cerevisiae alcohol dehydrogenase immobilized on magnetic nanoparticles. International Journal of Biological Macromolecules 42: 405~412
Li H P, Hu Z, Yuan J L, Fan H D, Chen W, Wang S J, Zheng S S, Zheng Z L and Zou G L. 2007. A Novel Extracellular Protease with Fibrinolytic Activity from the Culture Supernatant of Cordyceps sinensis: Purification and Characterization. Phytother. Res. 21: 1234~1241
Linder V, Verpoorte E, Thormann W, Rooij N F, Sigrist H. 2001. Surface biopassivation of replicated poly(dimethylsiloxane) microfluidic channels and application to heterogeneous immunoreaction with on-chip fluorescence detection. Analytical Chemistry, 73(17): 4181~4189
Lim C T, Zhang Y. 2007. Bead-based microfluidic immunoassays: The next generation. Biosensors and Bioelectronics, 22: 1197~1204
Mackman N. 2008. Triggers, targets and treatments for thrombosis. NATURE, 451: 914~918
Matsuda M. 2000. Structure and function of fibrinogen inferred from hereditary dysfibrinogens. Fibrinolysis & Proteolysis,14(2/3): 187~197
Mosesson M W. 2000. Fibrinogen functions and fibrin assembly. Fibrinolysis & Proteolysis, 14(2/3): 182~186
Martinoia S, Bove M, Tedesco M, Margesin B, Grattarola M. 1999. A simple microfluidic system for patterning populations of neurons on silicon micromachined substrates. J-Neurosci-Methods. 87(l): 35~44
Mutch N J, Moore N R, Wang E and Booth N A. 2003. Thrombus lysis by uPA, scuPA and tPA is regulated by plasma TAFI. Journal of Thrombosis and Haemestasis, 1: 2000~2007
Mizuno T, Sugimoto M, Matsui H, Hamada M, Shida Y, Yoshioka A. 2008. Visual evaluation of blood coagulation during mural thrombogenesis under high shear blood flow. Thrombosis Research, 121: 855~864
Matsui H, Sugimoto M, Mizuno T, Tsuji S, Miyata S, Matsuda M, and Yoshioka A. 2002. Distinct and concerted functions of vonWillebrand factor and fibrinogen in mural thrombus growth under high shear flow. BLOOD, 100(10): 3604~3610
Moresco R N, Vargas L C R, Silla L. 2007. Estimation of the levels of D-dimer by use of an alternative method based in the reaction time of fibrinogen/fibrin degradation products assay. J Thromb Thrombolysis, 24: 73~76
Mine Y, Wong A H K and Jiang B. 2005. Fibrinolytic enzyme in Asian traditional fermented foods. Food Res.Int., 38:243~250
Novokhatny V. 2008. Structure and activity of plasmin and other direct thrombolytic agents. Thrombosis Research, 122: S3~S8
Ngaboni Okassa L, Marchais H, Douziech-Eyrolles L, Cohen-Jonathan S, SoucéM, Dubois P, Chourpa I. 2005. Development and characterization of sub-micron poly particles loaded with magnetite/ maghemite nanoparticles. International Journal of Pharmaceutics, 302: 187~196
Olkhov R V, Shaw A M. 2008. Label-free antibody–antigen binding detection by optical sensor array based on surface-synthesized gold nanoparticles. Biosensors and Bioelectronics, 23: 1298~1302
Pihl J, Karlsson M and Chiu D T. 2005. Microfluidic technologies in drug discovery. DDT, 10: 1377~1383
Park Y D, Kim J W, Min B G, Seo J W, and Jeong J M. 1998. Rapid purification and biochemical characteristics of lumbrokinase III from earthworm for use as a fibrinolytic agent. Biotechnology Letters, 20(2): 169~172
Peng Y, Yang X J, Zhang Y Z. 2005. Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo. Appl Microbiol Biotechnol, 69: 126~132
Packer L, Tristram S, Herz J M, Russell C and Boraers C L. 1979. Chemical modification of purple membranes: role of arginine and carboxylic acid residues in bacteriorhodopsin. FEBS Letters, 108:243~248
Razzacki S Z, Thwar P K, Yang M, Ugaz V M, Burns M A. 2004. Integrated microsystems for controlled drug delivery. Advanced Drug Delivery Reviews, 56:185~198
Sarkar N K. 1960. Mechanism of Clot Lysis. Nature 185:624~625
Sorger P K. 2008. Microfluidics closes in on point-of-care assays. Nature Biotechnology, 26(12): 1345~1346
Smith A A, Jacobson L J, Miller B I, Hathaway W E, Manco-Johnson M J. 2003. A new euglobulin clot lysis assay for global fibrinolysis.Thrombosis Research, 112: 329~337
Song H, Li H W, Munson M S, Van Ha T G, and Ismagilov R F. 2006. On-Chip Titration of an Anticoagulant Argatroban and Determination of the Clotting Time within Whole Blood or Plasma Using a Plug-Based Microfluidic System. Anal. Chem., 78: 4839~4849
Sugimoto M, Matsui H, Mizuno T, Tsuji S, Miyata S, Matsumoto M, Matsuda M, Fujimura Y, and Yoshioka A. 2003. Mural thrombus generation in type 2Aand 2B vonWillebrand disease under flow conditions. BLOOD, 101(3): 915~920
Sun Y K, Ma M, Zhang Y, Gu N. 2004. Synthesis of nanometer-size maghemite particles from magnetite. Colloids and Surfaces A: Physicochem. Eng. Aspects, 245: 15~19
Torno M D, Kaminski M D, Xie Y M, Meyers R E, Mertz C J, Liu X Q, O'Brien Jr W D, Rosengart A J 2008. Improvement of in vitro thrombolysis employing magnetically-guided microspheres. Thrombosis Research, 121: 799~811
Tovar-Lopez F J, Rosengarten G, Westein E, Khoshmanesh K, Jackson S P, Mitchell A and Nesbitt W S . 2010. A microfluidics device to monitor platelet aggregation dynamics in response to strain rate micro-gradients in flowing blood. Lab Chip, DOI: 10.1039/b916757a
Tsuji S, Sugimoto M, Miyata S, Kuwahara M, Kinoshita S, and Yoshioka A.1999. Real-Time Analysis of Mural Thrombus Formation in Various Platelet Aggregation Disorders: Distinct Shear-Dependent Roles of Platelet Receptors and Adhesive Proteins under Flow. Blood, 94(3): 968~975
Ueda M, Kubo T, Miyatake K, Nakamura T. 2007. Purification and characterization of fibrinolytic alkaline protease from Fusarium sp. BLBAppl Microbiol Biotechnol, 74: 331~338
Whitesides G M. 2006. The origins and the future of microfluidics. NATURE, 442(27): 368~373
Wang C T, Ji B P, Li B, Nout R, Li P L, Ji H, Chen L F. 2006. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. J Ind Microbiol Biotechnol, 33: 750~758
Wang Y J, Wang X H, Luo G S, Dai Y Y. 2008. Adsorption of bovin serum albumin (BSA) onto the magnetic chitosan nanoparticles prepared by a microemulsion system. Bioresource Technology, 99: 3881~3884
Wang F, Wang C, Li M, Gui L L, Zhang J P, Chang W R. 2003. Purification, characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia fetida. Biotechnology Letters, 25: 1105~1109
Walker J M, Rapley R. 2008. Second edition. Molecular biomethods handbook. Humana press: Chapter 48: 851~859
Xu Z R, Fang Z L. 2004. Composite poly(dimethylsiloxane)/glass microfluidic system with an immobilized enzymatic particle-bed reactor and sequential sample injection for chemiluminescencedeterminations. Analytica Chimica Acta, 507: 129~135
Yoshiok K, Mizuno S, Miyata H, and Maki S. 1982. Distinction Between Fibrinogen and Fibrin Degradation Products Produced During Disseminated Intravascular Coagulation in Childhood. Eur. Pediatr, 138: 46~48
Zacharowski K, Zacharowski P, Reingruber S, Petzelbauer P. 2006. Fibrin(ogen) and its fragments in the pathophysiology and treatment of myocardial infarction. J Mol Med, 84: 469~477
Zhao X M, Wu Y P, Cai H X, Wei R, Lisman T, Han J J, Xia Z L, Groot P G. 2008. The influence of the pulsatility of the blood flow on the extent of platelet adhesion. Thrombosis Research 121: 821~825