四通道骨唾液蛋白压电免疫传感器阵列的研制  

Design of four-channel piezoelectric immunosensor array to detect bone sialoprotein

作　　者：陈钰[1,2] 刘仲明[2] 王捷[2] 

机构地区：[1]华南理工大学生物科学与工程学院,广东省广州市510006 [2]解放军广州军区广州总医院医学实验科,广东省广州市510010

出　　处：《中国组织工程研究与临床康复》2009年第43期8492-8495,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广东省科技计划项目(2009B030801280);广东省自然科学基金重点项目(06104396)~~

摘　　要：背景:骨唾液蛋白是细胞外基质中的一种高度磷酸化和糖基化的分泌性蛋白,其血清表达水平的检测在评估骨代谢水平方面具有重要的临床检测价值。目的:以AT切型、10Hz双面镀金的石英晶体为换能器,拟制作一种4通道压电蛋白芯片阵列,用于检验临床血清中骨唾液蛋白的表达水平。设计、时间及地点:观察实验,于2008-12/2009-07在解放军广州军区广州总医院医学实验科生物传感器实验室完成。材料:实验所用压电免疫传感器包括包被过的石英晶体、振荡器和频率计等。骨质疏松症患者血清(n=10)、正常人血清(n=9)、参考标准血清由解放军广州军区广州总医院检验科、血液中心提供。小牛血清、胎牛血清为Hyclone公司产品。方法:石英晶体的厚度切片模式采用AT切割,10MHz,大小为8.00mm×8.00mm×0.15mm,在石英晶片的双面采用真空镀膜的方法形成金电极,该阵列由弹簧支架以及4个石英晶体组成。采用双功能试剂Sulfo-LC-SPDP将鼠抗人骨唾液蛋白单克隆抗体巯基化,固定于石英晶体的金电极表面,通过免疫反应检测血清中骨唾液蛋白的表达。主要观察指标:不同抗体浓度与抗体固定量之间的关系,临床患者血清与正常人血清之间骨唾液蛋白表达的差异。结果:①随着抗体浓度的增加,石英晶体频率响应值也随之增大。实验以80mg/L为抗体固定浓度,检测稀释度为1∶4的参考标准血清,结果显示传感器阵列检测的最佳pH值条件在7.4左右。对稀释度分别为1∶9,1∶8,1∶7,1∶6,1∶5,1∶4,1∶3,1∶2的参考标准血清进行检测,结果显示在血清稀释度1∶9～1∶2的范围内,构建的骨唾液蛋白传感器阵列对血清检测的响应呈较好的线性关系,线性方程为△F=521.54c-46.548,相关系数为0.9703。②骨质疏松症患者血清中骨唾液蛋白的表达水平明显高于正常人血清(t=5.702,P<0.05)。结论:实验研制的骨唾液蛋白压电�BACKGROUND： Bone sialoprotein （BSP） is a highly phosphorylated, glycosylated secretory protein. It is clinically important for bone metabolism evaluation to assess the expression of serum BSP. OBJECTIVE： A four-channel chip microarray was designed using AT-cut 10 MHz quartz crystal to detect BSP expression from clinical serum. DESIGN, TIME AND SETTING： Observation experiment was performed at the Bisensor Laboratory of Department of Medical Laboratory, General Hospital of Guangzhou Military Command from December 2008 to July 2009. MATERIALS： Piezoelectric immunosensor included coated quartz crystal, oscillator, and frequency meter. Serum from osteoporosis patients （n=10）, normal people （n=9） and reference serum were provided by Blood Center and Department of Medical Laboratory, General Hospital of Guangzhou Military Command. Calf serum and fetal calf serum were purchased from Hyclone. METHODS： Quartz crystal thickness sections （8.00mm×8.00 mm×0.15 mm） were cut by AT cut, 10 MHz. Gold-membrane electrode was formed by vacuum coating. The microarray comprised spring bracket and four quartz crystals. Monoclonal antibody of BSP was thiolated by heterobifunctional cross-linkers Sulfo-LC-SPDP, and the hydrosulfuryl-antibody was fixed on the face of gold-membrane electrode to make a bio-sensitive membrane, which constructed the piezoelectric immunity chips. The serum BSP was detected after immunoreaction. MAIN OUTCOME MEASURES： The relationship between frequency shift of biosensor and concentration of antibody; Expression differences of BSP between osteoporosis and healthy human serum. RESULTS： With increasing antibody concentration, frequency shift of biosensor increased. With 80 mg/L as constant concentration of antibody and serum at dilution of 1 ： 4 as reference serum, the best pH value of immunosensor array detection was 7.4. The detection of reference standard serum at dilution of 1 ： 9, 1 ： 8, 1 ： 7, 1 ： 6, 1 ： 5, 1 ： 4, 1 ： 3, and 1 ： 2 showed that the serum diluti

关 键 词：压电免疫传感器 阵列 骨唾液蛋白 临床诊断 

分 类 号：R318[医药卫生—生物医学工程]