【作者】 闫慧慧；

【导师】 府伟灵；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2007， 硕士

【摘要】 目的1.对自行研制的压电石英生物传感器检测平台进行改进。2.用凝胶迁移率实验（Electrophoretic mobility shift assay,EMSA）验证NF-κB顺式元件（DNA）探针与特异性蛋白NF-κB的p65亚基和核蛋白的特异性结合。3.建立基于蛋白质—DNA相互作用的新型压电生物传感器及其影响因素探讨,并优化出最佳反应条件。方法1.利用精细微加工法制作石英晶体,在其表面光刻金膜电极,石英晶体AT切型、13×13mm、基频10MHz、金膜电极直径4.0mm,与检测池组装后形成传感器检测单元,传感器检测单元在检测仪上构成2×5型拔插式传感器阵列。频率记录分析软件从PESA-2.0升级到PESA-4.0,新软件采用Visual C++语言设计,增加多项功能使其更适用于实验研究和临床检测的需要。对构建好的压电传感器检测平台进行气液相稳定性实验及阵列检测稳定性实验。2.用EMSA验证NF-κB顺式元件（DNA）探针与特异性蛋白NF-κB的结合特异性。3.将NF-κB顺式元件（DNA）探针通过巯基化法固定在传感器的金膜表面,并与特异性蛋白NF-κB的p65亚基进行结合反应,通过计算机采集分析传感器频率数据,比较不同探针浓度、不同pH值、不同离子浓度下频率的变化,经SPSS软件进行单因素分析选择最佳反应条件。4.以不同浓度p65蛋白为标准品,绘制p65检测标准曲线,并对核蛋白提取物进行检测。同时,通过更换不同序列的巯基化探针,探讨传感器检测的特异性。结果1.AT切型石英晶体液相起振能力强、频率稳定适合压电生物传感器检测要求,传感器组装后,气、液相检测稳定性好,调稳后10分钟内气相频率变化不超过±1Hz、液相不超过±2Hz。2.为了满足临床检测的需要,新编PESA-4.0软件增加了如病人信息录入、历史结果查阅、曲线拟合、定标等多项功能,其中频率自动更新和图像编辑等功能的添加对实验室研究也有较大帮助。3.EMSA电泳结果显示标记了同位素的探针与NF-κB的p65亚基和核蛋白提取物的结合较空白对照产生了滞后的电泳条带,说明了探针和蛋白结合的特异性。样品空白泳道未加入蛋白固没有特异性结合条带。4.运用构建的压电石英晶体传感器检测p65标准蛋白,探针固定浓度为2.0μmol/L,pH值为7.5,MgCl₂离子强度为50mmol/L时结合反应条件最佳。并确定结合反应最终反应时间为15min。5.压电生物传感器对不同浓度p65进行检测,在10-40ng/μl浓度范围p65浓度与频率下降呈线性相关,同时对传感器的非特异性响应信号进行检测,非特异性信号响应小,不超过同浓度特异性信号的5%。结论1.构建的压电石英晶体传感器检测系统硬件设计较完善,工作稳定,软件稳定易用,功能较完善,整个检测系统可以应用于检测和研究。为临床检测开发的PESA-4.0分析软件对实验研究也有较大帮助。2.压电传感器检测取得了较好的结果,检测时间确定为15min,非特异性信号小,说明构建的传感器特异性好,选择性高,显示该平台在研究反式作用因子方面有较好的应用前景。3.EMSA电泳实验验证说明本实验设计的特异性探针与NF-κB能发生特异性结合反应,表明本研究构建的传感器具有良好的检测特异性。4.本研究构建的传感器检测平台较传统的EMSA方法有一定的优势,无需同位素标记,反应时间短,能高通量实时在线观察蛋白质和DNA的相互作用,为研究真核基因表达调控提供新型技术平台。更多还原

【Abstract】 Objective1.To improve the performance of piezoelectric quartz crystal biosensor detection system made by us.2.To confirm the specific reaction between NF-κB cis-acting element（DNA） probe and the specific protein NF-κB p65 subunit and the nucleoprotein,by the EMSA method.3.To discuss the influencing factors of the novel protein-DNA piezoelectric biosensor and optimize the reaction condition.Methods:1.Fine processing technique was adopted to fabricate single quartz crystal wafer（AT cut 13×13mm） with a fundamental frequency of 10MHz and to put the gold electrode of 4.0mm in diameter on the both two sides of quartz crystal.The single sensor was arranged to construct 2×5 insert-plug model array.Detecting software applied to record and analyze frequency changes was updated from PESA-2.0 to PESA-4.0.The new software was designed based on Visual C++,and added many useful functions suitable for laboratory research and clinical detection.In addition,the stability of system was tested in gas and liquid phase respectively.2.The cis-acting element probe of NF-κB modified by thiol group were immobilized on the gold membrane surface of the quartz crystal sensor by thiol-self-assembly technique, and was hybridized with the specific protein NF-κB p65 subunit.When some parameters, such as probe concentration,pH value and ion concentration,were changed,the corresponding changes of the frequency of each sensor were recorded and analyzed through the computer simultaneouly.The reaction condition was optimized by SPSS statistic software.3.The combination specific between NF-κB cis-acting element（DNA） probe and the specific protein NF-κB was confirmed with EMSA method. 4.The different concentrations of p65 were used as standard substance to draw detection calibration curve of p65 and determinate the level of the nucleoprotein.In addition,the detection specifity of the biosensor was probed by different sequence thiol group probe immobilized on the surface of piezoelectric crystal.Results:1.AT-cut quartz crystal has strong vibration ability and stable frequency both in gas and liquid phase.In ten minutes,the changes of frequency were less than±1Hz in gas phase and±2Hz in liquid phase respectively.2.The software of PESA- 4.0 which is designed for clinical detection mainly has been added some useful functions such as recording the information of patients,recalling history records,curve fitting,and calibrating,etc.Moreover,the functions of image editing and automatic updating of frequency were helpful for laboratory research.3.In the EMSA electrophoresis results,a lag electrophoresis band could be observed, which probe marked by the isotope combined with NF-κB p65 subunit and the nucleoprotein union resulted in the lag electrophoresis band,compared with the blank control group.But the binding band could not be observed in the blank control group.This phenomenon demonstrated that the probe and the protein combined specificity.4.The p65 subunit was detected by using the piezoelectric quartz crystal sensor.The optimized reaction condition was 2.0μmol/L probe concentration,pH 7.5,50mmol/L MgCl₂ and 15min for reaction time,respectively.5.Piezoelectric Biosensors different concentration p65 was detected in the 10-40ng/ul concentration of p65 concentration and frequency decreased linearly related,Non-specific signals of this sensor were less than 5%of specific signals of p65 at the same concentration.Conclusions:1.The hardware of piezoelectic quartz crystal sensor detection system was designed perfect relatively.It works stably and the software of the system used easily and stably.The studies imply that the detection system can be used for detection and research.In addition the PESA-4.0 software designed originally for clinical detection was also helpful for laboratory research.2.The piezoelectric examination has obtained the good result and the examination time is 15 min.The non-specificity signal is small,which indicates the specificity of sensor is ideal and the selectivity is high.All the data demonstrated that this platform has the good application prospect in the trans-acting-factor examination.3.The EMSA electrophoresis experiment implied that the specific probe designed in our experiment have the specific combination both with NF-κB p65 standard protein and the nucleoprotein.It was indicated that this sensor detection system is a good examination specificity.4.This sensor detection system has more advantages compares with traditional EMSA method.For example,it doesn’t need isotope and the reaction time is shorter.Furthermore, it can online observe and record the corresponding data with high flux.And it provide a new technical platform for the eukaryotic gene expression regulation study.更多还原