【作者】 姚春艳；

【导师】 府伟灵；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2008， 博士

【摘要】 目的:通过建立压电石英晶体振荡频率检测平台,利用适配子作为压电生物传感器的生物识别分子,构建适配子型压电石英晶体生物传感器,并用其对人免疫球蛋白E进行检测,对各项检测参数进行研究,并与抗体型压电免疫生物传感器进行比较。方法:1.利用精细微加工工艺制作的石英晶体作为换能器,并在此基础上制备生物传感器微阵列,以构建压电石英晶体生物传感器检测平台。2 .采用生物素-亲和素法将抗人免疫球蛋白适配子固定于石英晶体表面,用于人免疫球蛋白E的标准血清及临床标本的检测。根据频率变化和IgE浓度之间的关系绘制标准曲线。3.探索适配子型压电生物传感器进行实际检测的准确性、最低检测限等检测参数,并对其非特异结合反应、再生条件、保存时间等进行研究。4.分别用适配子型压电生物传感器和抗体型压电免疫传感器对IgE标准品和血清标本进行检测,并对两者的检测范围、最低检测限、检测时间、非特异性结合实验、再生实验、保存实验等进行研究,以比较适配子型压电生物传感器和抗体型压电免疫传感器进行实际检测的优越性。结果:1.所构建的适配子型压电生物传感器的检测范围为2.5-200μg/L,最低检测值为3μg/L,与化学发光法相比相关系数为0.992,具有良好的检测性能。2.适配子型压电生物传感器对BSA、HSA、IgG、IgM等干扰蛋白的检测信号显著低于特异性IgE的检测信号(<5%)。3.在选用的再生试剂中,EDTA表现了最好的再生效果,适配子芯片经过10次再生后仍可保留80%的生物学活性。4.对适配子芯片进行保存实验,在7天内稳定性几乎没有改变,随后稳定性逐渐下降,3周后检测活性约为初始检测的90%。5.适配子型压电生物传感器和抗体型免疫传感器均可在15min内完成检测,都具有良好的检测灵敏度、线性范围和特异性,准确性与化学发光法相当,对结果进行回归分析吻合性较好。6.适配子型生物传感器的再生和保存性能优于抗体型免疫传感器。结论:1.所构建的适配子型压电生物传感器和化学发光法进行人血清标本IgE检测的相关系数为0.992。适配子型压电生物传感器可以对复杂生物样本中的IgE进行高灵敏度、高特异性的检测。2.适配子型压电生物传感器和抗体型免疫传感器均可在15分钟内完成对IgE的检测,可为患者提供更快速的诊断,同时操作更加简便。与传统的检测方法相比,传感器方法具有无需标记,无需对标本进行预处理的特点。3 .适配子型压电生物传感器在最低检测限、结合特异性及准确性方面均优于抗体型免疫传感器。4.适配子型压电生物传感器具备优良的再生和保存性能,可进一步降低临床检测应用中的成本。5.适配子型压电石英晶体传感器作为一种新型生物传感器,具有快速、灵敏、实时检测、无需标记等优点,因此具有重要的科学意义和广阔的应用前景。更多还原

【Abstract】 Objectives:The aim of this study was to develop a rapid method to measure IgE in human serum by use of a direct aptamer-based biosensor based on a quartz crystal microbalance (QCM). The comparison with the antibody-based QCM immunosensor was also explored.Methods:1. We designed and fabricated aptamer-based piezoelectric quartz crystal microbalance biosensor. Self-oscillating TTL circuit and self-made optimized PESA version 4.0 software applied to record and analyze frequency changes.2. An avidin monolayer prepared by the covalently method was applied to immobilize biotinylated single-stranded DNA aptamers specific for IgE on the gold surface of the quartz crystal. The frequency shifts (FS) of the QCM were measured and related to IgE concentrations. The dose-curve was drawn according to the relationship betweenΔF and IgE concentrations.3. The sensitivity, detection limit, nonspecific response and regeneration ability of piezoelectric QCM aptamer biosensor were observed. At last, clinical samples were detected in order to explore the possibility about the detection of clinical samples. The aptamer piezoelectric biosensor used no labeled reagent and no pretreatment of samples.4. The sensitivity, detection limit, nonspecific response and regeneration ability of the QCM aptamer biosensor also compared with the antibody-based QCM immunosensor.Results:1. A linear relationship existed between the FS (Hz) and the IgE concentrations from2.5 to 200μg/L in buffer and human serum. The correlation coefficient between QCM method and chemiluminescence method was 0.992 for determination of IgE in 50 clinical human serum samples.2. The cross-reactivity of the aptamer was seen to be less than 5% for all the interference proteins.3. The aptamer receptors tolerated repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. Using 30 mmol/L EDTA as regeneration reagent gave the best result. More than 80% of the original response was maintained after 10 assays.4. The stability of the aptamer biosensor was almost fully maintained at the first 7 days and then started to decline. When stored for 3 weeks, the FS were all >90% of those on the response at the first day.5. The aptamer biosensor and QCM immunosensor were able to detect IgE with high specificity and sensitivity in 15 min. The results of them were compared with chemiluminescence method, the correlation coefficients were good.6. The regeneration ability and long-term stability of QCM aptamer biosensor were better than that of piezoelectric QCM immunosensor.Conclusions:1. The correlation coefficient between QCM aptamer biosensor and chemiluminescence was 0.992 for determination of IgE in the clinical human serum samples. The results showed that the aptamers was able to detect IgE with high sensitivity and selectivity, even in a complex biological sample such as serum.2. The QCM aptamer biosensor and QCM immunosensor capable of detecting IgE within 15 minutes, provides patients with a faster diagnosis due to a shorter detection time, and the use of relatively easy-to-use devices. Moreover, the biosensor method has shown its advantages over other conventional methods due to its label-free property. The direct biosensor system measured IgE without the need of a labeled reagent and pretreatment of samples.3. The precision, detection limit, specificity and accuracy of the aptamer biosensor were better than that of immunosensor.4. The reusability and long-term stability of the aptamer biosensor were well, which can reduce the cost of detection.5. The QCM biosensor can measure IgE and offer advantages of high specificity, reusability, low detection limit, no label or sample pretreatment, and shorter detection time. The aptamer QCM biosensor was suitable for sensitive and specific protein detection, representing an innovative tool for future proteomics.更多还原