声表面波传感器及其在肺癌标志物与毒素快速检测中的应用研究

发布时间：2018-08-11 21:42

【摘要】：声表面波传感器是微机电系统和微加工工艺发展下研制出的一种新型传感器,主要包括瑞利波传感器、水平剪切波传感器和乐甫波传感器等。其中乐甫波传感器由于其能在液体环境下稳定工作和高灵敏度的优点,近年来成为了研究的热点,并被逐渐应用于临床医学、环境、食品分析等领域。本论文以乐甫型声表面波传感器为研究对象,结合了微加工技术、信号检测技术以及生化检测技术,构建了一种乐甫型声表面波传感器并用于肺癌标志物和毒素检测。本论文设计并研制了一种高灵敏度的乐甫型声表面波传感器,并结合微流控芯片技术,搭建了多通道便携式乐甫型声表面波传感器流动检测和细胞检测系统。本论文利用了抗原抗体特异性结合的免疫检测技术,实现了对呼出气体冷凝物中肺癌生物标志物蛋白和海洋生物毒素中贝类毒素的检测分析。另外,还使用了细胞作为传感敏感元件构建出一种新型细胞传感器,并用于贝类毒素的检测。随着乐甫型声表面波传感器的发展,该检测技术及其便携式检测系统将会为生化检测提供快速高灵敏的分析测试平台。本论文的主要创新性研究工作在于:1.提出了乐甫型声表面波器件结合微流控芯片的传感器设计方法,设计了检测声表面波传感器幅值和相位信号的电路检测系统,保证了传感器的高通量检测精度。基于微加工工艺,设计并制造了 ST切石英为压电基底、SiO2为波导层、Au为敏感层的乐甫型声表面波传感器芯片,结合根据传感器参数设计了特定的微流控芯片,在保证传感器传输特性的同时,提高了乐甫型声表面波传感器用于生化检测的可靠性。针对乐甫型声表面波传感器,开发了拥有自动进样、检测电路和移动端软件的多路检测系统,实现了对传感器的自动化高通量检测。2.提出了结合纳米金的质量信号增强方法的乐甫型声表面波免疫传感器,成功实现了对呼出气体冷凝物中肺癌生物标志物癌胚抗原(CEA)的高灵敏检测。为了实现传感器对CEA的特异性检测,首先在传感器表面固定蛋白A,CEA的包被抗体可以利用蛋白A导向性地固定在传感器的敏感区域,之后利用包被抗体捕获样本溶液中的CEA。此外,为了提高传感器质量灵敏度,利用了纳米金颗粒对检测抗体进行标记,并在传感器表面构建"三明治"结构免疫反应。最后再通过金染色液,溶液中的金离子会被纳米金催化析出并沉积在纳米金表面,传感器质量信号被放大。该传感器用于呼出气体冷凝物中CEA检测的检出限为0.967ng/mL,比肺癌初筛的诊断阈值要低,表明该方法可以有效用于临床肺癌的筛查。3.提出了基于免疫竞争法的乐甫型声表面波免疫传感器,实现了对大田软海绵酸(OA)的特异性检测。本研究采用竞争免疫分析技术来检测小分子的OA含量。检测时同样使用了纳米金颗粒和金染色液技术用于放大传感器的检测信号,增加检测灵敏度。同时,该传感器在10-150ng/mL浓度范围内具有很好的线性,检出限为5.45ng/mL。并且通过检测实际加标样本以及与ELISA方法的对比实验,说明该方法具有很好的特异性和准确性,可以有效地用于贝肉实际样品中OA毒素的检测。4.提出了基于HepG2肝癌细胞的乐甫型声表面波传感器,并实现了对大田软海绵酸(OA)的便捷检测。本研究设计了一种基于HepG2的细胞传感器,并将其用于OA的检测。对毒素检测时的细胞培养密度进行了优化,最佳细胞密度为10000个/孔。当毒素浓度在10-100 ng/mL时,乐甫型声表面传感器所测得的插入损耗变化值与OA浓度有良好的线性关系,其相关系数为0.9834,其检出限为10.91 ng/mL。而且对其他毒素进行了测试,结果说明了该检测方法具有良好的特异性和重复性。该检测技术无需传统免疫检测手段所需要的各类试剂,并且相对于动物实验具有操作简单和快速等的特点。
[Abstract]:Surface acoustic wave (SAW) sensor is a new type of sensor developed under the development of micro-electromechanical system and micro-machining technology. It mainly includes Rayleigh wave sensor, horizontal shear wave sensor and Love wave sensor. In this dissertation, a novel surface acoustic wave (SAW) sensor based on the combination of microprocessing technology, signal detection technology and biochemical detection technology has been developed for the detection of lung cancer markers and toxins. A highly sensitive surface acoustic wave (SAW) sensor was developed and a multi-channel portable flow detection and cell detection system based on the microfluidic chip technology was built. In this paper, the specific binding immunoassay technology of antigen and antibody was used to detect lung cancer in exhaled gas condensate. Detection and analysis of shellfish toxins in biomarker proteins and marine biological toxins. In addition, a novel cell sensor was constructed using cells as sensing elements for the detection of shellfish toxins. The main innovative research work of this paper is as follows: 1. The sensor design method of surface acoustic wave device combined with microfluidic chip is proposed, and the circuit detection system for detecting amplitude and phase signals of surface acoustic wave sensor is designed to ensure the high-throughput detection accuracy of the sensor. Based on the processing technology, we designed and fabricated the surface acoustic wave sensor chip with ST-cut quartz as piezoelectric substrate, SiO2 as waveguide layer, Au as sensitive layer, and designed a specific microfluidic chip according to the sensor parameters. This chip not only ensures the transmission characteristics of the sensor, but also improves the reliability of the surface acoustic wave sensor for biochemical detection. Aiming at the surface acoustic wave (SAW) sensor of Lefu type, a multi-channel detection system with automatic sampling, detection circuit and mobile terminal software is developed to realize automatic and high-throughput detection of the sensor. 2. A novel surface acoustic wave (SAW) immunosensor of Lefu type is proposed, which combines the quality signal enhancement method of nano-gold and successfully realizes the condensation of exhaled gas. High sensitivity detection of carcinoembryonic antigen (CEA), a biomarker of lung cancer, in this study. In order to achieve the specific detection of CEA by the sensor, the coated antibody of CEA can be used to fix protein A on the surface of the sensor to the sensitive region of the sensor, and then capture CEA in the sample solution by the coated antibody. With high quality sensitivity, gold nanoparticles are used to label the antibody and a sandwich structure is constructed on the surface of the sensor. Finally, gold ions in the solution are precipitated and deposited on the surface of the nano-gold through the gold staining solution, and the mass signal of the sensor is amplified. The detection limit of CEA in body condensate is 0.967ng/mL, which is lower than the threshold of lung cancer screening. It shows that this method can be effectively used in clinical lung cancer screening. At the same time, the sensor has a good linearity in the concentration range of 10-150 ng/mL, and the detection limit is 5.45 ng/mL. Moreover, it can be used to detect the actual sample and ELIS. The comparative experiment of A method shows that this method has good specificity and accuracy, and can be effectively used for the detection of OA toxin in shellfish meat samples. 4. A Lefu-type surface acoustic wave sensor based on HepG2 hepatoma cells is proposed, and a convenient method for the detection of field soft spongy acid (OA) is realized. When the concentration of the toxin was 10-100 ng/mL, the change of insertion loss measured by the surface acoustic sensor had a good linear relationship with the OA concentration. The correlation coefficient was 0.9834, and the detection limit was 0.9834. 10.91 ng/mL. Other toxins were tested. The results showed that the method had good specificity and reproducibility. The method did not need all kinds of reagents needed by traditional immunoassay methods, and was simple and rapid compared with animal experiments.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TP212;R734.2

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