【摘要】：聲表面波傳感器是微機電系統(tǒng)和微加工工藝發(fā)展下研制出的一種新型傳感器,主要包括瑞利波傳感器、水平剪切波傳感器和樂甫波傳感器等。其中樂甫波傳感器由于其能在液體環(huán)境下穩(wěn)定工作和高靈敏度的優(yōu)點,近年來成為了研究的熱點,并被逐漸應用于臨床醫(yī)學、環(huán)境、食品分析等領(lǐng)域。本論文以樂甫型聲表面波傳感器為研究對象,結(jié)合了微加工技術(shù)、信號檢測技術(shù)以及生化檢測技術(shù),構(gòu)建了一種樂甫型聲表面波傳感器并用于肺癌標志物和毒素檢測。本論文設計并研制了一種高靈敏度的樂甫型聲表面波傳感器,并結(jié)合微流控芯片技術(shù),搭建了多通道便攜式樂甫型聲表面波傳感器流動檢測和細胞檢測系統(tǒng)。本論文利用了抗原抗體特異性結(jié)合的免疫檢測技術(shù),實現(xiàn)了對呼出氣體冷凝物中肺癌生物標志物蛋白和海洋生物毒素中貝類毒素的檢測分析。另外,還使用了細胞作為傳感敏感元件構(gòu)建出一種新型細胞傳感器,并用于貝類毒素的檢測。隨著樂甫型聲表面波傳感器的發(fā)展,該檢測技術(shù)及其便攜式檢測系統(tǒng)將會為生化檢測提供快速高靈敏的分析測試平臺。本論文的主要創(chuàng)新性研究工作在于:1.提出了樂甫型聲表面波器件結(jié)合微流控芯片的傳感器設計方法,設計了檢測聲表面波傳感器幅值和相位信號的電路檢測系統(tǒng),保證了傳感器的高通量檢測精度�；谖⒓庸すに�,設計并制造了 ST切石英為壓電基底、SiO2為波導層、Au為敏感層的樂甫型聲表面波傳感器芯片,結(jié)合根據(jù)傳感器參數(shù)設計了特定的微流控芯片,在保證傳感器傳輸特性的同時,提高了樂甫型聲表面波傳感器用于生化檢測的可靠性。針對樂甫型聲表面波傳感器,開發(fā)了擁有自動進樣、檢測電路和移動端軟件的多路檢測系統(tǒng),實現(xiàn)了對傳感器的自動化高通量檢測。2.提出了結(jié)合納米金的質(zhì)量信號增強方法的樂甫型聲表面波免疫傳感器,成功實現(xiàn)了對呼出氣體冷凝物中肺癌生物標志物癌胚抗原(CEA)的高靈敏檢測。為了實現(xiàn)傳感器對CEA的特異性檢測,首先在傳感器表面固定蛋白A,CEA的包被抗體可以利用蛋白A導向性地固定在傳感器的敏感區(qū)域,之后利用包被抗體捕獲樣本溶液中的CEA。此外,為了提高傳感器質(zhì)量靈敏度,利用了納米金顆粒對檢測抗體進行標記,并在傳感器表面構(gòu)建"三明治"結(jié)構(gòu)免疫反應。最后再通過金染色液,溶液中的金離子會被納米金催化析出并沉積在納米金表面,傳感器質(zhì)量信號被放大。該傳感器用于呼出氣體冷凝物中CEA檢測的檢出限為0.967ng/mL,比肺癌初篩的診斷閾值要低,表明該方法可以有效用于臨床肺癌的篩查。3.提出了基于免疫競爭法的樂甫型聲表面波免疫傳感器,實現(xiàn)了對大田軟海綿酸(OA)的特異性檢測。本研究采用競爭免疫分析技術(shù)來檢測小分子的OA含量。檢測時同樣使用了納米金顆粒和金染色液技術(shù)用于放大傳感器的檢測信號,增加檢測靈敏度。同時,該傳感器在10-150ng/mL濃度范圍內(nèi)具有很好的線性,檢出限為5.45ng/mL。并且通過檢測實際加標樣本以及與ELISA方法的對比實驗,說明該方法具有很好的特異性和準確性,可以有效地用于貝肉實際樣品中OA毒素的檢測。4.提出了基于HepG2肝癌細胞的樂甫型聲表面波傳感器,并實現(xiàn)了對大田軟海綿酸(OA)的便捷檢測。本研究設計了一種基于HepG2的細胞傳感器,并將其用于OA的檢測。對毒素檢測時的細胞培養(yǎng)密度進行了優(yōu)化,最佳細胞密度為10000個/孔。當毒素濃度在10-100 ng/mL時,樂甫型聲表面?zhèn)鞲衅魉鶞y得的插入損耗變化值與OA濃度有良好的線性關(guān)系,其相關(guān)系數(shù)為0.9834,其檢出限為10.91 ng/mL。而且對其他毒素進行了測試,結(jié)果說明了該檢測方法具有良好的特異性和重復性。該檢測技術(shù)無需傳統(tǒng)免疫檢測手段所需要的各類試劑,并且相對于動物實驗具有操作簡單和快速等的特點。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TP212;R734.2

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