近红外纳米生物传感器的构建及其全血检测的应用研究

发布时间：2018-03-20 22:39

本文选题：上转换　切入点：能量传递　出处：《中国科学院长春光学精密机械与物理研究所》2016年博士论文　论文类型：学位论文

【摘要】：癌症由于其临床治愈率非常低,已经成为威胁人类健康的一大杀手。究其原因,目前的诊断方法诊断结果基本都为中晚期,错过了最佳的治疗时机,增大了癌症的死亡率。而对于癌症早期发现的病人而言,其治愈率可达80%。因此,发展癌症的早期诊断技术对于维护人类生命健康具有重要的意义。血液免疫检测是医学临床疾病诊断的重要手段。其中,荧光免疫检测技术由于其检测相对简单并且具有较高的灵敏度引起了人们的广泛关注。然而,目前普遍应用的荧光探针的激发光均为紫外或可见光,血液中检测时,会激发血液产生强烈的荧光背景,湮灭探针的荧光信号,无法实现对分析物的检测。因此,目前临床应用的荧光方法,都必须预先进行血清/血浆分离,而多次的分离及清洗过程不可避免地会对待测生物分子的结构或构象造成破坏,从而对检测的准确性造成影响,导致错误的治疗方案,其后果极其严重。目前基于近红外光激发的纳米材料引起了人们的广泛关注。由于其激发光处于生物近红外窗口区,几乎不激发血液体系产生生物自荧光,从而可以有效地降低荧光背景的干扰,为全血的直接生物检测提供了新希望。本论文的出发点是针对目前荧光免疫检测技术难以实现全血直接检测的研究难点,以固相光纤和玻片为生物传感载体的生物传感器研究为基础,在生物标记新型探针和传感方法等方面开展了创新的研究,主要包括生物传感方式、近红外新型探针和新型供受体对的设计和制备、固相生物传感器的构建和生物检测,尤其是全血生物检测中的应用开展了分析表征和评价研究,旨在发展一种用于重大疾病早期诊断和预警的新技术和新方法。主要创新性研究成果如下:1.细胞内ph值是了解细胞内各种活动(如新陈代谢、增殖、凋亡等)的基础,更是许多重大疾病(如癌症、阿尔茨海默病等)诊断的重要依据。然而,细胞内ph值的精确量化检测一直是分子生物学和医学领域的重大难题之一。据此,本论文利用nayf4:yb~(3+),tm~(3+)上转换纳米粒子(ucnps)作为能量传递(et)的供体,以fitc分子作为et的受体,创新地以ucnps中475nm的上转换发光作为ph的响应信号,645nm的发光强度为量化检测自参考信号,发展了一种精确高灵敏的定量检测细胞内ph值的新方法。实现了细胞ph在3.0-7.0范围内的高准确性定量检测,检测灵敏度高达3.56单位/ph,误差小于0.4单位/ph。2.荧光免疫检测是医学临床诊断中应用最为广泛的检测技术,主要分为非均相检测(如elisa)和均相检测(如et基的传感方法)。然而,对于商业应用的非均相免疫检测而言,受目前荧光生物探针固有性质的限制,均需要预先血清/血浆分离以及后序复杂的清洗过程,严重影响了检测的准确性;对于均相免疫检测而言,荧光标记探针粒子的聚集及其引起的光散射,严重影响了检测灵敏度和可靠性。针对上述方法在血液检测中所面临的挑战性难题,本论文创新地构建了以ucnps为et供体、以金纳米粒子(gnps)为et受体的的固相生物传感器,发展了一种原位检测的新技术,并开展了其在全血检测中的应用研究。该方法解决了均相和非均相检测中的挑战性难题,实现了全血中igg一步、原位、高灵敏的检测。在20倍稀释的血液中检测灵敏度达3.4nm,首次实现了全血样品5-400nm的宽范围检测。3.高灵敏的全血直接检测对于实现诸如癌症和艾滋病等重大疾病的早期诊断和预警具有重要意义。然而,受制于受荧光生物探针固有性质以及血液复杂环境中强烈光散射、吸收和自荧光背景,全血样品的直接检测一直是一个挑战性的难题。针对上述问题,本论文创新地构建了基于近红外纳米sers标记物ag@4mba@sio2的光纤生物传感器。以4-mba分子和ag纳米粒子所得到的sers信号作为检测信号通过二氧化硅包覆增强sers纳米探针在复杂体系中的稳定性。将构建的新型SERS纳米探针与光纤倏逝波生物传感技术相结合,利用近红外光作为激发光,有效地降低了全血中荧光背景及各组分光散射对检测信号的影响,实现了全血中甲胎蛋白AFP的高灵敏及准确性检测,检测线性范围达50-500 ng/m L。本论文在近红外纳米生物探针与多种传感方式相结合的生物医学检测技术的研究方面,获得了一些可喜的结果,有效地解决了荧光免疫检测的两大领域性(均相检测与非均相检测)难题,发展了全血原位免疫检测的新技术与新方法,为癌症等重大疾病的临床早期诊断奠定了基础。在现有工作的基础上,综合材料学、物理学、化学、分子生物学、生物医学和临床等多学科、多领域,研发多通道、高灵敏的全血检测方法及全血原位在体检测技术将是未来生物医学研究的主要方向。
[Abstract]:Cancer due to its clinical cure rate is very low, has become a major threat to human health. The reason, diagnosis test results at present basically in the late stage, missed the best treatment time, increased cancer mortality. And for the early detection of cancer patients, the cure rate of up to 80%. so early diagnosis of cancer, technology development is of great significance for the maintenance of human health. Blood immune detection is an important means of medical clinical diagnosis. The fluorescence immunoassay for detection sensitivity due to its relatively simple and has high has attracted widespread attention. However, the excitation light fluorescent probes are widely used ultraviolet or visible light, blood test, can stimulate blood to produce strong fluorescence background, fluorescence signal annihilation probe, can not be achieved for the analyte detection Measured. Therefore, the current clinical application of fluorescence method, must carry on the serum / plasma separation in advance, and the inevitable separation and cleaning process repeatedly to cause damage to test the structure or conformation of biological molecules, thus the accuracy of detection of the impact, resulting in the wrong treatment, the consequence is extremely serious. The nano material near based on infrared excitation has attracted widespread attention. Because of its luminescence in biological near infrared window area, almost no blood system since the biological fluorescence excitation, which can effectively reduce the interference of background fluorescence, provides a new hope for the whole blood direct biological detection. The starting point of this paper is to present fluorescence immunoassay to realize the direct detection of difficulty of blood, with solid glass fiber and biosensor biosensor carrier based on biomarkers To carry out innovative research on new probe and sensing methods, including biological sensing mode, near infrared probe and a new model for receptor design and system on preparation, construction and detection of biological solid biological sensors, especially to carry out analysis on characterization and evaluation of application of blood biological detection, aims to develop a new technologies and new methods for disease early diagnosis and early warning. The main achievements are as follows: 1.. The intracellular pH is to understand the various activities within the cell (such as Chen Daixie, proliferation, apoptosis and so on) the basis, but many major diseases (such as cancer, Alzheimer's disease) an important basis for diagnosis. However, precise quantitative detection of intracellular pH value has been one of the major problems in the field of molecular biology and medicine. Therefore, this paper uses nayf4:yb~ (3+), tm~ (3+) upconversion nanoparticles (ucnps) as energy Transfer (ET) donor to FITC molecules as ET receptor, ucnps 475nm innovation to upconversion luminescence as a response to the pH signal intensity of 645nm for quantitative detection of self reference signal, the development of a new quantitative method for accurate sensitive detection of intracellular pH value to achieve a high accuracy. Quantitative in the range of 3.0-7.0 cell pH detection, high detection sensitivity up to 3.56 /ph, the error is less than 0.4 /ph.2. fluorescence immunoassay is medical diagnosis the detection technology is widely applied, mainly divided into non homogeneous detection (such as ELISA) and homogeneous (sensing detection methods such as et radicals). However for commercial applications, heterogeneous immunoassay, the fluorescent probe inherent limitations, both prior to serum / plasma separation and subsequent complex cleaning process, seriously affect the accuracy of detection for homogeneous immune detection; Measurement, fluorescent probe particle aggregation and light scattering caused by the serious impact on the sensitivity and reliability of detection. According to the above method in the blood tests the challenges facing the problem, this paper innovatively constructed using ucnps as et donor, using gold nanoparticles (GNPs) as the solid biosensor ET receptor. The development of a new technique for in situ detection, and carry out its application in whole blood test. The method solves the problem of homogeneous and heterogeneous detection of challenging problems, realize the whole blood IgG step, in situ, highly sensitive detection. In 20 times diluted blood detection sensitivity of 3.4nm, for the first time can achieve a wide range of whole blood samples to detect.3. 5-400nm high sensitive blood direct detection has important significance for early diagnosis and early warning such as cancer and AIDS and other major diseases. However, the fluorescence from the subject to the students The strong light scattering properties and inherent geophysical needle blood in complex environment, self absorption and fluorescence background, direct detection of whole blood samples is always a challenging problem. Aiming at the above problems, this paper constructs a novel optical fiber biosensor near infrared nano SERS markers based on ag@4mba@sio2. SERS signal to 4-MBA molecules and Ag the resulting nanoparticles as the detection signal through the enhanced stability of silica coated SERS nano probe in the complex system. Combining the construction of the new SERS nano probe and optical fiber evanescent wave biosensor technology, using near infrared light as excitation light, effectively reduce the influence of background fluorescence and light scattering were prepared in the detection signal high sensitivity and accuracy, detection of blood AFP AFP in the linear range of detection was 50-500 ng/m L. in the near infrared nano bio probe The research of biomedical detection needle with multiple sensing mode combination, obtained some encouraging results, effectively solve the two major areas of fluorescence immunoassay (homogeneous detection and non homogeneous detection) problem, the development of new technology and new method of whole blood in situ immune detection, which laid the foundation for the clinical early diagnosis of cancer and other major diseases. On the basis of the existing work, comprehensive material science, physics, chemistry, molecular biology, multidisciplinary, biomedical and clinical fields, research and development of multi channel detection method, whole blood and blood in situ high sensitive in detection technology will be the main direction of biomedical research in the future.

【学位授予单位】：中国科学院长春光学精密机械与物理研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.4;TP212.3

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