本文選題：上轉(zhuǎn)換　切入點：能量傳遞　出處：《中國科學院長春光學精密機械與物理研究所》2016年博士論文　論文類型：學位論文

【摘要】：癌癥由于其臨床治愈率非常低,已經(jīng)成為威脅人類健康的一大殺手。究其原因,目前的診斷方法診斷結(jié)果基本都為中晚期,錯過了最佳的治療時機,增大了癌癥的死亡率。而對于癌癥早期發(fā)現(xiàn)的病人而言,其治愈率可達80%。因此,發(fā)展癌癥的早期診斷技術(shù)對于維護人類生命健康具有重要的意義。血液免疫檢測是醫(yī)學臨床疾病診斷的重要手段。其中,熒光免疫檢測技術(shù)由于其檢測相對簡單并且具有較高的靈敏度引起了人們的廣泛關注。然而,目前普遍應用的熒光探針的激發(fā)光均為紫外或可見光,血液中檢測時,會激發(fā)血液產(chǎn)生強烈的熒光背景,湮滅探針的熒光信號,無法實現(xiàn)對分析物的檢測。因此,目前臨床應用的熒光方法,都必須預先進行血清/血漿分離,而多次的分離及清洗過程不可避免地會對待測生物分子的結(jié)構(gòu)或構(gòu)象造成破壞,從而對檢測的準確性造成影響,導致錯誤的治療方案,其后果極其嚴重。目前基于近紅外光激發(fā)的納米材料引起了人們的廣泛關注。由于其激發(fā)光處于生物近紅外窗口區(qū),幾乎不激發(fā)血液體系產(chǎn)生生物自熒光,從而可以有效地降低熒光背景的干擾,為全血的直接生物檢測提供了新希望。本論文的出發(fā)點是針對目前熒光免疫檢測技術(shù)難以實現(xiàn)全血直接檢測的研究難點,以固相光纖和玻片為生物傳感載體的生物傳感器研究為基礎,在生物標記新型探針和傳感方法等方面開展了創(chuàng)新的研究,主要包括生物傳感方式、近紅外新型探針和新型供受體對的設計和制備、固相生物傳感器的構(gòu)建和生物檢測,尤其是全血生物檢測中的應用開展了分析表征和評價研究,旨在發(fā)展一種用于重大疾病早期診斷和預警的新技術(shù)和新方法。主要創(chuàng)新性研究成果如下:1.細胞內(nèi)ph值是了解細胞內(nèi)各種活動(如新陳代謝、增殖、凋亡等)的基礎,更是許多重大疾病(如癌癥、阿爾茨海默病等)診斷的重要依據(jù)。然而,細胞內(nèi)ph值的精確量化檢測一直是分子生物學和醫(yī)學領域的重大難題之一。據(jù)此,本論文利用nayf4:yb~(3+),tm~(3+)上轉(zhuǎn)換納米粒子(ucnps)作為能量傳遞(et)的供體,以fitc分子作為et的受體,創(chuàng)新地以ucnps中475nm的上轉(zhuǎn)換發(fā)光作為ph的響應信號,645nm的發(fā)光強度為量化檢測自參考信號,發(fā)展了一種精確高靈敏的定量檢測細胞內(nèi)ph值的新方法。實現(xiàn)了細胞ph在3.0-7.0范圍內(nèi)的高準確性定量檢測,檢測靈敏度高達3.56單位/ph,誤差小于0.4單位/ph。2.熒光免疫檢測是醫(yī)學臨床診斷中應用最為廣泛的檢測技術(shù),主要分為非均相檢測(如elisa)和均相檢測(如et基的傳感方法)。然而,對于商業(yè)應用的非均相免疫檢測而言,受目前熒光生物探針固有性質(zhì)的限制,均需要預先血清/血漿分離以及后序復雜的清洗過程,嚴重影響了檢測的準確性;對于均相免疫檢測而言,熒光標記探針粒子的聚集及其引起的光散射,嚴重影響了檢測靈敏度和可靠性。針對上述方法在血液檢測中所面臨的挑戰(zhàn)性難題,本論文創(chuàng)新地構(gòu)建了以ucnps為et供體、以金納米粒子(gnps)為et受體的的固相生物傳感器,發(fā)展了一種原位檢測的新技術(shù),并開展了其在全血檢測中的應用研究。該方法解決了均相和非均相檢測中的挑戰(zhàn)性難題,實現(xiàn)了全血中igg一步、原位、高靈敏的檢測。在20倍稀釋的血液中檢測靈敏度達3.4nm,首次實現(xiàn)了全血樣品5-400nm的寬范圍檢測。3.高靈敏的全血直接檢測對于實現(xiàn)諸如癌癥和艾滋病等重大疾病的早期診斷和預警具有重要意義。然而,受制于受熒光生物探針固有性質(zhì)以及血液復雜環(huán)境中強烈光散射、吸收和自熒光背景,全血樣品的直接檢測一直是一個挑戰(zhàn)性的難題。針對上述問題,本論文創(chuàng)新地構(gòu)建了基于近紅外納米sers標記物ag@4mba@sio2的光纖生物傳感器。以4-mba分子和ag納米粒子所得到的sers信號作為檢測信號通過二氧化硅包覆增強sers納米探針在復雜體系中的穩(wěn)定性。將構(gòu)建的新型SERS納米探針與光纖倏逝波生物傳感技術(shù)相結(jié)合,利用近紅外光作為激發(fā)光,有效地降低了全血中熒光背景及各組分光散射對檢測信號的影響,實現(xiàn)了全血中甲胎蛋白AFP的高靈敏及準確性檢測,檢測線性范圍達50-500 ng/m L。本論文在近紅外納米生物探針與多種傳感方式相結(jié)合的生物醫(yī)學檢測技術(shù)的研究方面,獲得了一些可喜的結(jié)果,有效地解決了熒光免疫檢測的兩大領域性(均相檢測與非均相檢測)難題,發(fā)展了全血原位免疫檢測的新技術(shù)與新方法,為癌癥等重大疾病的臨床早期診斷奠定了基礎。在現(xiàn)有工作的基礎上,綜合材料學、物理學、化學、分子生物學、生物醫(yī)學和臨床等多學科、多領域,研發(fā)多通道、高靈敏的全血檢測方法及全血原位在體檢測技術(shù)將是未來生物醫(yī)學研究的主要方向。
[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.

【學位授予單位】：中國科學院長春光學精密機械與物理研究所
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R730.4;TP212.3

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