基于多巴胺適配體標記的碳點和納米石墨的熒光生物傳感平臺的構(gòu)建及其應(yīng)用
發(fā)布時間：2018-11-26 15:19
【摘要】：多巴胺(Dopamine, DA)作為中樞神經(jīng)系統(tǒng)一種重要的單胺類神經(jīng)遞質(zhì),對心血管系統(tǒng)、中樞神經(jīng)系統(tǒng)、內(nèi)分泌系統(tǒng)等具有廣泛而重要的影響。體內(nèi)DA含量缺乏可能會引起多種神經(jīng)系統(tǒng)性疾病,如阿爾茨海默癥、精神分裂癥、帕金森綜合征等；而體內(nèi)DA含量過高則會導(dǎo)致機體處于興奮或焦慮狀態(tài)。體內(nèi)DA含量的快速、準確檢測在神經(jīng)系統(tǒng)性疾病的臨床診斷以及病理機制研究方面具有十分重要的作用。但由于生物基質(zhì)的構(gòu)成復(fù)雜,且DA含量非常低,所以構(gòu)建高靈敏度、高選擇性的DA快速分析方法具有十分重要的現(xiàn)實意義。碳點(Carbon dots, CDs)又稱熒光碳納米顆粒,是一種以碳為骨架結(jié)構(gòu),粒徑小于10 nm的類球形熒光納米顆粒。CDs不僅具有傳統(tǒng)半導(dǎo)體量子點的光學(xué)性能(如激發(fā)譜帶寬、發(fā)射譜帶窄、熒光強度高、光化學(xué)穩(wěn)定性好等),還具有高量子產(chǎn)率,低細胞毒性,高生物相容性,發(fā)射譜激發(fā)波長依賴性等特性。目前以CDs為熒光探針構(gòu)建的生物傳感平臺多基于熒光猝滅原理,但此類探針的最大缺陷在于：在較強的熒光背景下測定熒光猝滅的程度,將會極大地影響探針檢測的靈敏度。因此,為了進一步提高檢測的靈敏度,越來越多的研究者開始關(guān)注增強型即on-off-on型熒光CDs生物傳感平臺的構(gòu)建。本論文以生物樣品中痕量DA的檢測為目的,以DA核酸適配體標記的CDs(Aptamer-CDs)作為待測物的熒光識別基團,同時以納米石墨(Nano-graphite.NG)作為熒光猝滅劑構(gòu)建了能夠高選擇性、高靈敏度地檢測生物樣品中痕量DA的熒光生物傳感平臺,并將其成功應(yīng)用于生物樣品中痕量DA的檢測。概要如下：1. Aptamer-CDs/NG生物傳感平臺的構(gòu)建CDs表面的羧基經(jīng)活化后與DA aptamer核酸鏈上修飾的氨基相作用生成酰胺鍵,成功合成Aptamer-CDs復(fù)合物,然后在體系中引入猝滅劑NG,Aptamer-CDs的熒光被猝滅。當體系中沒有DA存在的條件下,Aptamer-CDs的熒光被猝滅；當體系中存在DA時,Aptamer-CDs可以與DA特異性結(jié)合形成特定的球狀鏈結(jié)構(gòu),此結(jié)構(gòu)從NG表面脫落,Aptamer-CDs的熒光恢復(fù)。根據(jù)Aptamer-CDs的熒光恢復(fù)程度與DA濃度之間的線性關(guān)系實現(xiàn)對樣品中DA含量的定量檢測。試驗優(yōu)化了Aptamer-CDs復(fù)合物合成以及傳感平臺應(yīng)用的反應(yīng)物比例、溫度、時間等條件,最后通過傅立葉紅外光譜,瓊脂糖凝膠電泳以及高分辨透射電鏡對所構(gòu)建的熒光傳感平臺進行了結(jié)構(gòu)和形貌表征,證明Aptamer-CDs/NG生物傳感平臺的成功構(gòu)建。2. Aptamer-CDs/NG生物傳感平臺的應(yīng)用基于Aptamer-CDs和NG,我們構(gòu)建了新型的on-off-on型熒光生物傳感平臺,并成功實現(xiàn)對尿液中痕量DA含量的高靈敏度、高選擇性測定。干擾試驗表明,Aptamer-CDs/NG生物傳感平臺具有良好的選擇性。研究結(jié)果表明,在最佳試驗條件下,當DA濃度在0.10～5.00 nM范圍內(nèi),構(gòu)建的生物傳感平臺的熒光強度的相對變化值與DA濃度的自然對數(shù)值呈良好的線性關(guān)系,相關(guān)系數(shù)為0.9995,檢測限為0.055 nM。在該方法建立的基礎(chǔ)上,采用標準加入法,測得健康實驗室志愿者的尿液(稀釋60倍)中DA的含量分別為0.75 nM和0.80 nM,DA的加標回收率均在96.0%～105.2%之間,且相對標準偏差小于18.7%(n=3)。試驗結(jié)果表明構(gòu)建的on-off-on型熒光生物傳感平臺能夠滿足復(fù)雜生物樣品中DA的分析測定要求。本論文設(shè)計的熒光生物傳感平臺操作綠色便捷,測定復(fù)雜生物樣品時僅需進行稀釋處理便可實現(xiàn)對樣品中所含DA的快速分析測定。
[Abstract]:Dopamine (DA), as an important monoamine neurotransmitter in the central nervous system, has a wide and important influence on the cardiovascular system, the central nervous system and the endocrine system. The lack of in vivo DA content may cause multiple nervous system diseases, such as Alzheimer's disease, schizophrenia, Parkinson's syndrome, etc., while the high in vivo DA content can cause the body to be in an excited or anxious state. The rapid and accurate detection of the content of DA in vivo has a very important role in the clinical diagnosis of nervous system diseases and the research of the pathological mechanism. However, because of the complex composition of the biological matrix, and the content of DA is very low, it is of great practical significance to construct a high-sensitivity and high-selectivity DA rapid analysis method. Carbon dot (CDs), also called fluorescent carbon nanoparticles, is a kind of spherical fluorescent nanoparticles with carbon skeleton structure and particle size less than 10 nm. The CDs not only has the optical properties of the conventional semiconductor quantum dots (such as the excitation spectrum bandwidth, narrow emission spectrum band, high fluorescence intensity, good photochemical stability, etc.), but also has the characteristics of high quantum yield, low cell toxicity, high biocompatibility, emission spectrum excitation wavelength dependence, and the like. The biological sensing platform, which is constructed with CDs as a fluorescent probe, is based on the principle of fluorescence excitation, but the maximum defect of such a probe is that the degree of fluorescence extinction is measured in a stronger fluorescence background, and the sensitivity of the probe detection will be greatly affected. Therefore, in order to further improve the sensitivity of detection, more and more researchers have begun to focus on the construction of the enhancement-on-off-on-type fluorescence CDs biological sensing platform. The purpose of this paper is to detect the trace DA in the biological sample, and the DA nucleic acid aptamer-labeled CDs (Aptamer-CDs) is used as the fluorescent recognition group of the object to be tested, and the nano-graphite (Nano-graphite. NG) is used as the fluorescent quenching agent to construct the high-selectivity. The fluorescence biosensor platform of the trace DA in the biological sample is detected with high sensitivity and is successfully applied to the detection of trace DA in the biological sample. The summary is as follows: 1. Aptamer-CDs/ NG bio-sensing platform constructs the amine bond with the amino phase of the modified amino phase on the DA aptamer nucleic acid chain after activation, and successfully synthesizes the Aptamer-CDs complex, and then introduces the quencher NG in the system, and the fluorescence of the Aptamer-CDs is destroyed. When DA is not present in the system, the fluorescence of Aptamer-CDs is destroyed; when DA is present in the system, Aptamer-CDs can be specifically combined with DA to form a specific spherical chain structure, and the structure is detached from the NG surface, and the fluorescence of Aptamer-CDs is recovered. The quantitative detection of DA content in the sample was achieved according to the linear relationship between the degree of fluorescence recovery and the DA concentration of Aptamer-CDs. The synthesis of Aptamer-CDs and the proportion of reactants, temperature and time of the application of the sensing platform were optimized, and the structure and morphology of the constructed fluorescence sensing platform were characterized by the Fourier infrared spectrum, the agarose gel electrophoresis and the high-resolution transmission electron microscope. The success of the Aptamer-CDs/ NG Biosensing Platform was demonstrated. The application of the Aptamer-CDs/ NG Biosensing Platform is based on Aptamer-CDs and NG, and we construct a new type of on-off-on-type fluorescence bio-sensing platform, and successfully realize high sensitivity and high selectivity for trace DA content in urine. The interference test shows that the Aptamer-CDs/ NG bio-sensing platform has good selectivity. The results show that, under the best test condition, the relative change of the fluorescence intensity of the constructed biosensing platform and the natural logarithm of the DA concentration in the range of 0. 10-5. 00 nM have a good linear relationship with the natural logarithm of the DA concentration, and the correlation coefficient is 0.9995 and the detection limit is 0.055 nM. On the basis of the establishment of this method, the content of DA in the urine (60-fold dilution) of healthy laboratory volunteers was 0. 75 nM and 0. 80 nM, respectively, and the relative standard deviation was less than 18.7% (n = 3). The results show that the on-off-on-type fluorescence biosensor can meet the requirement of the analysis and determination of DA in complex biological samples. The fluorescence bio-sensing platform designed in this paper is simple and convenient to operate, and the rapid analysis and determination of the DA in the sample can be realized only by the dilution treatment when the complex biological sample is determined.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：O657.3;R446.1
本文編號：2358923
[Abstract]:Dopamine (DA), as an important monoamine neurotransmitter in the central nervous system, has a wide and important influence on the cardiovascular system, the central nervous system and the endocrine system. The lack of in vivo DA content may cause multiple nervous system diseases, such as Alzheimer's disease, schizophrenia, Parkinson's syndrome, etc., while the high in vivo DA content can cause the body to be in an excited or anxious state. The rapid and accurate detection of the content of DA in vivo has a very important role in the clinical diagnosis of nervous system diseases and the research of the pathological mechanism. However, because of the complex composition of the biological matrix, and the content of DA is very low, it is of great practical significance to construct a high-sensitivity and high-selectivity DA rapid analysis method. Carbon dot (CDs), also called fluorescent carbon nanoparticles, is a kind of spherical fluorescent nanoparticles with carbon skeleton structure and particle size less than 10 nm. The CDs not only has the optical properties of the conventional semiconductor quantum dots (such as the excitation spectrum bandwidth, narrow emission spectrum band, high fluorescence intensity, good photochemical stability, etc.), but also has the characteristics of high quantum yield, low cell toxicity, high biocompatibility, emission spectrum excitation wavelength dependence, and the like. The biological sensing platform, which is constructed with CDs as a fluorescent probe, is based on the principle of fluorescence excitation, but the maximum defect of such a probe is that the degree of fluorescence extinction is measured in a stronger fluorescence background, and the sensitivity of the probe detection will be greatly affected. Therefore, in order to further improve the sensitivity of detection, more and more researchers have begun to focus on the construction of the enhancement-on-off-on-type fluorescence CDs biological sensing platform. The purpose of this paper is to detect the trace DA in the biological sample, and the DA nucleic acid aptamer-labeled CDs (Aptamer-CDs) is used as the fluorescent recognition group of the object to be tested, and the nano-graphite (Nano-graphite. NG) is used as the fluorescent quenching agent to construct the high-selectivity. The fluorescence biosensor platform of the trace DA in the biological sample is detected with high sensitivity and is successfully applied to the detection of trace DA in the biological sample. The summary is as follows: 1. Aptamer-CDs/ NG bio-sensing platform constructs the amine bond with the amino phase of the modified amino phase on the DA aptamer nucleic acid chain after activation, and successfully synthesizes the Aptamer-CDs complex, and then introduces the quencher NG in the system, and the fluorescence of the Aptamer-CDs is destroyed. When DA is not present in the system, the fluorescence of Aptamer-CDs is destroyed; when DA is present in the system, Aptamer-CDs can be specifically combined with DA to form a specific spherical chain structure, and the structure is detached from the NG surface, and the fluorescence of Aptamer-CDs is recovered. The quantitative detection of DA content in the sample was achieved according to the linear relationship between the degree of fluorescence recovery and the DA concentration of Aptamer-CDs. The synthesis of Aptamer-CDs and the proportion of reactants, temperature and time of the application of the sensing platform were optimized, and the structure and morphology of the constructed fluorescence sensing platform were characterized by the Fourier infrared spectrum, the agarose gel electrophoresis and the high-resolution transmission electron microscope. The success of the Aptamer-CDs/ NG Biosensing Platform was demonstrated. The application of the Aptamer-CDs/ NG Biosensing Platform is based on Aptamer-CDs and NG, and we construct a new type of on-off-on-type fluorescence bio-sensing platform, and successfully realize high sensitivity and high selectivity for trace DA content in urine. The interference test shows that the Aptamer-CDs/ NG bio-sensing platform has good selectivity. The results show that, under the best test condition, the relative change of the fluorescence intensity of the constructed biosensing platform and the natural logarithm of the DA concentration in the range of 0. 10-5. 00 nM have a good linear relationship with the natural logarithm of the DA concentration, and the correlation coefficient is 0.9995 and the detection limit is 0.055 nM. On the basis of the establishment of this method, the content of DA in the urine (60-fold dilution) of healthy laboratory volunteers was 0. 75 nM and 0. 80 nM, respectively, and the relative standard deviation was less than 18.7% (n = 3). The results show that the on-off-on-type fluorescence biosensor can meet the requirement of the analysis and determination of DA in complex biological samples. The fluorescence bio-sensing platform designed in this paper is simple and convenient to operate, and the rapid analysis and determination of the DA in the sample can be realized only by the dilution treatment when the complex biological sample is determined.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：O657.3;R446.1
【參考文獻】
相關(guān)碩士學(xué)位論文 前2條
1 劉威;基于量子點熒光探針的腫瘤標志物GP73和腺苷的分析方法研究[D];南京醫(yī)科大學(xué);2015年
2 郭艷;碳量子點的結(jié)構(gòu)對熒光發(fā)射行為的影響[D];中北大學(xué);2012年
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