基于上转换纳米材料的生物传感新方法

发布时间：2018-08-19 10:43

【摘要】：随着纳米技术的不断发展和新型纳米材料的不断涌现,纳米科学为生物传感器的发展注入了新的活力,开辟了新的发展思路。近年来,越来越多的新型纳米发光材料因其独特的光学性能而成为当前研究的热点,尤其是上转换纳米发光材料(UCNPs),因其能够通过多光子机制将近红外光(通常为980nm)转换成强烈的紫外、可见光,具有激发光能量低、背景低、量子产率高、光漂白小、发光强度高且稳定、发射波长可通过控制其组成进行调节等优点而在生物检测中得到了广泛的应用,其毒性低、生物相容性好、组织穿透能力强等优点更是让其在细胞、肿瘤成像诊断和治疗中得到了广泛的研究。基于此,本文围绕新型的上转换纳米材料开展了以下三个方面的工作:一、利用溶剂热法制备了发射蓝色荧光的NaYF_4:Yb,Tm/NaYF_4上转换纳米颗粒。结合UCNPs的光学性能和氧化石墨烯独特的电子学性质发展了一个简便的、超灵敏的生物传感器用于S1核酸内切酶的检测。其原理为:DNA的5’端修饰磷酸基团,由于磷酸基团与镧系离子之间有更强的结合力从而使油相制备的UCNPs表面的油酸在液液界面通过配体交换的形式被置换下来,DNA组装到UCNPs表面,上转换纳米颗粒表面的DNA能够通过π-π共轭作用和疏水作用被吸附到石墨烯的表面,从而拉近UCNPs与石墨烯之间的距离,两者发生荧光共振能量转移,UCNPs荧光被猝灭。当有S1核酸内切酶存在时,上转换纳米颗粒表面的DNA会被S1核酸内切酶切割成单一的或短小的寡核苷酸片段,从而导致UCNPs与石墨烯之间作用力减弱,UCNPs远离氧化石墨烯,荧光信号得到恢复。本方法有很好的选择性,比以往的检测S1核酸内切酶的方法灵敏,可以灵敏检测到1×10~(-4) units m L~(-1)的S1核酸内切酶,并且探究了S1核酸酶抑制试验。这种创新的方法提供了一个探索上转换荧光共振能量转移性质的范例,扩展了上转换纳米材料在广泛的领域(如生物学、生物医学、生物/化学传感)的应用机会。二、利用溶剂热法制备了发射绿色荧光的NaYF_4:Yb,Er上转换纳米颗粒。结合UCNPs的光学性能、Exo Ⅲ特殊的酶切活性和氧化石墨烯独特的电子学性质发展了一种新颖的、简便的、灵敏度高的分析技术用于乳腺癌基因(BRCA1)的放大检测。其原理为:DNA的5’端修饰磷酸基团,由于磷酸基团与镧系离子之间有更强的结合力从而使油相制备的UCNPs表面的油酸在液液界面通过配体交换的形式被置换下来,DNA组装到UCNPs表面,当加入石墨烯和Exo Ⅲ时,上转换纳米颗粒表面的DNA能够通过π-π共轭作用和疏水作用被吸附到石墨烯的表面,从而拉近UCNPs与石墨烯之间的距离,两者发生荧光共振能量转移,UCNPs荧光被猝灭。。当有目标DNA乳腺癌基因(BRCA1)存在时,目标DNA与UCNPs表面的捕获DNA结合成双链,从而激活Exo Ⅲ切割捕获DNA成单一的或短小的寡核苷酸片段,从而释放目标DNA进行循环放大,UCNPs与石墨烯之间的作用力减弱,UCNPs远离氧化石墨烯,荧光信号得到恢复。本方法灵敏度高,易于操作,对对BRCA1的检测动态线性范围为0.02 nM～0.9 nM。三、利用溶剂热法制备了发射蓝色荧光的NaYF_4:Yb,Tm/NaYF_4上转换纳米颗粒。结合UCNPs的光学性能和Hg的电子学性质设计了一个新颖的、灵敏的生物传感器用于Hg~(2+)的检测。其原理为:DNA的5’端修饰磷酸基团,由于磷酸基团与镧系离子之间有更强的结合力从而使油相制备的UCNPs表面的油酸在液液界面通过配体交换的形式被置换下来,DNA组装到UCNPs表面,当有Hg~(2+)存在时,Hg~(2+)通过有效的电子转移过程促进UCNPs无辐射电子/空穴复合湮灭,从而使上转换纳米的荧光被猝灭。本方法在10 nM～10mM呈良好的线性关系,在水溶液中的检测限为5 nM。在实际样品的检测中,回收率在97%～102%范围之内,相对标准偏差约6%。表明该传感器能够有效地降低复杂生物样品中的背景干扰,在实际样品的定量分析中可以获得满意的结果。
[Abstract]:With the continuous development of nanotechnology and the emergence of new nanomaterials, nanoscience has injected new vitality into the development of biosensors and opened up new ideas for development. In recent years, more and more new nanoluminescent materials have become the focus of current research because of their unique optical properties, especially upconversion nanoluminescent materials. Ultraviolet and visible light can be converted from near infrared (usually 980 nm) to intense ultraviolet (UV) by multi-photon mechanism. Ultraviolet and visible light has the advantages of low excitation energy, low background, high quantum yield, low photobleaching, high and stable luminescence intensity, and emission wavelength can be adjusted by controlling its composition. Because of its low toxicity, good biocompatibility and strong tissue penetration ability, it has been extensively studied in cell, tumor imaging diagnosis and treatment. Based on this, this paper focuses on the new upconversion nanomaterials to carry out the following three aspects: First, the preparation of blue fluorescent NaYF_4:Yb, Tm/Na by solvothermal method. YF_4 upconversion nanoparticles. Combining the optical properties of UCNPs with the unique electronic properties of graphene oxide, a simple, ultra-sensitive biosensor has been developed for the detection of S1 nucleic acid endonuclease. The principle is that the 5'-terminal of DNA modifies the phosphoryl group, which has a stronger binding force with lanthanide ions, and thus makes the oil phase oil. Oleic acid on the surface of UCNPs is replaced by ligand exchange at the liquid-liquid interface. DNA is assembled on the surface of UCNPs. The DNA on the surface of UCNPs can be adsorbed to the surface of graphene through the conjugation and hydrophobicity of pi-pi, thus shortening the distance between UCNPs and graphene. The fluorescence resonance energy transfer occurs between them. In the presence of S1 endonuclease, the DNA on the surface of up-converted nanoparticles is cleaved into a single or short oligonucleotide fragment by S1 endonuclease, resulting in the weakening of the interaction between UCNPs and graphene, and the UCNPs are far away from graphene oxide, thus the fluorescence signal is recovered. Compared with previous methods for detecting S1 endonuclease, the method is more sensitive to detect S1 endonuclease with a sensitivity of 1 (-4) units m L (-1) and explores S1 nuclease inhibition test. Opportunities for applications such as biology, biomedicine, bio/chemical sensing. 2. Solvothermal synthesis of green fluorescent NaYF_4:Yb, Er upconversion nanoparticles. Combining the optical properties of UCNPs, the special enzyme digestion activity of Exo III and the unique electronic properties of graphene oxide, a novel, simple and highly sensitive fraction has been developed. The principle of BRCA1 amplification is that the 5'-terminal of DNA modifies the phosphate group, and the oleic acid on the surface of the oil-phase UCNPs is replaced by ligand exchange at the liquid-liquid interface because of the stronger binding force between the phosphate group and lanthanide ions. The DNA is assembled on the surface of the UCNPs when graphite is added. When EN and Exo III are present, the DNA on the surface of up-converted nanoparticles can be adsorbed to the surface of graphene through the conjugation and hydrophobic interaction of pi-pi, thus closing the distance between UCNPs and graphene. The fluorescence resonance energy transfer occurs between UCNPs and graphene, and the fluorescence of UCNPs is quenched. This method is sensitive and easy to operate, and can be used to detect BRCA1. NaYF_4:Yb, Tm/NaYF_4 up-conversion nanoparticles emitting blue fluorescence were prepared by solvothermal method in the range of 0.02 nM~0.9 nM.3. A novel, sensitive biosensor was designed for the detection of Hg~ (2+) based on the optical properties of UCNPs and the electronic properties of Hg. In the presence of Hg ~ (2 +), Hg ~ (2 +) promotes the non-radiation electron/hole annihilation of UCNPs through an effective electron transfer process. This method has a good linear relationship between 10 nM and 10 mM, and the detection limit in aqueous solution is 5 nM. In the detection of real samples, the recovery rate is in the range of 97%-102%, and the relative standard deviation is about 6%. It shows that the sensor can effectively reduce the background interference in complex biological samples, and the detection limit is 5 nM. Satisfactory results can be obtained in quantitative analysis.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1;TP212.3

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