基于细胞色素c和纳米发光材料的生物传感新方法研究
发布时间：2018-09-18 10:02
【摘要】：近年来,生物传感器因其准确度高、分析速度快、成本低、选择好等特点,已广泛应用于临床检测、药物筛选和医学研究中。光学检测技术具有背景噪音低、操作简便、灵敏度高等优点,在生物传感器领域广泛受到科研工作者的关注。此外,新型生物材料和金属纳米材料的发展为许多基础研究提供了全新的传感设计思路和平台。本论文基于细胞色素c和纳米发光材料在生物传感和生化分析应用领域的优势,致力于传感分析的研究热点,建立了多种生物传感新方法用于疾病相关的蛋白酶活性及其抑制剂检测、蛋白和小分子的检测。和传统方法相比,本论文所建立的方法操作简便、灵敏度高且分析成本低,同时初步显示了某些实际应用方面的检测能力。其具体内容如下：激酶催化的蛋白磷酸化过程在许多细胞生理过程中起到重要作用。在第2章中,基于磷酸化多肽对羧肽酶Y消化的抑制作用及细胞色素c强猝灭能力的特点,构建了一种新颖的荧光多肽／细胞色素c传感平台用于检测酪蛋白激酶Ⅱ(CK2)活性和抑制作用。实验中,多肽未被磷酸化时,羧肽酶逐个水解荧光团标记的多肽,导致荧光团的释放。加入细胞色素c后对其荧光无明显影响。然而磷酸化的氨基酸能阻碍羧肽酶的水解,导致磷酸化多肽片段吸附在细胞色素c表面。由于荧光团能与细胞色素c之间发生电子转移,造成荧光信号的下降。该方法能成功检测CK2活性和抑制。同时,在复杂环境中,该方法同样表现出了良好的分析性能,细胞裂解液样品中的回收率实验结果令人满意。此外,我们建立的方法可通用于周期蛋白依赖性激酶(CDK1)活性的测定。在第3章中,基于适配体与溶菌酶之间强特异性作用,成功构建了一种新型的适配体荧光传感器用于溶菌酶含量的免标记检测。我们首先通过一步法直接合成得到适配体功能化的碲化镉量子点(DNA-CdTe QDs),并利用其作为荧光探针。在本实验条件下,细胞色素c带大量正电荷,而DNA-CdTe QDs表面带负电荷,因而DNA-CdTe QDs能够很快通过静电吸附作用结合在细胞色素c上,量子点的荧光被猝灭。由于DNA-CdTe QDs与细胞色素c中的血红素辅因子之间的电子转移作用,量子点荧光被细胞色素c有效地猝灭。当体系中加入有溶菌酶时,由于量子点表面的适配体能够与溶菌酶特异性地结合,使得量子点与细胞色素c分离,量子点荧光从而恢复。由此可知,荧光强度的变化与溶液中溶菌酶的加入量直接相关,可以实现对溶菌酶的定量检测。该检测方法不需要进行任何荧光标记,整个实验过程操作简便,成本低廉。并且该方法具有较好的选择性和灵敏度。此外,通过替换适配体序列,该方法可扩展到其他蛋白质的检测。p-分泌酶(BACE1)在神经毒性的淀粉样蛋白(Aβ)的产生过程中起着很关键的作用,被认为是治疗阿尔默茨病的一个重要靶点。在第4章中,我们提出了一种基于酶水解产物原位合成量子点的策略用于BACE1活性及抑制的免标记荧光检测。在实验中,我们设计了一条双巯基的底物肽探针。当存在BACE1时,多肽探针会被BACE1水解成两条单巯基序列,由于单巯基多肽可以作为有效的配体或模板合成硫化镉量子点,从而能产生一个强的荧光信号。相反,当不存在BACE1或有抑制剂存在时,由于底物肽中的两个巯基与镉离子之间的双齿螯合作用,使得底物肽与晶体表面形成很强的亲和力从而抑制量子点的形成,几乎检测不到荧光。与以前报道的基于FRET技术的方法相比,该方法操作简单、分析成本低,同时,不需要任何荧光团标记和复杂的多肽探针设计糖尿病是一组普遍的代谢性疾病,经常监控和严格控制血糖含量的变化对于有效诊断和管理糖尿病有着十分重要的意义。在第5章中,我们发展了一种新型的由DNA为模板的银纳米颗粒(DNA-AgNPs)和NaYF4:Yb/Tm@NaYF4核壳上转换纳米颗粒(UCNPs)组成的上转换纳米复合物用于双氧水和葡萄糖的检测。该复合物是基于表面裸露UCNPs和DNA-AgNPs之间的发光能量转移(LRET)原理。在研究中,我们在富胞嘧啶的DNA序列一端连接上一段富腺嘌呤序列,并利用这条DNA作为模板一步合成银纳米颗粒。DNA-AgNPs首次被报道能直接自组装在UCNPs裸表面。该LRET过程主要是依靠DNA-AgNPs上磷酸骨架与UNCPs表面所暴露的镧系金属之间的配位作用,使得供体的上转换荧光被猝灭。当体系中存在双氧水时,双氧水能够将银纳米颗粒刻蚀并产生银离子,因此能量转移过程被抑制,上转换的荧光得以恢复。基于GOx能将葡萄糖转换为双氧水的原理,我们所构建的DNA-AgNPs/ UCNP复合物能进一步用于检测血样中的葡萄糖。该检测方法无需其他辅助酶,从而使整个实验成本低廉。并且该方法选择性较好,灵敏度高,双氧水和葡萄糖的检测下限分别为1.08和1.41μM。同时该检测方法在复杂体系中也具有较好的分析性能。碱性磷酸酶(ALP)活性的检测对于提供相关疾病的临床诊断及评估具有很重要的指导意义。在第6章中,我们以DNA为模板合成的银纳米簇作为荧光指示剂,利用富G序列靠近银纳米簇时,荧光大大增强的原理,发展了一种的荧光传感器用于检测ALP的活性。在本实验中,我们设计了两条DNA链,其中5’磷酸化的G-DNA作为ALP的水解底物,A-DNA包含一段银纳米簇合成的模板链以及一段与G-DNA杂交互补的序列。λ外切酶(λexo)能够将磷酸化的G-DNA迅速降解成单核苷酸,由于缺少富G序列的靠近,银纳米簇的荧光信号很弱；当体系中引入ALP时,ALP能够将磷酸化的G-DNA去磷酸化为羟基从而有效阻止λexo的酶切作用。由于银簇的模版链能与G-DNA一段序列杂交互补配对,使得银纳米簇的荧光强度因富G序列的靠近而大大增强。由于银簇荧光的信号强弱程度则依赖于ALP的浓度。因此,通过检测银簇的荧光强度变化便可实现ALP的检测。该方法不仅可用于ALP抑制剂的筛选,在复杂体系中也具有良好的分析性能。
[Abstract]:In recent years, biosensors have been widely used in clinical detection, drug screening and medical research because of their high accuracy, fast analysis speed, low cost and good selection. Optical detection technology has the advantages of low background noise, simple operation and high sensitivity, and has been widely concerned by researchers in the field of biosensors. Based on the advantages of cytochrome c and Nano-luminescent materials in the fields of biosensor and biochemical analysis, this paper is devoted to the research hotspot of biosensor analysis, and has established a variety of new biosensor methods for diseases. Compared with the traditional methods, the method proposed in this paper is simple, sensitive and low-cost. At the same time, it preliminarily demonstrates the detection ability in some practical applications. The specific contents are as follows: kinase-catalyzed protein phosphorylation process has many advantages. In Chapter 2, a novel fluorescent peptide/cytochrome C sensing platform was constructed to detect the activity and inhibition of casein kinase II (CK2) based on the inhibition of phosphopeptides on the digestion of carboxypeptidase Y and the strong quenching ability of cytochrome c. However, phosphorylated amino acids can hinder the hydrolysis of carboxypeptidase and lead to the adsorption of phosphorylated peptides on the surface of cytochrome c. The method can be used to detect the activity of cyclin-dependent kinase (CDK1). In complex environment, the method also shows good analytical performance. The results of recovery in cell lysate samples are satisfactory. In addition, our method can be used to determine the activity of cyclin-dependent kinase (CDK1). In Chapter 3, based on the strong specific interaction between aptamers and lysozyme, a novel aptamer fluorescence sensor was successfully constructed for the label-free detection of lysozyme content. We first synthesized aptamer-functionalized cadmium telluride quantum dots (DNA-CdTe QDs) directly by one-step method and used them as fluorescent probes. Under the condition of electrostatic adsorption, DNA-CdTe QDs can bind to cytochrome c quickly, and the fluorescence of QDs is quenched. Because of the electron transfer between DNA-CdTe QDs and heme cofactors in cytochrome c, the fluorescence of QDs is colored. When lysozyme is added to the system, the QDs can be separated from cytochrome C and the fluorescence of QDs can be restored due to the specific binding of the aptamers on the surface of QDs to lysozyme. Quantitative detection. The method does not require any fluorescent labeling. The whole experimental procedure is simple and inexpensive, and the method has good selectivity and sensitivity. In addition, by substituting aptamer sequences, the method can be extended to the detection of other proteins. p-secretase (BACE1) in neurotoxic amyloid protein (Abet) In Chapter 4, we propose a strategy for in situ synthesis of quantum dots based on enzymatic hydrolysates for label-free fluorescence detection of BACE1 activity and inhibition. In the presence of BACE1, the polypeptide probe is hydrolyzed into two single thiol sequences, which can be used as an effective ligand or template to synthesize CdS quantum dots and produce a strong fluorescence signal. Conversely, when BACE1 is not present or inhibitors are present, the double thiol groups in the substrate peptides are associated with CdS ions. Compared with the previously reported FRET-based methods, this method is simple to operate and has low analytical cost. At the same time, it does not require any fluorescent labels and complex peptide probes to design diabetes mellitus. In Chapter 5, we developed a novel upconversion composed of DNA-AgNPs and NaYF4:Yb/Tm@NaYF4 core-shell upconversion nanoparticles (UCNPs). The nanocomposite is based on the principle of luminescent energy transfer (LRET) between exposed UCNPs and DNA-AgNPs. In the study, we linked a adenine-rich sequence at one end of the cytosine-rich DNA sequence and used this DNA as a template to synthesize silver nanoparticles in one step. The LRET process mainly relies on the coordination between the phosphoric acid skeleton on DNA-AgNPs and the lanthanide metals exposed on the surface of UNCPs to quench the up-conversion fluorescence of the donor. Based on the principle that GOx can convert glucose to hydrogen peroxide, our DNA-AgNPs/UCNP complex can be further used to detect glucose in blood samples. The detection limits of hydrogen peroxide and glucose were 1.08 and 1.41 mu M, respectively. The detection of alkaline phosphatase (ALP) activity was very important for clinical diagnosis and evaluation of related diseases. In Chapter 6, DNA was synthesized as template. A fluorescence sensor was developed to detect the activity of ALP using silver nanoclusters as fluorescent indicators. In this experiment, we designed two DNA strands, in which 5'-phosphorylated G-DNA was used as the hydrolysis substrate of ALP and A-DNA contained a model of silver nanoclusters synthesis. Lambda exo can rapidly degrade phosphorylated G-DNA into mononucleotides, and the fluorescence signal of silver nanoclusters is very weak due to the lack of close proximity of G-rich sequences. When ALP is introduced into the system, ALP can dephosphorylate phosphorylated G-DNA into hydroxyl groups, thus effectively preventing the digestion of lambda exo. The fluorescence intensity of silver nanoclusters is greatly enhanced by the close proximity of G-rich sequences because the template chains of silver clusters can be complemented by the hybridization of G-DNA sequences. Because the fluorescence intensity of silver clusters depends on the concentration of ALP, the detection of ALP can be realized by detecting the fluorescence intensity of silver clusters. The screening of ALP inhibitors also has good analytical performance in complex systems.
【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TP212.3
本文编号：2247557
[Abstract]:In recent years, biosensors have been widely used in clinical detection, drug screening and medical research because of their high accuracy, fast analysis speed, low cost and good selection. Optical detection technology has the advantages of low background noise, simple operation and high sensitivity, and has been widely concerned by researchers in the field of biosensors. Based on the advantages of cytochrome c and Nano-luminescent materials in the fields of biosensor and biochemical analysis, this paper is devoted to the research hotspot of biosensor analysis, and has established a variety of new biosensor methods for diseases. Compared with the traditional methods, the method proposed in this paper is simple, sensitive and low-cost. At the same time, it preliminarily demonstrates the detection ability in some practical applications. The specific contents are as follows: kinase-catalyzed protein phosphorylation process has many advantages. In Chapter 2, a novel fluorescent peptide/cytochrome C sensing platform was constructed to detect the activity and inhibition of casein kinase II (CK2) based on the inhibition of phosphopeptides on the digestion of carboxypeptidase Y and the strong quenching ability of cytochrome c. However, phosphorylated amino acids can hinder the hydrolysis of carboxypeptidase and lead to the adsorption of phosphorylated peptides on the surface of cytochrome c. The method can be used to detect the activity of cyclin-dependent kinase (CDK1). In complex environment, the method also shows good analytical performance. The results of recovery in cell lysate samples are satisfactory. In addition, our method can be used to determine the activity of cyclin-dependent kinase (CDK1). In Chapter 3, based on the strong specific interaction between aptamers and lysozyme, a novel aptamer fluorescence sensor was successfully constructed for the label-free detection of lysozyme content. We first synthesized aptamer-functionalized cadmium telluride quantum dots (DNA-CdTe QDs) directly by one-step method and used them as fluorescent probes. Under the condition of electrostatic adsorption, DNA-CdTe QDs can bind to cytochrome c quickly, and the fluorescence of QDs is quenched. Because of the electron transfer between DNA-CdTe QDs and heme cofactors in cytochrome c, the fluorescence of QDs is colored. When lysozyme is added to the system, the QDs can be separated from cytochrome C and the fluorescence of QDs can be restored due to the specific binding of the aptamers on the surface of QDs to lysozyme. Quantitative detection. The method does not require any fluorescent labeling. The whole experimental procedure is simple and inexpensive, and the method has good selectivity and sensitivity. In addition, by substituting aptamer sequences, the method can be extended to the detection of other proteins. p-secretase (BACE1) in neurotoxic amyloid protein (Abet) In Chapter 4, we propose a strategy for in situ synthesis of quantum dots based on enzymatic hydrolysates for label-free fluorescence detection of BACE1 activity and inhibition. In the presence of BACE1, the polypeptide probe is hydrolyzed into two single thiol sequences, which can be used as an effective ligand or template to synthesize CdS quantum dots and produce a strong fluorescence signal. Conversely, when BACE1 is not present or inhibitors are present, the double thiol groups in the substrate peptides are associated with CdS ions. Compared with the previously reported FRET-based methods, this method is simple to operate and has low analytical cost. At the same time, it does not require any fluorescent labels and complex peptide probes to design diabetes mellitus. In Chapter 5, we developed a novel upconversion composed of DNA-AgNPs and NaYF4:Yb/Tm@NaYF4 core-shell upconversion nanoparticles (UCNPs). The nanocomposite is based on the principle of luminescent energy transfer (LRET) between exposed UCNPs and DNA-AgNPs. In the study, we linked a adenine-rich sequence at one end of the cytosine-rich DNA sequence and used this DNA as a template to synthesize silver nanoparticles in one step. The LRET process mainly relies on the coordination between the phosphoric acid skeleton on DNA-AgNPs and the lanthanide metals exposed on the surface of UNCPs to quench the up-conversion fluorescence of the donor. Based on the principle that GOx can convert glucose to hydrogen peroxide, our DNA-AgNPs/UCNP complex can be further used to detect glucose in blood samples. The detection limits of hydrogen peroxide and glucose were 1.08 and 1.41 mu M, respectively. The detection of alkaline phosphatase (ALP) activity was very important for clinical diagnosis and evaluation of related diseases. In Chapter 6, DNA was synthesized as template. A fluorescence sensor was developed to detect the activity of ALP using silver nanoclusters as fluorescent indicators. In this experiment, we designed two DNA strands, in which 5'-phosphorylated G-DNA was used as the hydrolysis substrate of ALP and A-DNA contained a model of silver nanoclusters synthesis. Lambda exo can rapidly degrade phosphorylated G-DNA into mononucleotides, and the fluorescence signal of silver nanoclusters is very weak due to the lack of close proximity of G-rich sequences. When ALP is introduced into the system, ALP can dephosphorylate phosphorylated G-DNA into hydroxyl groups, thus effectively preventing the digestion of lambda exo. The fluorescence intensity of silver nanoclusters is greatly enhanced by the close proximity of G-rich sequences because the template chains of silver clusters can be complemented by the hybridization of G-DNA sequences. Because the fluorescence intensity of silver clusters depends on the concentration of ALP, the detection of ALP can be realized by detecting the fluorescence intensity of silver clusters. The screening of ALP inhibitors also has good analytical performance in complex systems.
【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TP212.3
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