【摘要】：近年來(lái),生物傳感器因其準(zhǔn)確度高、分析速度快、成本低、選擇好等特點(diǎn),已廣泛應(yīng)用于臨床檢測(cè)、藥物篩選和醫(yī)學(xué)研究中。光學(xué)檢測(cè)技術(shù)具有背景噪音低、操作簡(jiǎn)便、靈敏度高等優(yōu)點(diǎn),在生物傳感器領(lǐng)域廣泛受到科研工作者的關(guān)注。此外,新型生物材料和金屬納米材料的發(fā)展為許多基礎(chǔ)研究提供了全新的傳感設(shè)計(jì)思路和平臺(tái)。本論文基于細(xì)胞色素c和納米發(fā)光材料在生物傳感和生化分析應(yīng)用領(lǐng)域的優(yōu)勢(shì),致力于傳感分析的研究熱點(diǎn),建立了多種生物傳感新方法用于疾病相關(guān)的蛋白酶活性及其抑制劑檢測(cè)、蛋白和小分子的檢測(cè)。和傳統(tǒng)方法相比,本論文所建立的方法操作簡(jiǎn)便、靈敏度高且分析成本低,同時(shí)初步顯示了某些實(shí)際應(yīng)用方面的檢測(cè)能力。其具體內(nèi)容如下：激酶催化的蛋白磷酸化過(guò)程在許多細(xì)胞生理過(guò)程中起到重要作用。在第2章中,基于磷酸化多肽對(duì)羧肽酶Y消化的抑制作用及細(xì)胞色素c強(qiáng)猝滅能力的特點(diǎn),構(gòu)建了一種新穎的熒光多肽／細(xì)胞色素c傳感平臺(tái)用于檢測(cè)酪蛋白激酶Ⅱ(CK2)活性和抑制作用。實(shí)驗(yàn)中,多肽未被磷酸化時(shí),羧肽酶逐個(gè)水解熒光團(tuán)標(biāo)記的多肽,導(dǎo)致熒光團(tuán)的釋放。加入細(xì)胞色素c后對(duì)其熒光無(wú)明顯影響。然而磷酸化的氨基酸能阻礙羧肽酶的水解,導(dǎo)致磷酸化多肽片段吸附在細(xì)胞色素c表面。由于熒光團(tuán)能與細(xì)胞色素c之間發(fā)生電子轉(zhuǎn)移,造成熒光信號(hào)的下降。該方法能成功檢測(cè)CK2活性和抑制。同時(shí),在復(fù)雜環(huán)境中,該方法同樣表現(xiàn)出了良好的分析性能,細(xì)胞裂解液樣品中的回收率實(shí)驗(yàn)結(jié)果令人滿意。此外,我們建立的方法可通用于周期蛋白依賴性激酶(CDK1)活性的測(cè)定。在第3章中,基于適配體與溶菌酶之間強(qiáng)特異性作用,成功構(gòu)建了一種新型的適配體熒光傳感器用于溶菌酶含量的免標(biāo)記檢測(cè)。我們首先通過(guò)一步法直接合成得到適配體功能化的碲化鎘量子點(diǎn)(DNA-CdTe QDs),并利用其作為熒光探針。在本實(shí)驗(yàn)條件下,細(xì)胞色素c帶大量正電荷,而DNA-CdTe QDs表面帶負(fù)電荷,因而DNA-CdTe QDs能夠很快通過(guò)靜電吸附作用結(jié)合在細(xì)胞色素c上,量子點(diǎn)的熒光被猝滅。由于DNA-CdTe QDs與細(xì)胞色素c中的血紅素輔因子之間的電子轉(zhuǎn)移作用,量子點(diǎn)熒光被細(xì)胞色素c有效地猝滅。當(dāng)體系中加入有溶菌酶時(shí),由于量子點(diǎn)表面的適配體能夠與溶菌酶特異性地結(jié)合,使得量子點(diǎn)與細(xì)胞色素c分離,量子點(diǎn)熒光從而恢復(fù)。由此可知,熒光強(qiáng)度的變化與溶液中溶菌酶的加入量直接相關(guān),可以實(shí)現(xiàn)對(duì)溶菌酶的定量檢測(cè)。該檢測(cè)方法不需要進(jìn)行任何熒光標(biāo)記,整個(gè)實(shí)驗(yàn)過(guò)程操作簡(jiǎn)便,成本低廉。并且該方法具有較好的選擇性和靈敏度。此外,通過(guò)替換適配體序列,該方法可擴(kuò)展到其他蛋白質(zhì)的檢測(cè)。p-分泌酶(BACE1)在神經(jīng)毒性的淀粉樣蛋白(Aβ)的產(chǎn)生過(guò)程中起著很關(guān)鍵的作用,被認(rèn)為是治療阿爾默茨病的一個(gè)重要靶點(diǎn)。在第4章中,我們提出了一種基于酶水解產(chǎn)物原位合成量子點(diǎn)的策略用于BACE1活性及抑制的免標(biāo)記熒光檢測(cè)。在實(shí)驗(yàn)中,我們?cè)O(shè)計(jì)了一條雙巰基的底物肽探針。當(dāng)存在BACE1時(shí),多肽探針會(huì)被BACE1水解成兩條單巰基序列,由于單巰基多肽可以作為有效的配體或模板合成硫化鎘量子點(diǎn),從而能產(chǎn)生一個(gè)強(qiáng)的熒光信號(hào)。相反,當(dāng)不存在BACE1或有抑制劑存在時(shí),由于底物肽中的兩個(gè)巰基與鎘離子之間的雙齒螯合作用,使得底物肽與晶體表面形成很強(qiáng)的親和力從而抑制量子點(diǎn)的形成,幾乎檢測(cè)不到熒光。與以前報(bào)道的基于FRET技術(shù)的方法相比,該方法操作簡(jiǎn)單、分析成本低,同時(shí),不需要任何熒光團(tuán)標(biāo)記和復(fù)雜的多肽探針設(shè)計(jì)糖尿病是一組普遍的代謝性疾病,經(jīng)常監(jiān)控和嚴(yán)格控制血糖含量的變化對(duì)于有效診斷和管理糖尿病有著十分重要的意義。在第5章中,我們發(fā)展了一種新型的由DNA為模板的銀納米顆粒(DNA-AgNPs)和NaYF4:Yb/Tm@NaYF4核殼上轉(zhuǎn)換納米顆粒(UCNPs)組成的上轉(zhuǎn)換納米復(fù)合物用于雙氧水和葡萄糖的檢測(cè)。該復(fù)合物是基于表面裸露UCNPs和DNA-AgNPs之間的發(fā)光能量轉(zhuǎn)移(LRET)原理。在研究中,我們?cè)诟话奏さ腄NA序列一端連接上一段富腺嘌呤序列,并利用這條DNA作為模板一步合成銀納米顆粒。DNA-AgNPs首次被報(bào)道能直接自組裝在UCNPs裸表面。該LRET過(guò)程主要是依靠DNA-AgNPs上磷酸骨架與UNCPs表面所暴露的鑭系金屬之間的配位作用,使得供體的上轉(zhuǎn)換熒光被猝滅。當(dāng)體系中存在雙氧水時(shí),雙氧水能夠?qū)€y納米顆�？涛g并產(chǎn)生銀離子,因此能量轉(zhuǎn)移過(guò)程被抑制,上轉(zhuǎn)換的熒光得以恢復(fù)�；贕Ox能將葡萄糖轉(zhuǎn)換為雙氧水的原理,我們所構(gòu)建的DNA-AgNPs/ UCNP復(fù)合物能進(jìn)一步用于檢測(cè)血樣中的葡萄糖。該檢測(cè)方法無(wú)需其他輔助酶,從而使整個(gè)實(shí)驗(yàn)成本低廉。并且該方法選擇性較好,靈敏度高,雙氧水和葡萄糖的檢測(cè)下限分別為1.08和1.41μM。同時(shí)該檢測(cè)方法在復(fù)雜體系中也具有較好的分析性能。堿性磷酸酶(ALP)活性的檢測(cè)對(duì)于提供相關(guān)疾病的臨床診斷及評(píng)估具有很重要的指導(dǎo)意義。在第6章中,我們以DNA為模板合成的銀納米簇作為熒光指示劑,利用富G序列靠近銀納米簇時(shí),熒光大大增強(qiáng)的原理,發(fā)展了一種的熒光傳感器用于檢測(cè)ALP的活性。在本實(shí)驗(yàn)中,我們?cè)O(shè)計(jì)了兩條DNA鏈,其中5’磷酸化的G-DNA作為ALP的水解底物,A-DNA包含一段銀納米簇合成的模板鏈以及一段與G-DNA雜交互補(bǔ)的序列。λ外切酶(λexo)能夠?qū)⒘姿峄腉-DNA迅速降解成單核苷酸,由于缺少富G序列的靠近,銀納米簇的熒光信號(hào)很弱；當(dāng)體系中引入ALP時(shí),ALP能夠?qū)⒘姿峄腉-DNA去磷酸化為羥基從而有效阻止λexo的酶切作用。由于銀簇的模版鏈能與G-DNA一段序列雜交互補(bǔ)配對(duì),使得銀納米簇的熒光強(qiáng)度因富G序列的靠近而大大增強(qiáng)。由于銀簇?zé)晒獾男盘?hào)強(qiáng)弱程度則依賴于ALP的濃度。因此,通過(guò)檢測(cè)銀簇的熒光強(qiáng)度變化便可實(shí)現(xiàn)ALP的檢測(cè)。該方法不僅可用于ALP抑制劑的篩選,在復(fù)雜體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.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：TP212.3

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