【作者】 李伟；

【导师】 苏荣欣；

【作者基本信息】 天津大学 ， 化学工程， 2014， 硕士

【摘要】 荧光金属纳米簇是一类介于原子与纳米颗粒之间的材料,由几十至一百个原子组成的纳米簇,其尺寸接近电子的费米波长,因此具有电子的离散能及和由尺寸决定的荧光性质。由于荧光金属纳米簇具有超小尺寸、良好的生物相容性和荧光稳定性,成为一类理想的荧光标记物及生物检测探针。本文重点开展基于蛋白包覆、稳定与还原的金纳米簇制备及其蛋白酶检测应用,主要研究结论如下：(1)pH调控金纳米簇形成：重点考察了pH、反应时间等对纳米簇尺寸与荧光性质的影响规律,并分析它们对已形成的纳米簇的调控行为。实验结果表明,将体系pH升至11以后,荧光纳米簇尺寸逐渐增大,荧光发射光谱也由蓝色逐渐向红色转移。对于红色荧光纳米簇,当体系的pH调节成酸性后,其荧光强度逐渐下降。该结果有助于理解金纳米簇的结构和形成机理,也可应用于金纳米簇的调控制备中。(2)基于酸性荧光金纳米簇的胃蛋白酶检测：胃蛋白酶是生物体内可在酸性条件下具有消化活性的酶,已广泛应用于食品工业中制备生物活性肽。本文设计了一种简单的、基于酶剪切活性的胃蛋白酶检测方法,通过胃蛋白酶的剪切,使溶菌酶稳定的金纳米簇荧光降低。在酸性条件下检测,不仅保证了金纳米簇的荧光强度,还保证了胃蛋白酶额最大活性。实验证明,胃蛋白酶检测的线性区间为1-100μg/mL,检测限达到0.6ng/mL。(3)基于碱性荧光金纳米簇的碱性蛋白酶检测：碱性蛋白酶是一类在生化、食品、制药、洗涤业等行业中应用很广的蛋白酶。本文设计开发了一种在碱性条件下形成并稳定的荧光金纳米簇,并利用它实现对碱性蛋白酶的灵敏检测。其检测原理是碱性蛋白酶将稳定和包覆金纳米簇的溶菌酶进行水解,造成金纳米簇的荧光下降,再关联荧光下降程度与碱性蛋白酶浓度的关系,便可实现对碱性蛋白酶的检测。研究了金纳米簇与碱性蛋白酶溶液体积比,反应温度和反应时间对检测灵敏性的影响。结果表明：在金纳米簇与碱性蛋白酶溶液体积比为1：9,反应温度为40℃,反应时间为3小时的条件下,检测效果最好。该检测方法的线性范围可达2-2000μg/mL(即酶活检测范围为4×10-5～0.04unit/mL),检测限为0.1μg/mL(酶活检测限为2×10-6unit/mL)(S/N=3),且检测的专一性较好,有望应用于实际检测。更多还原

【Abstract】 Recent advances in nanotechnology have given rise to a new class of fluorescentlabels, i.e. fluorescent metal nanoclusters. These nanoclusters are of significantinterest because they can provide the missing link between atomic and nanoparticlebehavior in metals. Composed of a few to a hundred atoms, their sizes are comparableto the Fermi wavelength of electrons, resulting in molecule-like properties includingdiscrete electronic states and size-dependent fluorescence. Ultrasmall size, goodbiocompatibility and photostability make them ideal fluorescent lables andbiodetection probes. This thesis focused on the preparation and application of goldnanoclusters based on the coating, stability and reduction by lysozyme. The mainconclusions in this thesis are as follows.(1) Firstly, we investigated the effects of pH, reaction time on the size andfluorescence properties of gold nanoclusters, and also analyzed the influence of theseconditions on the regulation of nanoclusters during their formation. We studied thechanges in lysozyme stabilized AuNCs followed by pH adjustment. The result showsthat the bule-emitting AuNCs will gradually turn to red-emitting AuNCs when pHgoes up. When pH goes down, the fluorescence intensity of red-emitting AuNCsdrops quickly. The results of this research not only helps understanding of thesynthesis mechanism of gold nanoclusters, but also helps to control the size ofAuNCs.(2) Pepsin is an active digestive enzyme present in the acidic environment ofanimal stomachs, and has been widely used to prepare bioactive peptides in the foodindustry. In this work, a simple fluorescence sensor for scissor-based detection ofpepsin activity was developed by using lysozyme-stabilized gold nanoclusters(AuNCs@Lyz) in aqueous media. Under acidic conditions (pH3.0), enzymaticdigestion of AuNCs@Lyz with pepsin results in a signifcant decrease of fuorescenceintensity. Notably, its acidic environment not only helps maintain the maximumfluorescence of gold nanoclusters, but also ensures the highest pepsin enzymaticactivity. In addition to offering high selectivity because of the unique proteolyticaction of pepsin under acidic conditions, this facile method provides high sensitivity. With the sensing system, the linear range for pepsin detection is found to be1μg/mLto100μg/mL, with a detection limit of0.6ng/mL at a signal-to-noise ratio of3.(3) Alkaline is a kind of enzyme widely used in various fields includingbiochemical engineering, food production, drug and washing industry. In this paper, aquick and sensitive assay for alkaline protease has been established based on thehydrolysis activity of protease to the lysozyme which is used to coat and stabilize thegold nanoclusters (Au NCs). Through the experiments, we obtained the optimumreaction condition. The optimum volume ratio of Au NCs and alkaline proteasesolution is1:9, while the optimum reaction temperature and reaction time are40oCand3h, respectively. Under these conditions, the linear range of this method is from2μg/mL to2000μg/mL (4×10-5~0.04unit/mL) with a limit of detection of0.1μg/mL(2×10-6unit/mL) and good selectivity. Therefore, this fluorescence detection shows itspotential in the detection of real samples.更多还原