摘 要

疾病的早期发现和和及时诊断具有重要意义，而脂肪酶活性与许多疾病有关。成功检测人体脂肪酶活性可以实现对某些疾病的预防和诊断。在本项研究中，初步开发了一种基于荧光内滤效应（IFE）的简便灵敏的荧光检测方法来检测脂肪酶活性。石墨烯量子点（GQD）通过柠檬酸的热解制备并直接用作荧光团。预先生成吐温20作为姜黄素的增溶剂和脂肪酶的底物。在没有脂肪酶的情况下，由于吐温20-姜黄素聚合物胶束的吸收与GQD的激发和发射两者之间的互补重叠，新形成的吐温20-姜黄素聚合物胶束能够作为强力吸收剂以基于IFE猝灭GQD的荧光。在脂肪酶的存在下，脂肪酶可以催化吐温20中酯键的水解裂解并导致猝灭效应的降低。该方法从0.1 mg / mL到1.6 mg / mL（R² = 0.995）呈现良好的线性关系，检测下限为1.07×10^-2 mg / mL。该方法成功应用于商业购买的脂肪酶和血清样品的分析。这种新开发的方法具有许多优点，包括无毒，无GQD修饰和没有吸收剂和荧光剂之间的联系，是简单低廉，敏感和有选择性的。

关键词：脂肪酶 荧光内滤效应 石墨烯量子点 吐温20 姜黄素

Detection of lipase activity based on the fluorescence internal filter effect of Tween 20-curcumin micelles on graphene quantum dots

Abstract

Early detection and diagnosis of diseases is of greatly significant importance. Lipase activity is associated with many diseases. The successful detection of lipase activity in human bodies can achieve the prevention and diagnosis of some diseases. In this study, a facile and sensitive fluorescent assay based on inner filter effect (IFE) was first developed to detect lipase activity. Graphene quantum dots (GQDs) were prepared by pyrolysis of citric acid and directly used as a fluorophore. Tween 20 preformed as the solubilizer for curcumin and the substrate for lipase. In the absence of lipase, the newly formative Tween 20-curcumin polymeric micelles was able to act as the powerful absorber to turn off the fluorescence of GQDs based on IFE owing to the complementary overlap between the absorption of Tween 20-curcumin polymeric micelles and both the excitation and emission of GQDs. In the existence of lipase, lipase could catalyze the hydrolytic cleavage of the ester bond in Tween 20 and resulted in a decrease in quenching effect. The proposed method exhibited a good linear relationship from 0.1 mg/mL to 1.6 mg/mL (R2= 0.995) with a low detection limit of 1.07 × 10−2 mg/mL. This method was also successfully applied to the analysis of the commercially purchased lipases and serum samples. This developed method enjoyed a number of advantages including non-poisonous, no modification of GQDs and without the linking between the receptor and the fluorophore, which is simple, lost-cost, sensitive and selective.

Key Words: lipase; inner filter effect;grapheme quantum dots;Tween 20,;curcumin

目录

摘 要 Ⅰ

Abstract Ⅱ

第一章 绪论 1

1.1荧光内滤效应（IFE）简介 1

1.1.1荧光内滤效应（IFE）的定义 1

1.1.2荧光内滤效应(IFE)的应用 1

1.2石墨烯量子点（GQDs）简介 1

1.2.1石墨烯量子点(GQD)的定义和合成 1

1.2.2石墨烯量子点(GQD)的特点和应用 2

1.3 姜黄素（Cur）简介 2

1.3.1姜黄素（Cur）的简介 3

1.3.2姜黄素（Cur）的荧光吸收 3

1.4脂肪酶简介 4

第二章 实验综述 5

2.1化学原料和材料 5

2.2实验仪器 5

2.3石墨烯量子点（GQD）的合成 5

2.4基于荧光内滤效应（IFE）的脂肪酶荧光检测 6

2.5基于荧光内滤效应（IFE）的实际样本检测 6

第三章 实验讨论 7

3.1石墨烯量子点（GQD）的表征 7

3.2基于IFE的脂肪酶Novozyme435活性测定原理 9

3.3脂肪酶Novozyme435活性的实验条件的优化 12

3.4基于IFE的荧光检测的分析性能 12

3.5方案的实际应用 13

第四章 结论展望 15

参考文献 16

致谢 20

第一章 绪论

1.1荧光内滤效应（IFE）简介

1.1.1荧光内滤效应（IFE）的定义

荧光内滤效应（IFE）指当荧光体的浓度较大或与其他吸收光物质共存时，Y因为荧光体或其他吸光物质对于激发光或发射光一定程度的吸收而导致荧光减弱的现象。荧光内滤效应的主要是由于溶液浓度过高，导致入射光被样品池前部荧光物质大量吸收或溶液中杂质对入射光大量吸收，荧光强度明显下降^[1]。

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