基于表面增强拉曼散射光谱的氨基酸和多肽检测方法研究

发布时间：2018-01-03 20:10

本文关键词：基于表面增强拉曼散射光谱的氨基酸和多肽检测方法研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：本论文针对目前缺乏快速灵敏的氨基酸和多肽检测手段等问题,建立了几种基于表面增强拉曼散射(SERS)技术的分析方法。将SERS技术与化学衍生法结合,利用衍生反应物或生成物的SERS信号间接分析氨基酸和多肽;将SERS基底改进成银@二氧化钛(Ag@Ti O2)纳米材料,通过磷酸基团对Ti O2的亲和作用以及银的拉曼增强效应不仅对磷酸化氨基酸进行了富集而且实现了SERS检测。本论文的创新点主要包括:(1)将偶氮偶联反应和SERS技术结合,以对巯基苯胺(PATP)分子作为重氮组分,组氨酸(His)和酪氨酸(Tyr)作为偶联组分进行偶氮偶联反应,生成最大吸收在500 nm左右的偶氮生色分子。以表面增强共振拉曼散射(SERRS)检测代替吸光度检测,根据其SERRS信号进行His和Tyr的定量分析、混合物鉴别和实际样品分析。优点是无需复杂的样品前处理、实验过程快速简便、选择性好和灵敏度高。(2)利用上述基于SERRS技术和偶氮偶联反应的方法,以磺胺酸分子作为重氮组分,分别与His、Tyr和促甲状腺激素释放激素(TRH)偶联生成具有SERRS活性的偶氮分子,进行定量分析。运用该方法实现了正常人和甲亢患者血清中TRH的检测,该方法具有简单快速、超灵敏性、选择性以及实用性和可靠性的优势。(3)基于经典的氨基酸总量检测法-茚三酮比色法,将过量茚三酮试剂与混合氨基酸样品作用后的反应液负载在银纳米粒子(Ag NPs)自组装膜上进行SERS测试,过量茚三酮的SERS强度变化反映氨基酸总量变化。该化学衍生方法成熟可靠,操作简便,SERS技术的应用大大提高了其检测灵敏度。(4)基于半导体纳米材料Ti O2对磷酸基团的特异捕获能力,制备了新型双功能Ag@Ti O2核壳SERS活性基底,该纳米材料既能富集磷酸化氨基酸或多肽又能够增强待测分子的拉曼信号。该方法为实际样品中磷酸化多肽的检测及探究蛋白质磷酸化作用提供了新的思路和手段。
[Abstract]:This paper aims at the lack of rapid and sensitive detection methods for amino acids and peptides. Several analytical methods based on surface-enhanced Raman scattering (SERS) technique were established. The SERS technique was combined with chemical derivation method. Amino acids and peptides were analyzed indirectly by SERS signal of derivative reactants or products. The SERS substrate was modified to form silver @ titanium dioxide (AgTiO _ 2) nanomaterials. The affinity of phosphate group to TIO 2 and Raman enhancement of silver not only enriched phosphorylated amino acids but also achieved SERS detection. The azo coupling reaction was combined with SERS technique. The azo coupling reaction was carried out with p-mercaptoaniline PATP as diazo component, histidine Hisand tyrosine tyrosine tyrosine Tyr as coupling component. Azo chromogenic molecules with maximum absorption of about 500 nm were formed. Surface enhanced Resonance Raman scattering (SERRS) detection was used instead of absorbance detection. According to its SERRS signal, the quantitative analysis of His and Tyr, the identification of mixture and the analysis of actual samples are carried out. The advantages are that there is no need for complex sample pretreatment, and the experimental process is fast and simple. Based on SERRS technique and azo coupling reaction, sulfamic acid was used as diazo component, and His was used as a diazo component, respectively. Tyr was coupled with thyrotropin releasing hormone (TSH) to form azo molecules with SERRS activity. Quantitative analysis. Using this method to achieve the normal and hyperthyroidism patients serum TRH detection, this method is simple, rapid, hypersensitivity. Selectivity and practicability and reliability are based on the classical total amino acid determination method-ninhydrin colorimetry. The reaction solution loaded with excess ninhydrin reagent and mixed amino acid sample was tested by SERS on silver nanoparticles Ag NPs self-assembled film. The SERS intensity change of excess ninhydrin reflects the change of total amino acid content. The chemical derivation method is mature and reliable and easy to operate. The application of SERS technology greatly improved its detection sensitivity. 4) based on the semiconductor nano-material TIO _ 2, the specific capture ability of phosphate group was greatly improved. A novel bifunctional Ag@Ti O2 core-shell SERS active substrate was prepared. The nanomaterials can not only enrich phosphorylated amino acids or peptides, but also enhance the Raman signal of the molecules to be tested. This method provides a new idea and hand for the detection of phosphorylated peptides in actual samples and the exploration of protein phosphorylation. Paragraph
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.37

【相似文献】