【作者】 陈静；

【导师】 王玉枝；

【作者基本信息】 湖南大学 ， 分析化学， 2021， 博士

【摘要】 低共熔溶剂（Deep eutectic solvent,DES）因具有合成简单、毒性低、绿色环保等特性,在分离分析领域受到广泛推崇,被盛赞为当代新型“绿色溶剂”。DES主要由氢键供体和氢键受体按照一定摩尔比组成,可根据被分析物的理化性质、检测技术和分离方法的不同,设计并合成能解决目标需求的功能型DES。将含有多种官能团的DES对磁性纳米颗粒进行功能化修饰,并结合磁固相萃取（Magnetic solid-phase extraction,MSPE）技术用于对混合体系中目标分析物的分离,具有操作简单、吸附效率高等优点。具有类表面活性剂性质的DES,与双水相体系（Aqueous two-phase system,ATPS）萃取技术相结合,可以构建新型的双胶束两相分离体系。DES作为荧光增敏剂,可用于与金属纳米簇构建新型的荧光传感检测体系。将DES修饰在类酶活性的纳米材料上可构建基于DES调控的纳米酶比色探针。本论文设计合成了多种具有不同功能化和特殊属性的新型DES,并与MSPE、ATPS、荧光传感和比色传感测定技术相结合,构建了对复杂样品中目标分析物的分离分析新体系和新方法。主要研究内容如下:（1）基于三元巯基型低共熔溶剂修饰的磁性氧化石墨烯固相萃取分离水中汞离子设计合成了巯基功能化的三元低共熔溶剂（THS-DES）,并负载在磁性氧化石墨烯上,制备出基于巯基功能化的纳米复合材料（THS-DES@M-GO）。结合MSPE技术,将THS-DES@M-GO用于水溶液中Hg²⁺的分离分析。在最佳条件下,THS-DES@M-GO对溶液中Hg²⁺的R%高达99.91%。方法学考察显示本研究所建立的固相萃取分离方法具有良好的精密度、重复性和稳定性。热力学实验研究表明,THS-DES@M-GO对Hg²⁺的吸附模式与Langmuir模型吻合良好,最大吸附容量为215.1mg·g^-1。重复利用研究表明,94.94%的Hg²⁺能从吸附剂上洗脱下来,经过七次重复过程后,依旧保持对Hg²⁺有90.23%的去除率。所拟方法可用于去除实际含汞污水。（2）基于三元多官能团型低共熔溶剂修饰的磁性多壁碳纳米管固相萃取胰蛋白酶设计合成了三元多官能团型低共熔溶剂（TMFG-DES）,并将其修饰在磁性多壁碳纳米管上,制备出磁性纳米复合材料（Mn Fe₂O₄-MWCNTs@DES₁）。结合MSPE技术,将其用于胰蛋白酶（Try）的萃取分离分析。采用响应面优化法（RSM）及Box-Behnken Design模型对萃取吸附条件进行优化。在最佳条件下,Mn Fe₂O₄-MWCNTs@DES₁对Try的最大吸附容量为1020.1 mg·g^-1。吸附热力学和动力学研究显示,该吸附模型与Langmuir等温吸附模型和拟二阶动力学模型吻合。机理研究表明,静电相互作用、氢键作用和螯合作用是吸附的主要驱动力。对洗脱后的Try活性进行分析,结果表明,Try的酶催化活性、构象和二级结构都未出现显著改变。Mn Fe₂O₄-MWCNTs@DES₁可以从牛胰粗提物中提取分离Try。所拟方法在生物大分子分离纯化中具有较好的应用前景。（3）基于低共熔溶剂的双胶束两相体系用于萃取分离染料合成了具有类表面活性剂性能的两种类型DESs:DL/PEG400和[N₈₈₈₁]Cl/PEG400。结合ATPS技术,设计一种基于亲/疏水性DESs的双胶束两相体系（DES/DES-AMTPS）。选择三种典型的染料作为目标分析物以评估该体系在分离分析领域的应用。采用单因素和RSM法对萃取条件进行优化,在最佳条件下,超过95.00%的日落黄和99.75%的苏丹红（Ⅲ）分布在富含胶束相中,而其余的亚甲基蓝则分布在含胶束较少相。机理研究表明,物质之间的亲疏水性和静电相互作用是促使染料在DES/DES-AMTPS体系中分离的主要驱动力。本研究还实现了染料在两相间的反萃取和[N₈₈₈₁]Cl/PEG400的回收及重复使用。（4）基于低共熔溶剂荧光增强效应构建铜纳米簇荧光法测定铁离子设计合成了由酒石酸稳定的铜纳米团簇（Aci-Cu NCs）,基于DES的荧光增敏功能,研究得出Aci-Cu NCs在DES介质的荧光强度得到显著增强。实验验证了Aci-Cu NCs在DES介质中以胶束-簇集体形式存在。将基于DES增敏的Aci-Cu NCs@DES荧光检测体系用于测定溶液中Fe³⁺,在最佳条件下,定量检测浓度区间为1-20μM,最低检出限（LOD）为0.0374μM。选择性实验和实际应用研究结果显示,该体系对实际样品中Fe³⁺具有特异性识别能力。本研究结果为新型荧光检测体系的建立提供了新思路。（5）基于低共熔溶剂修饰的二氧化锰纳米片用于DNA的比色测定将DES修饰在二氧化锰纳米片上制备出具有类氧化酶活性的新型纳米材料（DES/Mn O₂）。该材料能够将无色的3,3’,5,5’-四甲基联苯胺（TMB）氧化成蓝色产物（ox TMB）,并在652 nm处有吸收峰。DNA可通过氢键和静电相互作用吸附在DES/Mn O₂表面,从而抑制DES/Mn O₂对TMB的氧化活性。根据上述原理,本研究建立了一种基于DES/Mn O₂-TMB比色传感体系并用于对DNA进行定量测定。在最佳条件下,DES/Mn O₂-TMB体系对DNA的定量检测浓度区间为10-100μg·m L^-1,LOD为0.37μg·m L^-1。选择性实验结果表明,该体系对DNA具有优良的选择性。实际样本分析结果显示,该方法可用于牛全血DNA的检测。本研究所建立的基于DES/Mn O₂-TMB体系在生物技术领域具有广泛的应用前景。更多还原

【Abstract】 Due to characteristics of simple synthesis,low toxicity and eco-friendlily,deep eutectic solvent（DES）is known as the new"green solvent"in the new century and has been widely used in the field of separation analysis.DES is mainly composed of hydrogen bond donor and hydrogen bond acceptor in a certain molar ratio.Therefore,functional DESs can be designed and synthesized according to the physical and chemical properties of the analyte,the difference in detection technology and separation method.Designing a new DES containing multiple functional groups to modify nano-magnetic particles and combining magnetic solid-phase extraction（MSPE）technology to separate target analytes in complex samples.It has the advantages of simple operation and high adsorption efficiency.DES with surfactant like properties can be used to build a new aqueous micellar two-phase system（AMTPS）with aqueous two-phase system（ATPS）extraction technology.Moreover,as a fluorescence sensitizer,DES can be applied to establish a new fluorescence sensing detection system with metal nanoclusters.At the same time,enzyme like nanomaterials modified with DES can be devoted to construct enzyme colorimetric probes for colorimetric sensing and detection.In this study,a series of new types of DESs with various functions and special properties were designed and synthesized.Combined with MSPE,ATPE,fluorescence and colorimetric sensing technology,several new systems and methods for the separation and analysis of target analytes in complex samples were constructed.The main research contents are as follows:（1）Magnetic solid-phase extraction for the removal of mercury from water with ternary hydrosulphonyl based deep eutectic solvent modified magnetic graphene oxideA ternary hydrosulphonyl based DES（THS-DES）was prepared and decorated on magnetic graphene oxide to synthesize the magnetic adsorbents（THS-DES@M-GO）.Combined with MSPE,the THS-DES@M-GO was used for the removal of mercury(Hg²⁺)from water.Under the optimal adsorption conditions,the R%of THS-DES@M-GO to Hg²⁺could achieve 99.91%.Methodological investigations showed that the method established in this research had excellent precision,repeatability and stability.Thermodynamic experimental studies revealed that the adsorption mode of Hg²⁺by THS-DES@M-GO fitted well with the Langmuir model,and the maximum adsorption capacity was 215.1 mg·g^-1.Reuse research showed that 94.94%of Hg²⁺could be eluted from the adsorbent.And after 7 repeated,THS-DES@M-GO still maintained a high removal rate of 90.23%for Hg²⁺.The MSPE technology established in this research could be used to remove actual mercury-containing wastewater.（2）Ternary multiple functional groups based deep eutectic solvent modified magnetic Mn Fe₂O₄-multiwalled carbon nanotubes for solid-phase adsorption of trypsinA ternary multiple functional groups DES（TMFG-DES）was prepared and decorated on magnetic multiwalled carbon nanotubes to prepare a multifunctional magnetic composite nano-materials（Mn Fe₂O₄-MWCNTs@DES₁）.Combined with MSPE,Mn Fe₂O₄-MWCNTs@DES₁ were utilized to the adsorb trypsin（Try）.The response surface methodology（RSM）experiments were prepared under Box-Behnken Design,which were applied to optimize the adsorption conditions for Try.RSM result showed that the practical maximum adsorption capacity for Try was 1020.1 mg·g^-1.The kinetics and adsorption isotherms results showed that the second-order kinetic model and Langmuir isotherm model were perfectly matched with the adsorption process.Mechanism exploration proved that electrostatic interaction,hydrogen bonding interaction and chelation interaction were the dominant force for Mn Fe₂O₄-MWCNTs@DES₁ to high-performance adsorption of Try.The activity of Try after elution had been analyzed,and the result illustrated that the enzyme activity,conformation and secondary structure of Try did not change significantly.The proposed method had effective practical applicability to isolation Try from crude bovine pancreas.As a result,the proposed method provided a potential value in the adsorption of biomacromolecule.（3）Aqueous micellar two-phase system composed by deep eutectic solvents for extraction of pigmentsTwo types of DESs with surfactant-like properties were synthesized:DL/PEG400and[N₈₈₈₁]Cl/PEG400.Combined with the ATPS technology,a novel AMTPS based on a hydrophilic/hydrophobic DESs was designed（DES/DES-AMTPS）.Three typical pigments were selected as target analytes to evaluate the application of the DES/DES-AMTPS.Through single factor and RSM,the extraction conditions were systematically optimized.Under the optimal conditions,it was found that almost 95.00%sunset yellow and 99.75%sudan（III）distributed in the micellar-rich phase,whereas nearly all of methylene blue stayed in the micellar-poor phase.Mechanism study proved that the formation of micelles between pigments and DESs by hydrophobic and electrostatic interaction were the dominant force in the separation.In addition,this research not only realized the transfer of the pigments between the two phases,but also recovered and reused of[N₈₈₈₁]Cl/PEG400 in the regeneration studies.（4）Fluorescence determination of iron with copper nanoclusters based on fluorescence enhancement effect of deep eutectic solventA kind of copper nanoclusters（Aci-Cu NCs）based on tartaric acid as protective agent was synthesized.It was found that the surfactant-like DES could form micellar co-aggregation with Aci-Cu NCs,resulting in an increase in fluorescence intensive of Aci-Cu NCs.The fluorescence sensing assay（Aci-Cu NCs@DES）based on DES sensitization was applied for the determination of Fe³⁺.Under the optimum conditions,the assay worked in the Fe³⁺concentration ranges from 1-20μM and had a detection limit（LOD）of 0.0374μM.Selective experiments and practical application research results showed that the Aci-Cu NCs@DES system had specific recognition of Fe³⁺in real water samples.The fluorescence assays offer a new idea for the establishment of a novel fluorescence detection system.（5）A composite prepared from Mn O₂ nanosheets and deep eutectic solvent as an oxidase mimic for the colorimetric determination of DNAA new type of nanomaterial（DES/Mn O₂）with oxidase-like activity was fabricated by modifying the DES on Mn O₂ nanosheets.Owing to the oxidase-like activity of DES/Mn O₂,it could oxidize the colorless 3,3’,5,5’-tetramethylbenzidine（TMB）into a blue product（ox TMB）with an absorption spectrum at 652 nm.DNA was easily adsorbed on the surface of the DES/Mn O₂ by hydrogen bonding and electrostatic interactions,which leading to the oxidation activity of DES/Mn O₂ on TMB was inhibited.Based on the above mechanism,a colorimetric sensing assay for the determination of DNA by DES/Mn O₂-TMB system was established.Under the optimum conditions,the DES/Mn O₂-TMB system could quantitatively detect DNA in a concentration range of 10-100μg·m L^-1 with a LOD of 0.37μg·m L^-1.The selectivity study showed the DES/Mn O₂-TMB system to be highly selective for DNA.The real sample analysis result illustrated that the proposed method could be used for the detection of DNA in bovine whole blood.The DES/Mn O₂-TMB system established in this study holds promising potential applications in sensing and biotechnology.更多还原