A Thermo-responsive Aqueous Two-phase Flotation System Containing Aptamer Motifs For Selective Separation And Enrichment Of Hg²⁺ Ions

Mercury is toxic substances that pollute the environment and are enriched in human beings,causing severe harm to human health.Therefore,the separation and enrichment of mercury in the environment is of great significance to the ecosystems and human health.The aqueous two-phase extraction technology which has been successfully applied to the separation and enrichment of metal ions is used for the separation and enrichment of mercury with complex matrix and very low content in environmental samples.The aqueous two-phase extraction technology still has low enrichment ratio,low efficiency of selective separation and so on.Therefore,it is a trend to seek a high enrichment multiple and selectivity for separation and enrichment method of mercury ions.This thesis aims to construct a thermo-responsive aqueous two-phase flotation system containing aptamer motifs for selective separation and enrichment of Hg²⁺ions.The detailed research contents are as follows:1.Construction of highly recyclable thermo-responsive diblock polymer and organic salts aqueous two-phase system and its application for separation and purification of bromelainIn view of the problems of high application cost and environmental pollution caused by the difficulty in recycling of phase-forming polymers in the current aqueous two-phase system,the reversible addition fragmentation chain transfer?RAFT?polymerization has synthesized a thermo-responsive diblock polymer PEG₁₁₃-b-PNIPAM₁₄₉ of controllable molecular weight and low low-critical solution temperature?LCST approx.28.5°C?and a combination of PEG₁₁₃-b-PNIPAM₁₄₉ with organic salts developed a aqueous two-phase system.The binodal data of PEG₁₁₃-b-PNIPAM₁₄₉ and organic salts at different temperatures were evaluated experimentally.The best fitting equation for the binodal data suitable for the thermo-responsive diblock polymer and organic salts aqueous two-phase system was obtained.The liquid-liquid phase equilibrium data of PEG₁₁₃-b-PNIPAM₁₄₉49 and organic salts at different temperatures were obtained by a combination of experimental determination and calculation.By plotting the phase diagrams of PEG₁₁₃-b-PNIPAM₁₄₉ and organic salts aqueous two-phase system,the effects of temperature and phase-forming salt on the phase diagrams of the system were studied in detail,and the capability of phase separation of different salts was evaluated.The aqueous two-phase system of PEG₁₁₃-b-PNIPAM₁₄₉ and organic salts were used for the separation and purification of bromelain from pineapple juice samples.The extraction rate and enzyme activity of one-time extraction of bromelain were 94.87%and 77.06%,which was better than that of EOPOEO-based aqueous two-phase system.Importantly,PEG₁₁₃-b-PNIPAM₁₄₉ still showed 92.16%recovery in five cycles.The proposed aqueous two-phase system realizes efficient recovery of the thermo-responsive diblock polymer and separation from the target,which reduces the cost and affords a new thought for the industrial application of aqueous two-phase system.2.Construction and application of mercury ions and cysteine recognition component based on thermo-responsive hydrophilic diblock polymerTo further improve the sensitivity and cycle utilization of the fluorescence sensor,a thermo-responsive fluorescence sensor PEG₁₁₃-b-P?NIPAM-co-HBMA?₉₀-P1 based the PNIPAM block-labeled nucleic acid aptamer P1 of the thermo-responsive diblock polymer PEG₁₁₃-b-P?NIPAM-co-HBMA?_m was developed for sequential detection of Hg²⁺ions and Cys.The thermo-responsive fluorescent sensor first exhibits a fluorescence quenching effect on Hg²⁺ions,and forms a thermo-responsive fluorescence sensor of"T-Hg²⁺-T"structure.Then,Cys is added to the mixed system to form a[Cys-Hg²⁺]complex to release a thermo-responsive fluorescent sensor to restore fluorescence.Based on fluorescence spectroscopy experiments,the thermo-responsive fluorescent sensor exhibits high selectivity,high sensitivity,and anti-interference properties.Based on the thermo-responsive properties of the fluorescent sensor,increasing the temperature causes the fluorescent response element P1 to accumulate in the micelle nucleus,further reducing the detection limit of Hg²⁺ions and Cys.Importantly,the thermo-responsive fluorescent sensor still showed excellent detection results after five cycles.The construction of the thermo-responsive fluorescent sensor lays a foundation for constructing a new type of fluorescent sensor that integrates high sensitivity and can be recycled.3.Construction of aqueous two-phase flotation system based on aptamer-based thermo-responsive polymer and its application for separation and enrichment of mercury ionsIn order to solve the problems of low enrichment ratio and low selectivity of aqueous two-phase system,a novel aqueous two-phase flotation system was constructed,and the thermo-responsive block hydrophilic polymer embedding of labeled aptamer motifs in the previous experiment was introduced.The segment polymer PEG₁₁₃-b-P?NIPAM-co-HBMA?_m-P1?TBC-P1?acts as a collecting agent to achieve high-efficiency,selective separation and enrichment of mercury ions.Firstly,the effects of five single factors?concentration of K₂HPO₄,flotation time,concentration of collector,concentration of PEG-10000,and flotation flow rate?on flotation efficiency and partition coefficient of Hg²⁺ions were investigated.Secondly,a five-factor and three-level response surface experimental model was established to optimize the factors affecting flotation efficiency and partition coefficient of Hg²⁺ions for selecting the optimal flotation conditions.At the same time,the recovery rate of the collecting agent TBC-P1 was 78%after five cycles.The establishment of a new aqueous two-phase flotation system has achieved high efficiency,selective separation and enrichment of mercury ions and recycling of collecting agent,and has shown important application value in the field of separation and concentration of environmental contaminants.