Study On The Application Of Novel Materials For The Detection Of Phthalates In Beverages And Vitamin D And E In Serum Samples

Backgrounds:With the rapid development of material chemistry,nanoscience and green chemistry,various novel materials are emerging and applied in the field of analytical chemistry.For establishing analytical methods for trace analytes in complex matrix samples,it is still a challenging work to separate,enrich and purify the analytes from complex samples more effectively.Based on various interactions between extractants and targets,the specificity and selectivity of the sample preparation method can be improved by applying materials with suitable physiochemical properties or specific recognition abilities to the targets.Green solvents can reduce the usage of toxic organic reagents and protect the environment and the analysts.Nanomaterials and nano composite materials have the large specific surface area and are easy to modify.They can improve extraction efficiencies and enrichment factors of the pretreatment methods.At the same time,these novel materials have been also applied in biosensors for improving their performances.In recent years,developing new sample preparation techniques and sensors based on novel materials has become one of the research hotspots in the field of analytical chemistry.However,these researches focus on the establishment of analytical methods,some of them are lack of the applications in real samples.The next step is to do more in-depth application research for the determination of different targets from complex matrixes,especially in the field of health inspection and quarantine.Phthalates are one group of the most widely used plasticizers in the world.However,they are not chemically bonded with plastics and are easy to be released into the environment.Phthalate’s pollution can affect human hormone secretion,damage reproductive and respiratory systems,and increase the risks of cardiovascular diseases and cancer.Vitamin E is a group of fat-soluble vitamins for maintaining normal human physiological functions and metabolism.It consists of eight isomers with different physiological functions and biological activities.Vitamin E deficiency can cause nervous and immune system diseases,and has a certain correlation with cancer,cardiovascular diseases,diabetes,and other chronic diseases.Vitamin D participates in calcium and phosphorus absorption and multiple metabolic pathways.The lack of vitamin D can lead to rickets and other bone diseases.It is also associated with a variety of chronic diseases.Vitamin D deficiency is now still common,especially in developing countries,as well as some specific populations like pregnant women,adolescents,and the elder people.To improve analytical methods for food phthalates detection and the diagnosis of vitamin E and D nutritional status,three novel materials,including magnetic nanoparticles,ionic liquids and aptamers,were chosen for the different targets from different matrices.Magnetic solid phase extraction method was established for the extraction of 11 phthalates from beverages.Ionic liquids based dispersive liquidliquid microextraction method was developed for the extraction of eight vitamin E isomers from human serum.Two aptamer based electrochemical sensing platforms were developed for the sensitive and specific detection of 25-hydroxyvitamin D3 in human serum.The applications of novel materials in sample preparation and biosensors for food and biological sample analysis were investigated in this study,which can provide technical support for food safety monitoring,clinical diagnostics,and updating the public health guidelines and standard analytical methods of food and biological samples.Methods:In this study,different green solvents and novel recognizing materials were chosen for the detection of different compounds from various matrices.Magnetic solid phase extraction-high performance liquid chromatography method,dispersive liquid-liquid microextraction-high performance liquid chromatography method and aptamer based electrochemical sensing platforms were established for the rapid and sensitive detection of phthalates,vitamin E isomers and 25-hydroxyvitamin D3,respectively.1.Three magnetic nanoparticles including magnetic Fe3O4,magnetic multiwalled carbon nanotubes,magnetic graphene oxide were synthesized by solvothermal method.The phthalates were adsorbed by magnetic nanoparticles from plastic bottled beverages by vortex assisting and then eluted with n-hexane-acetone(1:1,v/v)for chromatographic analysis.The separation was accomplished on a gradient program.The column temperature was 35℃,and the detection wavelength was set at 225 nm.The parameters affecting the chromatographic separation and magnetic solid phase extraction including the type and volume of adsorbents,elution solvent,and ion strength were optimized.The method performances were also investigated in this study,such as the detection limit,accuracy,and precision.The developed method was successfully applied for the detection of phthalates in four kinds of plastic bottled beverages.2.The ionic extractant was added into the diluted serum sample and vortexed for extracting targets from the sample.After centrifugation,the ionic phase was collected for chromatographic analysis.The mobile phase was methanol-acetonitrile(80:20,v/v)and the flow rate was 0.7 mL/min.The column temperature was 30℃.The parameters affecting the liquid-liquid microextraction were optimized,including the type and volume of the ionic liquids,dispersive solvent,vortex time,and salt addition.The method performances were also investigated in this study.The developed method was successfully applied for the simultaneous determination of eight vitamin E isomers in human serum.3.A novel electrochemical aptasensor was established for the detection of 25hydroxyvitamin D3 in human serum.The oligonucleotide aptamers were immobilized on bare gold surfaces using a disulfide linker.In presence of 25-hydroxyvitamin D3,the aptamer can specifically recognize the target which can cause the secondary structure and electrochemical signal changes.Circular dichroism and ultravioletvisible spectroscopy analysis were carried out to study the interactions between aptamers(VDBA14 and VDBA28)and 25-hydroxyvitamin D3.The binding abilities of the two ssDNA 25-hydroxyvitamin D3 aptamers on gold surface were confirmed and compared by electrochemical methods including cyclic voltammetry,square wave voltammetry,and electrochemical impedance spectroscopy.A conventional three-electrode system was employed with gold electrode as working electrode,Ag/AgCl as reference electrode,and platinum wire as counter electrode.The binding buffer containing 2 mmol/L K3[Fe(CN)6]-K4[Fe(CN)6](1:1,v/v)was used as the aqueous electrolyte.The method performance parameters including detection limit,linear range,sensitivity,selectively,reproductivity and stability were evaluated.The developed electrochemical aptasensor was applied to test the human serum sample.4.An electrochemical aptasensor based on catalytic hairpin self-assembly signal amplification strategy was established and applied to the determination of 25hydroxyvitamin D3 in human serum.HP1 contains the functional domain sequence of 25-hydroxyvitamin D3 aptamer.In the presence of 25-hydroxyvitamin D3,the HP1 opens and forms a double DNA strand with another hairpin on the top of DNA tetrahedron which modified on the gold surface,and the HP1 strand is replaced by HP2,then triggering the catalytic hairpin assembly reaction.The interaction between HP1 and 25-hydroxyvitamin D3 was verified by ultraviolet-visible spectroscopy.Cyclic voltammetry,square wave voltammetry and electron impedance spectroscopy were used to verify the electrochemical signal changes caused by catalytic hairpin assembly signal amplification reaction.The sensor performances including detection limit,linear range,sensitivity,selectively,reproductivity and stability were evaluated.Finally,the developed aptasensor was used to detect the real human serum samples.Results:1.As for most phthalates,the magnetic multiwalled carbon nanotubes showed the highest adsorption efficiencies among the three magnetic nanoparticles.The linear ranges of the magnetic multiwalled carbon nanotubes based magnetic solid phase extraction-high performance liquid chromatography method were 2-300 μg/L.The detection limits and quantification limits were in the range of(0.659-5.33)×102 μg/L and(2.20-17.8)× 10-2 μg/L,respectively.The recoveries of the method ranged from 79.8%to 114%,with the relative standard deviations less than 15.8%.The established method has been successfully applied for the determination of 11 phthalates in mineral water,carbonated soft drink,tea beverage,and fruit juice samples.Eleven phthalates were detected in these sample.There is one sample containing all the 11 analytes.The concentration of di(2-ethylhexyl)phthalate in seven samples was higher than the level recommended by the World Health Organization.2.The detection limits and quantification limits of the ionic liquid based dispersive liquid-liquid extraction coupled with high performance chromatography method was 0.857-4.16 ng/mL and 2.86-13.9 ng/mL for eight vitamin E isomers in serum sample.Acceptable recoveries ranging from 74.2%to 103%were achieved,with relative standard deviations less than 13.0%.The proposed method was successfully applied to 44 human serum sample from healthy people.The ranges of the analyte were 5.26×103-3.35×104 ng/mL for a-tocopherol,420-3.46×103 ng/mL for(β+γ)-tocopherol,8.53-459 ng/mL for 8-tocopherol,not detected-73.7 ng/mL for a-tocotrienol,not detected-117 ng/mL for β-tocotrienol,not detected-78.2 ng/mL for γ-tocotrienol,not detected-7.21 ng/mL for 8-tocotrienol.The concentrations of the analytes detected by the proposed method were within the range of reported results in other published methods.3.The interaction between two aptamers(VDBA14 and VDBA28)and the target analyte were studied and compared using spectroscopic and electrochemical methods.It showed that the VDBA14 can strongly recognize 25-hydroxyvitamin D3 while the disulfide linker is attached at 5’terminal of the aptamer.The developed electrochemical aptasensor exhibited a wide linear range of 1-1000 nmol/L and a limit of detection of 0.085 nmol/L.The sensitivity was 0.0366 kQ/(nmol/L· cm2).The established aptasensor had a good recovery of 92.7%-97.8%in human serum,which indicated its favorable potential working in the real samples.4.The developed electrochemical aptasensor exhibited a wide linear range of 0.11000 nmol/L and a limit of detection of 0.026 nmol/L.The sensitivity was 646.9Ω/(nmol/L·cm2).The established aptasensor had a good recovery of 93.0%-102%in human serum sample,and the relative standard deviations were 3.47%-8.69%.It showed that this method had favorable potential working in the real samples.Conclusions:1.The established magnetic solid phase extraction-high performance liquid chromatography method in this study is simple,rapid,green and has a good reproducibility.It can be applied to the simultaneous determination of 11 phthalates in plastic bottled beverages and can provide technical support for monitoring of food safety risks.2.An ionic liquid based disperse liquid-liquid microextraction coupled with high performance liquid chromatography method is proposed for the analysis of eight vitamin E isomers in serum samples.The sample pretreatment process is simple,fast,green,cheap and can be completed within 10 min.No protein precipitation,organic reagents and special equipment is required for this process.The established method has the advantages of good linearity,sensitivity,recoveries,precision,and has been successfully applied to the real human serum sample analysis.The ionic liquid is green,selective and has a broad scope in future applications of biological and food sample analysis as the novel extractants.3.The single-stranded DNA aptamer is used as the biometric element of electrochemical sensor in this study.The established sensor is rapid,easy,sensitive,stable,cost-effective.It can specifically recognize 25-hydroxyvitamin D3 without labels,enzymes,and signal amplification.It is suitable for batch human serum samples with the low detection limit and wide linear range and might become a potential alternative method for clinical diagnosis and biomedical analysis.4.The established electrochemical aptasensor based on DNA tetrahedron and catalytic hairpin assembly signal amplification is rapid,sensitive,cheap and have a wide linear range.It can be used to detect 25-hydroxyvitamin D3 in real human serum sample for clinic diagnosis and public health research.Main innovation points in this study:1.Magnetic carbon nanomaterials were used as adsorbents in food analysis,and a magnetic solid phase extraction-high performance liquid chromatography method was established for the simultaneous determination of 11 phthalates in plastic bottled beverages.The method has a better sensitivity and accuracy than other published methods and has been used for real sample analysis with satisfactory results.2.Taking advantages of environmental friendliness and good selectivity,ionic liquids were used for the biological sample analysis samples.A dispersive liquidliquid microextraction coupled with high performance liquid chromatography method was established for the simultaneous determination of eight vitamin E isomers in human serum.The developed method has been applied in real serum samples with satisfactory results.3.Specific aptamer was applied for the biological sample analysis,and two electrochemical aptamer sensors were constructed for the detection of 25hydroxy vitamin D3 in human serum.4.The catalytic hairpin assembly signal amplification strategy based on DNA tetrahedron was applied for the detection of small molecules for the first time.