Research On Nanomaterial-Enhanced Ultramicro Liquid-Phase Separation For Bioanalysis

Liquid-phase separation methods,including liquid-phase microextraction and liquid chromatography,are rapid,economic and convenient separation methods that commonly used in analytical chemistry field.With the continuous improvement of environmental awareness,the ultramicro liquid-phase separation method has become the mainstream.Ultramicro liquid-phase separation methods comprise single-drop microextraction(direct immersion,headspace)and ultramicro-liquid chromatography.Recent studies have found that various types of nanomaterials exhibit good properties,such as optical properties,catalytic properties,magnetic properties and adsorption properties.These nanomaterials can be used to improve the selectivity and sensitivity of ultramicro liquid chromatography and therefore are widely utilized in the field of analysis.This thesis proposed the liquid-phase separation method combined with magnetic catalytic nanomaterials or bimetallic optical nanomaterials for highly sensitive and selective detection of harmful volatile gases(H2S and formaldehyde)and nucleic acids(DNA and mi RNA).(1)Chapter 3 demonstrated the duplex specific nuclease(DSN)-assisted target amplification strategy combined with HPLC-fluorescence detection platform for selective separation and sensitive quantification of multiple mi RNAs.The proposed assay could alleviate the low sensitivity when conventional HPLC is used for nucleic acid detection.In order to separate the signals of different mi RNAs,DNA probes with different lengths and base sequences were immobilized on magnetic beads.After the DSN-assisted amplification,an effective magnetic separation was performed to minimize the background signal and extend the dynamic range.This assay achieved a detection limit of 0.39 f M for mi RNA-122,0.30 f M for mi RNA-155 and 0.26 f M for mi RNA-21,respectively.The proposed assay was validated by applying to detect mi RNA-122,mi RNA-155 and mi RNA-21 in serum samples from healthy persons and cervical cancer patients,which results were comparable with those of quantitative real-time polymerase chain reaction(q RT-PCR).This work coupled magnetic nanomaterial with long and short probe-based recycling amplification strategy to realize a highly sensitive assay for multiple mi RNAs on a conventional HPLC-fluorescence platform for the first time(without need of an expensive MS/MS system).(2)In Chapter 4,magnetic three-phase single-drop microextraction(MTP-SDME)technique was proposed to achieve the sensitive quantification of nucleic acids,includingmi RNA(mi RNAs-122)and DNA fragment of Hepatitis B virus(HBV-T).The method utilized a target recycling amplification strategy to constitute a magnetic hyperbranched DNA/Fe3O4 network,which displayed peroxidase-like catalytic activity towards the colorimetric TMB-oxidation reaction in the extractant droplet after magnetic separation,thereby providing a highly sensitive signal for nucleic acid quantification.This method achieved a detection limit of 0.147 a M and a linear calibration range between 0.5 a M and 1p M for mi RNA-122,as well as a detection limit of 0.34 a M and a linear calibration range between 1 a M and 1 p M for HBV-T DNA.Furthermore,the practicability of this method was validated by determining mi RNA-122 and HBV in serum sample of liver cancer patients,which results are comparable with those of q RT-PCR.In this work,the MTP-SDME technique was designed and applied for the first time.By integrating with hyperbranched catalytic networks of magnetic nanomaterials with minute quantity of solvent(6 μL),the signal intensities of target analytes were rapidly(6 seconds)enhanced,which realized further signal amplification aside from the traditional nucleic acid amplification methodology.(3)In Chapter 5,low cost,highly sensitive and efficient detection of hydrogen sulfide(H2S)was realized by the utility of silver-gold core-shell nanoprism(Ag@Au-np)combined with headspace-single drop microextraction(HS-SDME).Ag@Au-np was used as the extractant of HS-SDME and etched by H2 S,resulting in the change of the ultraviolet-visible(UV-vis)signal and thereby quantified the concentration of H2 S.After SDME,the rapid analysis and detection of H2 S were realized by UV-vis spectrophotometer and smartphone nano-colorimetry(SNC)respectively.The coating of the gold layer not only ensured the high stability of the nanomaterials,but also improved the selectivity towards H2 S.The HS-SDME method is simple to operate and requires only a droplet of solvent to complete the analysis.This HS-SDME-SNC approach exhibited a calibration linear range between 0.1and 100 μM,and a detection limit of 65 n M.The practicability of the proposed HS-SDME-SNC was successfully validated by determining H2 S in biosamples(egg and milk).This work realized the combination of nanomaterials,HS-SDME and SNC for the first time,which layed a foundation for sensitive and portable detection of volatile gas in the future.(4)In Chapter 6,low cost,highly sensitive and efficient detection of formaldehyde(FA)was realized by the utility of Au-np/Tollens’ reagent(Au-np/TR)combined with HS-SDME.When FA was extracted by the extractant and reduced by TR,Au@Ag-np will formed,resulting in the change of the UV-vis signal and thereby quantified the concentration of FA.The method has a great potential of naked eye detection.In addition,matrix interference could be effectively avoided by the headspace mode of SDME.This HS-SDME-SNC approach exhibited a calibration linear range between 0.1 and 100 μM,and a detection limit of 30 n M.The practicability of Au@Ag-np/TR-HS-SDME-SNC was successfully validated by determining FA in biosamples(octopus and chicken flesh).This work is the derivation of the application of methodology in Chapter 5,while makes further innovation in the construction of materials as well as the mechanism of signal generation.