The Application Of Novel Micro-extraction Methods And Fluorescent Biosensing Technology In The Analysis Of Biological Samples

Biological samples are complex,valuable,scarce and easily inactivated,which poses a great challenge to the analysis of biological samples.Compared with the requirements of conventional samples analysis,the sensitivity and selectivity are essential for analysis of biological samples.In addition,the analytical method should be simple,efficient,less sample consumption and less interference from substrates.The development of Solid phase micro-extraction has met these requirements.The combinations of solid phase micro-extraction?SPME?and chromatography are suitable for analysis of complex biological samples.Biosensors have the advantages of no sample separation,high sensitivity,fast analysis and so on,and are suitable for the analysis of characteristic biomolecules.In view of the two basic biological analysis problems of quantitative analysis of drugs and tumor biomarkers in biological samples,a SPME coupled with high performance liquid chromatography?HPLC?method and a fluorescent biosensor were established in this paper,respectively.The main research contents are as follows:1.A simple,miniaturized micropipette tip-based poly?MAA-co-EGDMA?monolith was prepared by in situ polymerization.Several parameters for monolith preparation were systematically optimized.Methacrylic acid?MAA?and ethylene glycol dimethacrylate?EGDMA?were selected as monomer and cross-linker with the ratio of 1:6.Methanol containing 30%DMSO was selected as porogens.Several factors such as washing solvents,flow rate of sampling,sample volume and pH of sample solutions which affect enrichment efficiency of yohimbine were also evaluated in this work.Under optimized conditions,good enrichment capacity and stability of poly?MAA-co-EGDMA?monolith were obtained.Finally,the prepared poly?MAA-co-EGDMA?monolith combined with HPLC was also used to analyze yohimbine in plasma samples,which exhibit good sensitivity with LOD as low as 0.5ng·mL^-1,excellent reproducibility with RSD?2.5%,and good recoveries in the range of 97.9%-103.8%of yohimbine in human plasma.2.The epithelial cell adhesion molecule?EpCAM?plays a significant role in tumorigenesis and tumor development.EpCAM is considered as one of the tumor signaling molecular for cancer diagnosis,prognosis and therapy.Herein,a enzyme-free and highly sensitive fluorescence biosensor on the basis of combination of aptamer-based EpCAM recognition and toehold-aided DNA recycling amplification strategy was developed for sensitive and specific fluorescence detection of EpCAM.Due to the highly specific binding between EpCAM and corresponding aptamer,the strand a released from the complex of aptamer/strand a in the presence of EpCAM which binded to corresponding aptamer,triggered the toehold-mediated strand displacement process.The amplified fluorescence signal was achieved on the basis of recycling use of strand a for ultrasensitive EpCAM detection with the detection limit low to 0.1 ng·mL^-1,which was comparable or superior to reported immunoassays and biosensor strategies.In addition,high selectivity towards EpCAM was exhibited when other proteins were selected as control proteins.Finally,this strategy was successfully used for ultrasensitive fluorescence detection of EpCAM in human serum samples with the recoveries of 109.2-114.8%.Importantly,the present strategy may be also expanded to detect other targets by using the corresponding aptamers.