Application Of Capillary Electrophoresis-Laser Induced Fluorescence Detection In Biomarker Analysis

The rapid development of molecular biology has put forward the higher requirements for biological analysis and detection technology.It is urgent to develop efficient and high-resolution separation tools to achieve rapid,sensitive and high-throughput detection of trace biomolecules.Capillary electrophoresis(CE)technology has gradually become a powerful tool for qualitative and quantitative analysis of trace components in complex biological samples due to its advantages of fast analysis,low sample size and strong versatility.However,CE has high requirements for detector’s sensitivity,resolution and response time due to the small inner diameter of the capillary and the small volume of the sample zone produced after separation.Laser-induced fluorescence(LIF)is the most sensitive one among CE detection methods,but its sensitivity and specificity still need to be improved for the detection of ultra-trace biomolecules in biological samples with complex matrixes.The purpose of this paper is to develop a new CE-LIF method with high sensitivity and specificity.Through the introduction of specific recognition reaction,signal amplification strategy,online preconcentration technology and efficient fluorescence collection unit,the rapid,sensitive and accurate analysis of ultra-low content biomarkers in complex biological samples can be realized.The main research work is as follows:1.Ultrasensitive assay of O-Glc NAc transferase based on capillary electrophoresis-laser induced fluorescenceAs one of the most important protein post-translational modifications,O-linked N-acetylglucosamine(O-Glc NAc)glycosylation plays critical regulatory roles in the precise regulation of key biological processes such as gene expression,signal transduction,cell differentiation,etc.O-Glc NAc transferase(OGT)is directly related to biomolecules and various diseases,which is expected to become a new potential therapeutic target for related diseases.In this work,a fast,reliable and sensitive OGT detection method based on capillary electrophoresis and laser induced fluorescence was established.We designed AF488 modified peptide containing serine active group as substrate for OGT catalyzed reaction and non-radioactive UDP-Glc NAc is employed as sugar donor to perform O-Glc NAc glycosylation modification.The enzyme activity of OGT is measured by quantitative determination of glycosylated peptide produced by the reaction.Large volume sample stacking technique for sample injection and a unique fluorescence collection system for LIF detection are adopted to greatly enhance the detection sensitivity,thus a low limit of detection down to 0.23 p M for OGT detection is achieved.This method can be used to study the kinetics of the enzymatic reactions and to screen the inhibitors.The method is successfully applied to detect OGT activity in clinical blood samples with satisfactory accuracy.This research has developed a simple,accurate and sensitive OGT analysis method in biological samples,which has potential application in biological clinical detection.2.Ultrasensitive online analysis of cancer-derived Micro RNAs based on capillary electrophoresis-laser induced fluorescenceMicroRNA(miRNA)is an endogenous single-stranded non-coding RNA with a length about 18-24 nucleotides.The abnormal expression of mi RNA is closely associated with many health-threatening malignant diseases.However,due to the relatively low level of mi RNA expression in cells,the abundance of mi RNA in biological fluid is extremely low,and the length of mi RNA base is relatively short,resulting in high sequence homology among family members.It is still a challenging task to establish a simple,rapid,sensitive and specific mi RNA detection method.In this work,combining the catalytic hairpin self-assembly(CHA)-exoenzyme III(Exo III)isothermal amplification strategy,an online signal amplification mi RNA detection method based on capillary electrophoresis-laser induced fluorescence detection technology is proposed.CHA amplification initiated by the target opens hairpin DNA(H2)on the magnetic beads,while the target mi RNA is released and hybridizes with another probe.Exo III plays a role of releasing single-stranded DNA from the concave end of the 3’-end,constantly cleaving H2 bound to hairpin DNA(H1),thus enabling the release of signal DNA with a fluorophore complementary to H2.The magnetic beads can be fixed inside the capillary by a magnet for in-line signal amplification.The target mi RNA transforms into a large number of released fluorescent signal molecules to achieve significant signal amplification.The released signal reporter is proportional to the amount of the target mi RNA,as a significant measurable signal for the target mi RNA-21.With this strategy,the target mi RNA can be detected as low as 32 f M.We quantify mi RNA-21 in two kinds of cancer cells,and verify the applicability of this method to complex actual samples.The mi RNA detection strategy shows high sensitivity and selectivity,and has broad application prospects in disease diagnosis and biomedical research.