Study On New Methods For The Detection Of SphK1 Activity

The phosphorylation reaction of lipids activated by kinases plays fundamental roles in various kinds of biological functions,including lipid metabolism,signal transduction and mass transportation,especially in cancer developments.So assays of key lipid kinases are of great necessity in both biological study and clinical research But there is little direct redox signal for lipid molecules as the catalysis of lipid kinases often occurs in the cell membrane environment.So most of available approaches proposed could only rely on phosphosite-specific antibodies or fluorescent Pro-Q Diamond dyes,which need complex and expensive steps as it is difficult to convert the phosphosite recognition events into detectable signals.As a result,it is highly required to develop new methods for lipid kinase analysis.Therefore,based on the catalytic environment simulation of lipid kinases,we have proposed two assay methods for the detection of lipid kinases utilizing the metal-coordinated ligand(NTA-Fe)as the recognition element to effectively simplify this analysis procedure.Based on this principle,we have fabricated two lipid kinase biosensors separately taking sphingosine kinase 1(SphK1)as an example while simulating the catalytic environment to realize the effective analysis of lipid kinases and even other lipid metabolites.1.An assay method to detect SphK1 activity based on bifunctional nanoprobes.In this work,we designed a bifunctional Fe(?)-coordinated nanoprobe for the first time to fabricate an assay method to detect lipid kinase activity in high sensitivity based on the lipid environment simulation.In this method,this bi-functional nanoprobe can simultaneously target phosphosites through the chelating effect and generate electrochemical signal through the H2O2 electrocatalysis by coordinated metal ions and AuNPs.By this way,phosphosite recognition and signal generation could both be realized,improving the detection accuracy to a great extent.Besides,sensitivity of the assay is significantly improved due to NTA-Fe(?)enrichment on the nanoparticle and continuous H2O2 electrocatalysis.This work gave a simple and highly-sensitive sphingosine kinase 1(SphKl)analysis as a sample method.As low as 7.404 pmol min-1 mg-1 limit of detection is achieved.So this work can act as a new method for SphK1 activity and also holds great promise in the biological detection and clinical diagnosis of other vital phosphorylation processes.2.An assay method to detect SphKl activity based on liposome signal amplification.In this work,we utilized the liposome structure to three-dimensionally simulate the catalytic environment of lipid kinases to detect lipid kinase activity more conveniently and efficiently.For this method,the lipid substrate is embedded on the surface of the liposome through hydrophobic interaction.After the phosphorylation reaction being catalyzed by lipid kinase,the phosphorylated lipid products anchored on liposomes are recognized and captured by Fe(?)-immobilized gold electrode.At the same time,the liposome is also utilized as a carrier to encapsulate signal molecules,so the signal could be transmitted to the electrode surface directly.Compared to present methods,this assay strategy is efficient without additional signal transduction steps.Besides,sensitivity of the assay is ensured owing to the enrichment of signal molecules encapsulated in liposomes for simultaneous electrochemical signal generation and amplification.Also for proof-of-concept,sphingosine kinase 1(SphK1)is detected in this work.The assay method gives a quite low detection limit down to 2.3318 pmol min-1 mg-1(S/N=3).Thus,this electrochemical method can be effectively utilized for SphK1 detection,which may also hold great promise in new detection methods of other vital lipid kinases and even other lipid metabolites.