Study On The Construction Of Electrochemical Aptasensor Based On Multiple Types For Endotoxin Detection

Endotoxin,also referred to lipopolysaccharide(LPS),is the amjor ingredient of the outer membrane of Gram-negative bacteria.Trace amounts of LPS can cause fever reaction and it is responsible for microcirculation disturbance,septic shock,vascular blood clotting and even ultimately death at high levels.So,the sensitive detection of LPS is especially critical and significant in the fields of medical and therapeutics and the level of LPS in pharmaceutical products is strictly controlled by relevant organizations all over the world.Therefore,two types of electrochemical aptasensor were designed based on the nanocomposites and avidin-biotin system for the ultrasensitive detection of LPS in this paper.The main research contents of this work are as follows:Part Ⅰ A signal-off electrochemical aptasensor for ultrasensitive detection of lipopolysaccharide based on the Ag-TNT/P-r GO nanocompositeObjective: To establish a signal-off electrochemical aptasensor based on the signal amplification strategy of nanocomposite and used for LPS detection.Method: The Ag nanoparticles(Ag NPs)decorated titanium dioxide nanotube(TNT)conjugated with polydiallyldimethylammonium chloride(PDDA)functionalized reduced graphene oxide(r GO)to form a new nanocomposite of Ag-TNT/P-r GO was prepared.The nanocomposite was dropped onto the clean gold electrode(GE)surface as the sensing platform,in which Ag nanoparticles not only served as the redox probe to generate the electrochemical signal,but also could immobilize LPS binding aptamer(LBA)through Ag-N bonds.Then,bovine serum albumin(BSA)was utilized to block the nonspecific binding sites.Finally,different concentrations of LPS solution were incubated on the modified electrode.The reduction of the electrochemical signal responses originated from the redox probe of Ag was measured by cyclic voltammetry(CV)to characterize the LPS concentration.Result: The aptasensor exhibited a wide linear range from 10 fg/m L to 100 ng/m L and the limit of detection(LOD)was as low as5 fg/m L,with the correlation coefficient of 0.9997.The intra-assay relative standard deviation(RSD)was about 2.7%,and the inter-assay RSD was about 0.2%.The corresponding average recovery percentages were in a range from 96% to 103% in the Vitamin B1 injections.Conclusion: A relatively simple electrochemical aptasensor based on the Ag-TNT/P-r GO nanocomposite was constructed for highly sensitive detection of LPS.The proposed aptasensor displayed satisfactory stability,reproducibility and selectivity.Part Ⅱ A signal-on sandwich-type electrochemical aptasensor for ultrasensitive detection of lipopolysaccharide using avidin-biotin system coupled with the MOF/Ag-P-N-CNTs nanocompositeObjective: To establish a signal-on sandwich-type electrochemical aptasensor based on avidin-biotin system coupled with the signal amplification strategy of nanocomposite and used for LPS detection.Method: The Au nanoparticles(Au NPs)were electrodeposited on the surface of the clean glassy carbon electrode to load streptavidin(SA).The modified electrode was followed by treating with biotin-modified capture probes(BCPs)solution to immobilize capture probes via avidin-biotin interactions.BSA solution was then incubated onto the modified electrode to block nonspecific binding sites.Afterwards,LPS solution with different concentration was dropped on the modified electrode.In addition,the nanocomposite of PDDA functionalized nitrogen-doped carbon nanotubes(N-CNTs)modified with Fe-based metal-organic framework(Fe-MOF)and Ag NPs(MOF/Ag-P-N-CNTs)was prepared as a signal amplification material.The thiol-modified signal probes(TSPs)were then integrated with MOF/Ag-P-N-CNTs through Ag-S bonds to obtain the tracer label.Finally,the modified electrode was incubated with tracer labels to fabricate the sandwich-type electrochemical aptasensor.Differential pulse voltammetry(DPV)was utilized to investigate the electrochemical signal responses to characterize the concentration of LPS.Result: The peak current was proportional to the LPS concentration with a linear range from 1 fg/m L to100 ng/m L with the LOD of 0.11 fg/m L and the correlation coefficient of0.9997.The intra-assay RSD was about 0.55%,and the inter-assay RSD was about 0.95%.The corresponding average recovery percentages were in a range from 95.5% to 104.7% in the Vitamin C injections.Conclusion: This proposed aptasensor has a wide linear range,low LOD value,excellent stability,reproducibility and selectivity,and shows satisfactory recovery in actual injection samples.