Singal Enhancement Immunoassay Based On Phospholipid Bilayer Membrane Mediated Redox Reaction

Immunoassay has broad application prospect in the field of analysis and detection because of its high specificity.However,due to the low molar absorption coefficient of organic chromogenic reagents,traditional enzyme-linked immunosorbent assay(ELISA)has a certain degree of low sensitivity,which limits its practical application.Therefore,the construction of an immunoassay platform to enhance the detection signal becomes a breakthrough point for improving the detection sensitivity of ELISA.Due to its excellent ability to encapsulate multiple signal molecules,liposome has become a carrier component for biosensor transduction and amplification detection signal,which is widely used in the fields of biology,chemistry,medicine,composite materials and so on.This study systematically demonstrated that a catalytic REDOX cycle system mediated by the vesicle structure formed by the lipid bilayers(liposome)can amplify the signal of low concentration of the small molecule p-aminophenol.Then application of the REDOX system to the enzyme-linked immunoassay platform has improved the detection sensitivity of target proteins The main research contents are as follows:(1)A signal enhancement colorimetric analysis method was developed based on liposome mediated REDOX reaction.Functional liposome containing TCEP molecules were synthesized and used as the signal cycle amplification center.The phenanthroline iron(Ⅲ)was used as the colorimetry probe to realize the highly sensitive detection of p-aminophenol.In this part,Sephadex G-50 micro-column centrifugal method was used to separate and purify liposome,and then the synthesis process and experimental conditions of liposome were optimized to improve the sensitivity of detection.The results showed that the absorbance was linear with the concentration of AFP in the range from 0.01-0.03 μM and 0.05-0.7 μM,respectively.The concentration of 4-AP that could be observed by naked eye was as low as 0.03 μM,and the detection limit was 6 nM.By contrast,the signal was amplified 38 times and the detection limit was reduced 50 times by liposome mediated REDOX colorimetric mmethod.The innovation of this method lies in the inclusion and non-splitting penetration strategy of liposome structure combined with REDOX cycle amplification technology to enhance the detection signal of small molecules of enzymatic products.(3)Alkaline phosphatase(ALP)was detected by catalytic REDOX reaction based on colorimetric analysis system mediated by liposome.Using the principle that the enzyme substrate p-aminophenyl phosphate sodium salt hydrate(PAPP)can generate the enzymic product p-aminophenol under the catalytic action of alkaline phosphatase(ALP),the REDOX color development system described in(1)can indirectly and highly sensitive detect ALP,and the detection limit of ALP is as low as 0.0025 U/L.Compared with the conventional enzyme detection method,the detection signal can be amplified 32 times.(3)An enzyme-linked immunoassay platform was constructed based on the principle of liposome mediated REDOX cycle.High sensitivity detection of liver cancer marker protein human alpha-fetoprotein(AFP)using sandwich immunoassay.The results showed that the concentration of AFP was as low as 1.8 pg/mL can be detected,which was nearly two orders of magnitude more sensitive than the traditional ELISA.The high selectivity,excellent reproducibility,and good recoveries show the developed ELISA can be used to for the detection of other antibodies,DNA and half antigen.