Construction And Application Of High Performance ELISA Based On Novel Signal Generation System

With the rapid growth of aging population,cancer has become the"number one killer"in the world.Sensitive and reliable detection is of great importance for early diagnosis of cancer and other dread diseases.In recent years,various emerging analytical techniques have been bringing out.Immunoassay has been widely used in clinical diagnosis due to its advantages such as simple operation,requirements on analytical instruments and low cost.This thesis aims to construct colorimetric and dual signal immunoassay platforms based on bio-enzymes and nanomaterials,improving resolution,reliability and accuracy.The main research contents and results are as follows:1.A high-resolution colorimetric immunoassay platform realized by coupling enzymatic multicolor generation with smartphone readoutThe traditional colorimetric immunoassay mostly depends on a single color change from light to dark or from dark to light.When the concentration of analytes is close to each other,it is difficult to distinguish the color with the naked eye.In order to solve this problem,a versatile colorimetric immunoassay is proposed by coupling of urease-catalyzed pH-induced multicolor generation with the typical ELISA.In the experiment,the indicator phenol red and Ag⁺were used as the chromogenic agent and urease inhibitor,respectively.The activity of urease was regulated by the ALP-catalyzed ascorbic acid（AA）production,as a consequence the indicator phenol red changed from yellow to orange to red within the pH range of 6.8⁸.4,achieving the qualitative and semi-quantitative detection by the naked eye.In this experiment,R IgG,a model analyte,was specifically recognized by monoclonal antibody and ALP modified secondary antibody.The activity of urease was controlled by Ag⁺,leading to the adjustment of color generation.With the increase of R IgG concentration,ALP catalyzes the generation of more AA to reduce Ag⁺into silver element,which has no obvious inhibition of urease activity.Thus,urease catalyzes urea to produce ammonia gas,leading to the increase of solution pH and color change.RIgG in human serum,a mimic clinical sample,was also successfully detected with a good performance,demonstrating the potential of the proposed strategies in practical application.Under the optimal condition,a limit of detection（LOD）of 1.73 ng mL^-11 and a dynamic range from 0 to 18 ng mL^-11 are achieved with a good linear relationship.A colored picture for each test is directly taken using a smartphone and then quantitatively analyzed with the free software Image J,eliminating the use of expensive and desktop equipment.2.In situ ascorbic acid-induced dual signal generation from metal-organic frameworks for a high-performance immunoassayIn this work,Fe（Ⅲ）based metal-organic frameworks nanoparticles were prepared by a hydrothermal method using a fluorescent organic ligand 2-amino terephthalic acid（BDC-NH₂）.The resulted Fe-MOFs show a very weak fluorescent emission,while a significant emission enhancement is observed in the presence of AA.Based on the AA-induced enhancement effect,AA detection with detection limit at 0.051μM was accomplished by use Fe-MOFs as an optical probe.In addition,Fe-MOFs also possess oxidase-like activity and catalyze TMB oxidation to produce a blue color.The above signal generation strategy was further combined with the typical ELISA to develop a dual-mode fluorescent and colorimetric immunoassay platform.Human prostate specific antigen（PSA）,a cancer biomarker,was used as a model analyte to evaluate the application potential of the proposed dual-mode platform.When PSA is present,the ALP-conjugated anti-PSA is introduced into the microplate wells through the specific binding of antigen-antibody.ALPcatalyzes AAO to produce AA,which induces fluorescent enhancement of Fe-MOFs and inhibits Fe-MOFs-catlyzed TMB coloration.A limit of detection for PSA is determined to be180 pg mL^-1,which is much lower than PSA in normal people’s blood,demonstating the great potential in practical application.The mechanism of AA-induced emission enhancement of Fe-MOFs was investigated systematically.In comparison with free organic ligand BDC-NH₂,luminescence behavior of Fe-MOFs is obviously weak due to the ligand-metal charge transfer（LMCT）.As expected,after treatment by AA,the fluorescence of Fe-MOFs is significantly enhanced,which is attributed the AA-induced inhibition of LMCT from the fluorescent ligand BDC-NH₂ to Fe（Ⅲ）.When Fe（Ⅲ）in Fe-MOF is reduced into Fe（Ⅱ）by AA,the LMCT is partially inhibited and the fluorescence of BDC-NH₂ is consequently recovered,thus resulting in a fluorescence enhancement of the Fe-MOFs.At the same time,the oxidase-like activity of Fe-MOFs is also inhibited by the AA-induceded Fe（Ⅲ）reduction.In conclusion,a high resolution colorimetric and a dual-mode immunoassay platform is developed based on urease and Fe-MOFs for the detection of tumor biomarker.The developed ELISA platform is also applicable to the detection of other biological macromolecules or small molecules.Thus this project opens a new way to develop analytical platforms for bioanalysis in different fields.