New Colorimetric Immunosensing Methods Based On The Chromogenic Reaction Of Copper Ion

Accurate detection of tumor biomarkers shows great importance in the early disease diagnosis field.The utilization of the immunosensors,which are developed by combination of the highly specific immunoreaction with the signal transduction through the methods such as fluorimetry,colorimetry,chemiluminescence,electroanalytical chemistry for the determination of tumor biomarkers,has recently gained great attentions.Among of them,colorimetry possesses better application value in this issue because of its cheaper instrument and more visualized and convenient signal transduction over other methods.To improve the analytical performance of the conventional colorimetric immunosensing methods further,this thesis combines the excellent properties of the magnetic bead?MB?-based assay platform and copper chromogenic reaction with the signal amplification strategies such as enzymatic reaction and nanoprobes,and has performed the following researches on the construction of new colorimetric immunosensing methods for CEA determination:1.Copper chromogenic reaction based new colorimetric immunoassay for the tumor biomarker detectionA new colorimetric immunoassay method was developed for the rapid and sensitive detection of a tumor biomarker of carcinoembryonic antigen?CEA?by combination of a MB-based sandwich immunoassay and a copper chromogenic reaction.The magnetic immunoassay platform was constructed through the covalent immobilization of the capture antibody on the surface of carboxylated magnetic beads.After immuno-recognition of CEA,signal antibody-functionalized copper oxide nanoparticle?CuO NP?probes were applied for sandwich immunoreaction to form an immunocomplex.The CuO NP labels quantitatively captured onto the immunocomplex were then dissolved in acid solution to release high-content copper ions.Based on the coordination of these ions with the newly synthesized chromogenic agent of 1,2-diphenyl-2-?2-?pyridin-2-yl?hydrazono?ethanon,a red complex was produced for the colorimetric signal readout,resulting in the successful construction of a sensitive immunoassay method for CEA detection.Under the optimal conditions,this method showed a wide linear range over three orders of magnitude and a low detection limit of 26 pg/mL.Besides,this method showed excellent performance with low cost,rapid and convenient operation as well as satisfactory reproducibility,stability and accuracy.2.Ultrasensitive colorimetric immunosensing based on enzyme-induced biomineralization of cupric subcarbonateHerein we combine the enzyme-nanoprobe induced biomineralization of cupric subcarbonate with a sensitive copper chromogenic reaction to develop an ultrasensitive colorimetric immunosensing method.The nanoprobe was prepared through the surface assembly of signal antibody and high-content urease on the nanocarrier of gold nanoparticle?Au NP?.Upon sandwich immunoreaction at a MB-based immunosensing platform,the analyte of carcinoembryonic antigen and the Au NP/urease nanoprobe were quantitatively captured onto the MB surface successively to form an immunocomplex.The enzyme reaction of the nanoprobe resulted in the catalyzed hydrolysis of the urea substrate to produce large amounts of OH^-and HCO₃^-.These ions were further combined with added Cu²⁺to form the biomineralization of cupric subcarbonate rapidly on the immunocomplex.Based on the acid-release of Cu²⁺from the biomineralized products for the chromogenic reaction,a red complex was obtained and employed for the ultrasensitive colorimetric signal transduction.Under the optimal conditions,a linear range with five orders of magnitude was obtained with a low detection limit of 0.45 pg/m L.In addition,this method features low cost,convenient operation as well as satisfactory reproducibility,stability and accuracy.3.Ultrasensitive colorimetric immunosensing based on the signal amplification by hybridization chain reaction and enzyme-induced biomineralizationBy combination of the hybridization chain amplification and enzyme-induced biomineralization of cupric subcarbonate at a MB platform,this work develops a copper chromogenic reaction-based new colorimetric immunosensing method.The CEA biorecognition at the MB-based assay platform and the following binding of its aptamer strand specifically can initiate the hybridization chain reaction between two hairpin oligonucleotides with featured base sequences.The biotin labels at the end of the hairpin DNA opened by the hybridization chain reaction can be used to specifically capture the streptavidin and high-content urease dually functionalized Au NP nanolabels.Based on the enzyme-induced biomineralization of cupric subcarbonate for the quantitative enrichment of copper ion and the subsequent release of copper ion for its chromogenic reaction,successful colorimetric signal transduction was thus achieved for this method.As all the hybridization chain reaction,nano signal amplification and enzyme-induced biomineralization greatly enhance the signal responses of the immunorecognition events,this method shows ultrahigh sensitivity.Under the optimal conditions,this method can be used for the accurate detection of CEA in a linear range over five orders of magnitude with a low detection limit of0.071 pg/mL.In addition,this also features the advantages including low cost,convenient operation as well as satisfactory reproducibility,stability and accuracy.