Study On The Nano And Necleic Acid Signal Amplification-based Disease Biomarker Biosensing

The sensitive and accurate detection of the biomarkers like proteins and nucleic acids is of very importantance for the disease diagnosis.Compared with the large equipment-based analytical methods used in the clinical diagnostic field,the biosensor has a promising application prospect for the detection of disease biomarkers because of its unique advantages including convenient operation,low cost,small sample consumption,fast analysis speed,as well as high selectivity and sensitivity.In order to realize the accurate detection of low-abundant biomarkers in complicated matrices to fulfil the needs of early disease diagnosis,how to combine various signal amplification techniques to construct highly sensitive signal transduction strategies for biosensors and thus effectively improve their analytical sensitivity has recently become a research hot in the field of analytical science.Hence,based on the specific immuno and nucleic acid biorecognitions and related nanomaterial and nucleic acid signal amplifications,this thesis has carried out two researches on the study of ultrasensitive biosensing strategies for disease biomarkers by using the carcinoembryonic antigen?CEA?protein and the micro RNA-21 nucleic acid as the model analytes,respectively,which is lised as follows:1. Enzyme-catalytic deposition of gold nanoparticles at vertically aligned carbon nanotubes for electrochemical immunosensing of proteinBy combination of the sandwich immunoreaction at a vertically aligned single-walled carbon nanotube?SWCNT?-based immunosensor with the enzymatically catalytic deposition of gold nanoparticles?Au NPs?by a gold nanoprobe,this work successfully develops a novel electrochemical immunosensing method.The vertically arranged nanostructure was prepared through the covalently linking of terminally carboxylated SWCNTs at an aryldiazonium-modified electrode.It not only provides an excellent platform for the high density immobilization of antibodies to obtain the immunosensor but also serves as useful molecular wires to accelerate the electron transfer during electrochemical immunosensing.Meanwhile,the enzymatic reaction of the nanoprobe prepared from surface functionalization of the nanocarrier of Au NP by high-content glucoamylases can catalyze the deposition of a large of Au NPs at the immunosensor.So the quantitative capture of the as-prepared enzyme-nanoprobes throught the sandwich immunoreaction and the electrochemical stripping analysis of these nanoparticles at the immunosensor enabled the convenient signal transduction of the method.Due to the sensitive gold stripping analysis at the vertically aligned SWCNTs and the multi-enzyme signal amplification of the nanoprobe,the electrochemical signal response was greatly enhanced.Thus,the method can be used for the ultrasensitive detection of the tumor biomarker of carcinoembryonic antigen in a wide linear range from 1 pg m L^-1to 100 ng m L^-1 with a low detection limit of 0.48 pg m L^-1.2. Homogeneous colorimetric biosensing of micro RNA based on the exonuclease and Mg²⁺-dependent DNAzyme-assisted signal amplificationBy combination of the dual signal amplification of exonuclease and metallic ion-dependent DNAzyme,this work successfully develops a new homogeneous biosensing method for the highly sensitive colorimetric detection of micro RNA-21?mi RNA-21?.Based on the nucleic acid hybridization reaction between the designed hairpin DNA and target analyte,the catalytic reaction of T7 exonuclease?T7 Exo?could be triggered off,which thus caused the cleavage release of the Mg²⁺-dependent DNAzyme sequence designed on the hairpin DNA.Meanwhile,the mi RNA-21analyte released during the catalytic reaction would re-participate in the hybridization reaction with the hairpin DNA to realize the T7 Exo-assisted target recycling.In this way,the high-content Mg²⁺-dependent DNAzyme sequences released during the above reaction would further assemble with their substrate chains immobilized on the surface of magnetic beads?MBs?to form the active secondary structure.So the formed DNAzyme would catalyze the release of the biotin-labeled DNA fragments from the substrate chain.By employment of the specific biorecognitions of the as-prepared horseradish peroxidase functionalized Au NP?Au NP/HRP?nanotags onto the MB platform through the biotin-avidin interaction and the catalytic chromogenic reaction of the nanotags,the convenient colorimetric signal transduction of the homogeneous biosensing method can be thus realized.As both the T7 Exo-assisted target recycling and the catalyzed cleavage reaction of the Mg²⁺-dependent DNAzyme can greatly enhance the Au NP/HRP nanotag-based colorimetric signal response,this method shows an ultrahigh analytical sensitivity.Under the optimum conditions,it can be used for the accurate detection of mi RNA-21 in a linear range from 0.01 pM to 1 nM with a detection limit down to 3.1 fM.