Studies On Amperometric Immunosensors Based On Assembly Of Nanoparticles And Novel Signal Amplificatory Strategy

Electrochemically based immunosensors are valuable analytical tools for monitoring of the antibody or antigen as the advantages of short response time,high sensitivity, high specificity and easy manipulation,which combined the merits of electrochemical technology and immunoassay.An immunosensor is an analytical device that responds to a suitable combination of a biological recognition system and an electrochenmical transducer,thus the platforms construction of the antigen or antibody biomolecules immobilization have been the vital step in successful development of.an immunosensor. Therefore,this research focuses on the preparation of multi-functionalized nanomaterials,the construction of the immunoreaction interface and the development of sensitivity enhancement electrochemical immunosensors.The detail contents are as follows:1.In the review section,after general introduction of immunoassay including its principle and the construction of immunoreaction interface,the application of nanotechnology into immunosensors was highlighted.Moreover,the research development of immunosensors based on novel signal amplificatory strategy was presented.Finally,the work and significance of this thesis was briefly introduced.2.Since most immune protein are not intrinsically able to act as redox partners in an electrochemical reaction,most amperometric immunoassay techniques are relied on the label of either antigen or antibody,which requires highly qualified personnel, tedious assay time,or Sophisticated instrumentation.In this experiment,we described a new strategy for the development of a novel reagentless amperometric immunosensor based on the carcinoembryonic antibody(anti-CEA) molecule that was coupled at high density to sandwich(nano-Au/SiO₂@Thionine/nano-Au) nanocomposite layers for efficient carcinoembryonic antigen(CEA) recognition. Herein,the immobilized SiO₂@Thionine may act as electrochemical redox probe to monitor the variety of the kinetic-barrier of the electrode interface.Thus,the detection mode is based on the change of the current response before and after the specific binding of anti-CEA to CEA,due to the immunocomplex inhibiting the access of redox probe to electrode.The assay format avoids the label of antibody or antigen,the competitive or sandwich formats,the addition of an electron transfer mediator to the solution and the separation step,This significantly simplifies the immunoassay,procedure and shortens assay times.3.This work describes a two-step conjugate synthesis of a porous organometallic nanostructured materials composed of ferrocenemonocarboxylic(Fc-COOH)and 3,4,9,10-perytenetetracarboxilic diarthydride.(PTCDA) and then a reagentless amperometric immunosensor prepared with positively charged gold nanoparticles (PGN) immobilized in this nanostructure Conductive film is developed.This nanostructured material comtaining ferrocenyt(PTC-Fc) coutd easily form stable film on the electrode surface with efficient redox-activity and excellent conductivity. Furthermore,with the negatively charged surface,this film can be used as an interface to adsorb the PGN,which were prepared in-organic solvents at relatively high concentrations with improved monodispersity compared to those prepared in aqueous solution.The presence of PGN provided a congenial microenvironment for adsorbed biomolecules and decreased the electron transfer impedance.Thus,with anti-CEA as a model antibody,the proposed immunosensor showed rapid and highly sensitive amperometric response to CEA with acceptable preparation reproducibility and stability.4.Although the label-free immunosensors are able to detect the physical changes during the immune complex formation,whereas the enzyme-labeled imrnunosensors use signal-generating labels which allow more sensitive and versatile detection modes when incorporated into the complex.Thus,a concept based on a novel redox-biocompatible composite protein membrane fabrication,double enzyme membrane modification technique and antibody immobilization,was exploited to develop a highly sensitive amperometric enzyme immunosensor for detection of CEA. In this concept,a solution of bovine serum albumin(BSA)Containing horseradish peroxidase(HRP) is coated on the gold electrode in such a way that the first enzyme membrane is achieved.Then Co(bpy)₃³⁺,as a redox probes,was embedded in BSA-HRP composite membrane vis the electrostatic force and hydrophobe functions. Moreover,the new strategy of the employment of HRP to block the possible remaining active sites and amplify the response of the antigen-antibody reaction was proposed.The immunosensor constructed with the double layer biocatalytic HRP membranes and the desirable Co(bpy)₃³⁺/BSA redox-biocompatible composite membrane performed high sensitivity and a wide linear response to CEA,as well as good Stability and long-term life.5.With their unique property-superparamagnetism,magnetic nanoparticles have become attractive for exploitation mainly in biology and medicine because they can simplify the process of proteins immobilization and separation.In this work,the preparation,characterization and application of a three-layer magnetic nanoparticle composed of a Fe₃O₄ magnetic core,a Prussian Blue(PB) interlayer and a gold shell (it can be abbreviated as Au-PB-Fe₃O₄) for an ultrasensitive and reproducible electrochemical immunosensing fabrication was described for the first time.With the employment of the Au-PB-Fe₃O₄ nanoparticle,a new signal amplification strategy was developed based on bienzyme(horseradish peroxidase and glucose oxidase) functionalized Au-PB-Fe₃O₄ nanoparticles for an electrochemical immunosensing fabrication by using CEA andα-fetoprotein(AFP) as model systems,respectively. The experiment results show that the multilabeled Au-PB-Fe₃O₄ nanoparticles exhibit satisfying redox electrochemical activity and highly enzyme catalysis activity,which predetermines their utility in high sensitivity antibody detection schemes. Furthermore,this immunosensor could be regenerated,by simply using an external magnetic field which ensured a reproducible immunosensor with high sensitivity.6.In this work,the multi-walledcarbon nanotubes(CNTs) were individually dispersed in an aqueous solution by a kind of natural proteins,bovine serum albumin(BSA),to obtain BSA molecules coated CNTs(BSA-CNTs).Then the gold colloids(nano-Au) were absorbed on the BSA-CNTs surface by the amido and disulfide groups of BSA. Later,a functionalized gold/carbon nanotube composite nanohybrid (DpAu/nano-Au/BSA-CNTs) modified electrode was developed based on electrochemical deposition of Au³⁺ onto nano-Au/BSA-MWNTs surface.Thus,a sensitive immunosensor for carbohydrate antigen 19-9(CA19-9) based on antibody immobilization on a functionalized gold/carbon nanotube composite nanohybrid has been constructed with the amplification response of antigen-antibody by the employment of Nation-coated SiO₂ nanoparticles labeled secondary antibody based on a sandwich immunoassay.More importantly,the special biomolecule-binding can not only cause the construction of the dielectric immunocomptex layer but also introduce the insulated Nation coated SiO₂ nanoparticles which demonstrate the relatively high resistance,resulting in a strong detection signal.Moreover,the extremely high stability of the functionalized gold/carbon nanotube composite nanohybrid monolayer allows the designed biosensing interface to obtain a good stability and long-term life.