Study On The Highly Sensitive Amperometric Immunosensor Based On Biomolecule Immobilized On Nanoparticles

Developing of sensitive immunoassay technology is one of the important tasks in immunoassay,which is crucial for realizing the detection of some vital diseases. Electrochemical apparatus are simple,sensitive and easy to be miniaturized.So it attracted substantial research and has been widely used for the assay of biological analytes.The electrochemical immunosensor uses specificity of the reaction of antigen and antibody to combine traditional method for immunoassay with the technology of modern sensor,which both has the high specificity of the immunochemical system and has the high sensitivity of electrochemical analysis and has been broadly applied in clinical diagnosis.Nanoparticles have excellent properties in electricity,magnetism,optics and predominant biocompatibility,it can be used as a platform to immobilize antibodies by adsorption,especially core-shell nanoparticles which exert crucial functions in the fields of biochemical immunoanalysis.This dissertation focuses on designing and fabricating novel biomimetic interface combining some kinds of nanoparticles with bifunctional carbon nanotubes,doped titania colloids,core-shell nano-Prussian,doped silica nanoparticles,and gold nanoparticles for the immobilization of biomolecules.Transmission electron microscopy,Scanning electron microscopy,Atomic force microscopy,UV-Vis spectra and electrochemical techniques are used as tools for studying the properties of interface.We discussed their applications in bioanalysis domain.This research focuses on the details as follow.1.In this work,a water-in-oil microemulsion method was used to develop ferrocenecarboxylic-doped SiO₂ nanoparticles(Fc-COOH-doped SNPs)in which the Fc-COOH were homogeneously dispersed and tightly captured in a three-dimensional cage of the silica network.Then,the surface of Fc-COOH-doped SNPs were functionalized with amino groups by 3-aminopropyltriethoxysilane (APTEOS)through Si-O-Si bonds.The Fc-COOH-doped SNPs were characterized by X-ray photoelectron spectroscopy(XPS)and transmission electron microscopy (TEM).The experimental results showed that the nanoparticles were uniform in size and the Fc-COOH did not leak out in aqueous solution.With amino groups on the surface,the nanoparticles were easily immobilized with cancer antigen 15-3 (CA 15-3)antibodies through covalent conjugation using glutaraldehyde.The developed immunosensor showed good linearity at the studied concentration range of 2.0 up to 240 U/mL and a detection limit of 0.64 U/mL at S/N=3.2.A newly functional nanoparticle has been prepared to immobilize the protein for the detection ofα-1-fetoprotein(AFP).Prussian blue(PB)nanoparticle was initially synthesized,then bovine serum albumin(BSA)was used to coat the PB nanoparticle to improve the stability of the PB nanoparticle as well as functionalize the surface of PB nanoparticle,and then gold colloids were loaded on the BSA-coafed PB nanoparticle to construct a core-shell-shell nanostructure.The functional three layer nanoparticles were composed of PB nanoparticles core and gold colloids shell with the middle conjunction layer of BSA.The core-shell-shell nanoparticle offer some advantages as follows:(1)protecting the PB NPs from agglomeration in a stable aqueous suspension;(2)providing bioactive functionalities throughout the nanoparticle surface for further biological interactions or couplings(with antibodies,for example).(3)The outer layer of gold colloids may improve the electrochemical activity and bio-compatibility because the nano-Au possessed high surface-to-volume ratio and good biocompatibility. The fabrication procedures of the immunosensor is simplicity.Under optimal conditions,the proposed immunosensor exhibited high sensitivity and a wide linear range from 0.02 to 200.0 ng/mL towards AFP with a detection limit of 0.006 ng/mL(S/N=3).3.A new electrochemical immunosensor for the detection ofα-1-fetoprotien(AFP) was developed based on AFP antibody(anti-AFP)-functionalized organic/inorganic hybrid nano-composite membrane.To fabricate such a hybrid membrane, 3,4,9,10-perylenetetracarboxylic acid-bound thionine molecules(PTCTH)were initially doped into titania colloids(TiO₂),and then gold nanoparticles and anti-AFP were immobilized onto the composite film in turn.In contrast with the electrode fabricated only with thionine not 3,4,9,10-perylenetetracarboxylic acid, the immunosensor with PTCTH exhibited high sensitivity and fast electron transfer. The presence of gold nanoparticles provided a good microenvironment for the immobilization of biomolecules,enhanced the surface coverage of protein,and improved the sensitivity of the immunosensor.Under optimal conditions,the proposed immunosensor exhibited a wide linear range from 2.5 to 200.0 ng/mL towards AFP with a detection limit of 0.5 ng/mL(S/N=3).4.One kind of composite with redox activity was prepared by ammonolysis of 3,4,9,10-perylenetetracarboxylic dianhydride(PTCDA),o-phenylenediamine and variable valence transition metal ions of Co²⁺.The composite which can be used as redox probe had good conductivity for electrons transfer and can be reacted with nano-Au for lots of amino groups.Carbon nanotubes and the composite were employed as electrode materials in this study,forming nanoparticle layers with large surface-to-volume ratio,good adsorption ability and excellent conductivity. Nano-Au was immobilized on the electrode according to Au-NH₂ covalent bond, and then the immunosensor was prepared by immobilizing antibody of a-1-fetoprotien(AFP)which can make bond with nano-Au.The immunosensor had good stability with a wide linear range of 0.20～120 ng/mL and a detection limit of 0.12 ng/mL(S/N=3).5.We have successfully developed a sensitivity of amperometric immunosensor based on avidin-biotin immobilization and amperometric signals amplified by avidin-labeled HRP-functionalized Pt nanoparticles and amplified again by HRP. The sandwich-type electrochemical immunosensor for the detection of AFP using enzyme multilayers and Pt nanoparticles(PtNPs)on avidin that associated with the nanohybnd film of gold nanoparticles/multi-wall carbon nanotubes (AuNP/MWCNTs)modified electrode is presented.The detection ofα-1-fetoprotein(AFP)immunological reaction is based on dual signal amplification:(1)a large amount of HRP introduced on the electrode surface through avidin-labeled HRP-functionalized PtNPs(2)H₂O₂ and biotm-labeled HRP induced catalytic oxidation,which results in great enhancement of current response.The immobilization of anti-AFP is accomplished by avidin-biotin immobilization technique,it offers various advantages such as it is extremely specific and strong noncovalent binding method;the avidin-biotin immobilization procedure generally maintains bioreceptor binding activity more successfully that other regular methods.At the same time,the nanohybrid film AuNP/MWCNTs can improve the response to the immunosensors due to its higher exposed surface area, facilitated to electron transfer and excellent biocompatibility.The detection limit for AFP is found to be 0.1 ng/mL at S/N=3 and the detection concentration range is from 0.25 to 100 ng/mL.