Construction Of Immune Biosensors Based On Upconversion Fluorescent Nanoprobe And Their Applications

Blood immunoassay is one of the important means of clinical diagnosis of modern medicine,which is of great significance to the maintenance of human life and health.In the current methods of blood immunoassay,optical immunoassay based on fluorescent marker has been widely used in the diagnosis and prognosis of clinical diseases because of its relatively simple detection method and high sensitivity.Traditional fluorescent markers are mostly excited by ultraviolet or visible light,which has shallow tissue penetration depth,biological light damage,and strong fluorescent background,reducing the signal to noise ratio.Rare earth ion-doped upconverting nano-luminescent materials can absorb long-wavelength near-infrared photons and emit short-wavelength ultraviolet or visible photons.The appearance and rapid development of this new luminescent material provide a new direction for design and preparation of fluorescent probes.Because the rare earth-doped upconversion nanoparticles are excited by near-infrared light,they have a series of unique advantages:low light damage to biological tissues,deep tissue penetration,low bioautofluorescence,little photobleaching effect,etc.Meanwhile,long luminescence lifetime,low biological toxicity,good chemical stability and tunable peak position of the spectrum with rare earth ion species,which make it an ideal probe material for fluorescence immunoassay.Fluorescent immunosensor is a new kind of biosensor combining the advantages of high sensitivity from fluorescent labeling technology and high specificity from immunological reaction.It is immunologically reacted with the analyte by the antigen?or antibody?immobilized on the surface of the solid-phase carrier.We performed the immune response signal into optical signals for testing.Compared with the traditional fluorescence immunoassay technology,it has the advantages of compact structure,low cost and easy operation.In this paper,we synthesized a high quality NaYF₄:Yb³⁺,Tm³⁺upconverting nanoparticles?UCNPs?as a fluorescent probe and used a silicon wafer as a solid phase support to construct a novel solid-phase immunobiosensor,which has been successfully detected goat anti-human IgG in PBS buffer solution.We studied the design and preparation of high quality NaYF₄:Yb³⁺,Tm³⁺upconversion fluorescent nano-probes,the modification of solid support and the application of biosensors.The main results are summarized as follows:?1?High-quality NaYF₄:Yb³⁺,Tm³⁺UCNPs were synthesized by thermal decomposition method using chloride as raw material.The diameter of nanoparticles was about 23 nm,the size distribution was uniform and dispersion was good.The crystal quality was high,which was pure hexagonal crystal.The nanoparticle emits a bright blue-violet light visible to the naked eye at a 980 nm laser excitation.At the same time,the effects of changing the dosage of NH₄F on the size and morphology of synthesized nanoparticles were simply studied.?2?The usage of removing ligand method to transfer nanoparticles from the oil phase to the aqueous phase,and introducting carboxyl through polyacrylic acid?PAA?,so that the nanoparticles have a very good hydrophilicity and biocompatibility,which brings the possibility for the further functional modifications in biological applications.The human IgG was coupled to the surface of UCNPs to construct the up-conversion fluorescent probe.Protein G was used as a bridge to modify rabbit anti-goat IgG onto the silicon surface,constructing a solid-phase support.Protein G enhanced the rabbit anti-goat IgG molecular activity and controled the orientation of its active side,thereby increasing the sensitivity of the sensor.?3?Detection of goat anti-human IgG in buffer was achieved using the constructed immunobiosensor.With the increase of goat anti-human IgG concentration in the solution,the upconversion luminescence of the solid support surface was gradually enhanced.The fluorescence intensity was in a good linear relationship with goat anti-human IgG concentration in the range of 5-400 nM,and the detection limit was 1.82 nM,and showed good detection specificity and stability.This work is expected to be used for the detection of multiple antigens,antibodies,and DNA in the blood,providing new detection methods and platforms which were of widely linear range and of high selectivity for the rapid immunoassay diagnosis of various major diseases.