| Cancer is one of the most important reasons threatening people's health and happiness.If early diagnosis and intervention can not be achieved,subsequent treatment or maintenance will face great difficulties.Accurate determination of serum tumor markers can help to improve the diagnosis rate of cancer,and also play a role in monitoring drug treatment or surgical prognosis.Immunoassay has been proved to be an ideal method for the determination of trace tumor markers,mainly due to the high reactivity and specificity during the formation of antigen-antibody immune complexes.Currently,magnetic polymer microspheres have gradually replaced the planar carriers used in traditional immunoassays.However,magnetic polymer microspheres generally have some problems,such as poor magnetic responsiveness,inhomogeneous coating,small specific surface area,strong non-specific adsorption,and fail to solve the problems of high background value,inconvenient cleaning and long time-consuming in traditional immunoassay.Magnetic micro-nanomaterials are usually a kind of materials with both micro-nano-characteristics and magnetic responsiveness.They have larger specific surface area and stronger magnetic responsiveness.They are expected to be used in immunoassay and replace magnetic polymer microspheres.In this paper,the influence of magnetic micro-nanomaterials on chemiluminescence immunoassay system was first studied,and then the chemiluminescence immunoassay method based on magnetic micro-nanomaterials was developed.In view of the problems existing in current immunoassay technology,a variety of new magnetic micro-nanomaterials were prepared and modified and new immunoassay schemes such as washing-free and label-free were proposed.The mechanism,influencing factors and application performance of these schemes were studied.The inhomogeneous coating on the surface of micro-nanoparticles may lead to the exposure of their contents.Therefore,a simulation system was established in this paper to study the effect of exposure conditions on chemiluminescence immunoassay using Fe3O4 magnetic micro-nanoparticles with exposed inorganic surfaces.Some factors such as the concentration of magnetic micro-nanoparticles,the coexistence of horseradish peroxidase(HRP),different particle sizes and various treatment methods were studied.It was confirmed that both the micro-nanoparticle body and the exposed metal oxide surface are closely related to the detection signal.This conclusion can provide a new explanation for the measurement deviation that occurs in chemiluminescence immunoassays.A chemiluminescence immunoassay system based on Fe3O4 magnetic submicron particles was developed and applied to the determination of human chorionic gonadotropin(HCG).Carboxyl functionalized magnetic submicron particles were modified with anti-alpha-HCG antibody and further used as solid-phase support in sandwich immunoassay.Under the optimum conditions,the detection limit of HCG was as low as 1.184 ng/mL,the linear response range was 2-100 ng/mL,and the correlation coefficient was 0.989.At the same time,this method can be used for the determination of HCG in serum samples,which has preliminary verified its potential for rapid and economical detection of biological samples.A novel chemiluminescence(CL)washing-free immunosensor was designed for chemiluminescence immunoassay of tumor markers represented by human alpha-fetoprotein(AFP).Firstly,active functional groups were initiated on the surface of Fe3O4 magnetic submicron particles prepared by solvothermal method.Bifunctional polyethylene glycol spacer arm molecules were coupled with active functional groups to reduce the non-specific adsorption capacity of the particles.Furthermore,N-(4-aminohexyl)-N-ethylisoluminol(AHEI)and anti-AFP antibodies were further immobilized on the particles by coupling the spacer arm molecules.After incubation with AFP and horseradish peroxidase(HRP)labeled anti-AFP antibody respectively,the formed sandwich-type immunocomplexes on the particle surface can lead to the close proximity between AHEI and HRP,which eventually leads to the enhancement of detected CL signal.Since a bound HRP labeled anti-AFP antibody produces much stronger CL signal than an unbound one,this approach eliminates the time-and labor-consuming washing steps usually required of such assays.Under the optimal conditions,this present method showed a wide quantitative range from 1 to 250 ng/mL with a detection limit of 1.751 ng/mL.The detection limit can meet the needs of physiological(0-20 ng/ml)and clinical(>20 ng/ml)AFP levels.This method can also be applied in serum samples,and also avoid the problem of particle aggregation caused by multiple separation and washing steps.A label-free colorimetric immunoassay was described for the pregnancy marker HCG.The assay is based on the use of MnO2 nanorods as a peroxidase mimic.The nanorods were prepared by a hydrothermal method using EDTA as a template.They exhibited excellent peroxidase-like activity and stability.The nanorods were immobilized in a chitosan matrix in the wells of a BSA-modified microplate which then were further modified with streptavidin and biotinylated capture antibodies.The specific recognition between HCG and the antibodies in wells inhibited the peroxidase-like activity of the nanorods.Hence,the substrate 3,3',5,5'-tetramethylbenzidine is less efficiently catalytically oxidized by H2O2 to form a blue colored product.This results in a decrease in the absorbance at 652 nm.Response was linear in the 0.5-400 mIU/mL HCG concentration range,and the detection limit was 0.36 mlU/mL under optimized conditions.The method is highly specific,acceptably reproducible and stable.It was applied to the determination of HCG concentration in serum samples,and results were in good agreement with data obtained by the reference method. |
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