Developing Of A Novel Clinical Kit For Assaying Of Alpha-fetoprotein Based On Luminescing And Magnetic Nano-materials

Hepatocellular carcinoma(HCC)is a highly malignant cancer,being the third cause of tumor-related death in the world.Thus,there must be excellent candidates for the rapid and inexpensive diagnosis of diseases.Recently,the usual way to predict HCC is detecting the concentration of AFP in serum and the most common approach in analyzing AFP is an enzyme-linked immunosorbent assay immunoprecipitation,immunoblotting techniques,and immunofluorescence.However,most of the usual methods are time-consuming,labor-intensive, and hazardous to health or require highly qualified personnel and sophisticated instrumentation.In the past decades,semiconductor nanocrystals(often known as quantum dots,QDs)have been attracted wide interests due to their various beneficial factors,which makes them take advantage over the convenient dyes,and more suitable for the usage in biology and medicine. Meanwhile,magnetic nanoparticles had been thoroughly studied due to their widespread biomedical applications.Up to now,however,there have been few researches in which QDs and magnetic nanoparticles were both used in practical application.In this paper,firstly,CdTe QDs were synthesized by aqueous phase method,and the preparation course was optimized.Then the CdTe QDs were conjugated with the bovine serum albumin(BSA).The preparation of CdTe was occurred in the aqueous phase by using L-Cysteine as stabilizer.The influence of reflux time,pH of precursor,and the mole ratio of reagent was investigated respectively to get the optimum reaction conditions.The CdTe QDs were conjugated with the BSA through electrostatic attrationand.The PL intensity of the complex was tested so as to ensure that the CdTe QDs were appropriate to be used as a protein probe.Secondly,Fe₃O₄-dextran magnetic nanoparticles with good paramagnetism were prepared and their monodispersity was checked by using transmission electron microscope.The amount of anthrone in the surface magnetic nanoparticles was determined by the anthrone colorimetry method.The result showed that about 390μg of dextran was absorbed in the surface of 1 mg of Fe₃O₄-dextran,suggesting that there was enough dextran in the surface of each magnetic nanoparticle.After the hydroxyl of dextran was oxidized into aldehyde group by KIO₄,the Fe₃O₄-dextran nanoparticles could easily conjugate with protein.Finally,the calibration curve and the working equation were obtained.Based on the equation, the human serums were tested.The processes were:(1)the Fe₃O₄-dextran nanoparticles were oxidized and conjugated with mouse anti-AFP antibody(the primary antibody)firstly.And then the complexes of primary antibody and magnetic nanoparticle were used to capture the antigen, AFP,so as to separate them.(2)The water soluble fluorescent nanoparticles were synthesized and conjugated with mouse anti-AFP antibody(the second antibody).(3)The sandwich immunoreaction occurred,and the superfluous QDs-anti-AFP antibody was washed away.The relationship between PL intensity of the complex and the concentration of AFP was determined, and the calibration equation—Y=k(X+4.5)/V—was obtained.(4)The human serum samples of 50 people were tasted.The result showed thatthe outcomes determined by this method were similar to those determined by Time-resolved Fluorescence when the concentration of AFP was high.When the concentration of AFP was low,the difference was slightly obvious.The reason may lie in that there were some molecules with the similar structure to AFP.Because of inevitable conditional restriction,the human serum could never be collected easily.Sothe pre-treatment of the serum should never be shook so as to reduce the interference causing by the protein molecules with the similar structure to AFP's if the proceeding research were carried out.At the same time,the preparation of nanoparticles waited to be further optimized. Through these ways,the CdTe QDs that had narrower half high,and Fe₃O₄-dextran magnetic nanoparticles better paramagnetism.More importantly,this new technique could be used in other antibody-antigen system if the antibody was changed.