Multiplexed Nanoparticle-based Detection Of Environment Estrogen By Highly Sensitive Fluorescence Immunoassay

Environmental estrogen is a category of exogenous estrogen compounds with thestructure and function similar to endogenous estrogen. After entering the living body fromoutside, the environmental estrogen can not only interfere with the normal physiologicalprocess and create many deleterious effects, but also can activate or inhibit the function ofthe endocrine system, thereby leading to breast cancer, prostate cancer, birth defect,infertility, precocious girls, diabetes. Most importantly, environmental estrogen can play animportant role in the existence of a biological species. Therefore, a new type analysismethod of detecting environment estrogen is established, having important significance inpractice on the study of environmental estrogens material.Nano-material has been paid special attention on the theory research and practicalapplication because of its unique quantum size effect, surface effect and the opticalproperties showed. Gold nanoparticles (GNPs) have several dominances as follows: thesimplely fast process of synthesis, narrow distribution of nanoparticles size. Besides, thiolor other biological ligands can be efficiently attached onto its surface. Such characteristicsmake GNPs become a kind of biological sensor, so that achieve the purpose of recognition.GNPs also can be coupled with many biological molecules, such as proteins, enzymes,antibodies, nucleic acid and so on. The detection sensitivity in immunoassay is improvedby applying the functional GNPs probes, which provide important means in somebiological systems. Magnetic nanoparticles (MMPs) has also characteristics of themagnetic orientation, good biocompatibility and reactive group except unique size effectof nanoparticles, which could be coupled with can cell, antibodies, enzymes, proteins andnucleic acids. Magnetic particles, as a kind of high performance magnetic carrier, makeimmunoassay possess these advantages of separation easily, simple operation, rapidseparation, enrichment, simple equipment, low toxicity in the magnetic field. Therefore,MMPs has been widely used in a variety of research fields such as immunology, biologyand medicine. Qualitative or semi-quantitative determination for the antigen or antibodysamples could be achieved by detection method of simple and fast, which have brought a revolutionary development in the immunoassay detection.Herein, in order to achieve higher sensitivity, a novel fluorescence immunoassaybased on double-codified nanotechnology was developed from competitive reaction usingMMPs and DC-GNPs for further signal amplification to detect trace hapten in samples.This present thesis consists of four parts as follows:1. Preparation of double-codified gold nanoparticles (DC-GNPs)Immune analysis platform for signal amplification was formed by combinessecondary antibody and DNA to the surface of gold nanoparticles, which had character ofgeneral. When GNP was used as a carrier of barcode DNA, barcode DNA strands could bedirectly anchored on a gold nanoparticle via thiol bonds. Bare sites of gold surface wereelectrostatic adsorbed to a secondary antibody. DC-GNPs were sequently achieved byhybridize with complementary sequences. At least, such a secondary antibody iscorresponding to many strands of DNA with fluorescence signals, leading to magnifyfluorescent signal many times.2. Multiplexed nanoparticle-based detection of bisphenol A by highly sensitivefluorescence immunoassayIn order to achieve higher sensitivity, a novel fluorescence immunoassay based ondouble-codified nanotechnology was developed from competitive reaction using MMPsand DC-GNPs for further signal amplification to detect free bisphenol (BPA) in samples.After optimizing the conditions of immunoreaction such as the concentration of theDC-GNPs, functional MMPs, DTT and FITC-A, we can get the standard working curve.△I(I0–I)=74.82lgC+2005.97(R=0.9964). For bisphenol A, the linear range was from1.0×10-4ng/mL to10.0ng/mL and the limit of detection was found to be0.039pg/mL. Thepercent recoveries are in the range of91.5%-105.7%in the plastic and the percentrecoveries are in the drinking water in the range of91.4%-116.6%.3. Multiplexed nanoparticle-based detection of E2by highly sensitive fluorescenceimmunoassayA novel fluorescence immunoassay based on double-codified nanotechnology wasdeveloped from competitive reaction using MMPs and DC-GNPs for further signal amplification to detect free E2in urine samples. With the optimized reaction conditions,nanoparticle-based fluorescence immunoassay method was applied to confirm the linearrange of E2. The standard curve showed a good response for E2at concentrations of1.0×10-5ng/mL-1.0ng/mL. And the linear correlation coefficient was R=0.9950.According to the definition, we obtained the detection limit of0.006pg mL1. Theproposed method was applied to the analysis of E2in urine samples, giving values ofrecovery in the range of96.5%-107.4%.4. Multiplexed nanoparticle-based detection of E3by highly sensitive fluorescenceimmunoassay in urine samplesA sensitive and rapid method for the determination of E3by fluorescenceimmunoassay (FIA) was established on the basis of competing mode immunoassay.DC-GNPs conjugated by DNA and gold nanoparticals were regarded as a probe in thissystem. After optimizing the conditions of immunoreaction such as the concentration ofthe DC-GNPs, functional MMPs, DTT and so on. Linear relationship between values offluorescence intensity and logarithm of mass concentration (lgc) of E3was obtained in therange of1.0×10-4ng/mL~10.0ng/mL with detection limit of0.045pg/mL. The proposedmethod was applied to the analysis of E3in urine samples, giving values of recovery in therange of93.0%-112.0%.