Sandwich-type Electrochemical Immunosensor Based On Ferro Magnetic Nanoparticles

The traditional enzyme-linked immunosorbent assay （ELISA） is the most widely usedimmunoassay method in clinical laboratory. ELISA tests are based on the principle of a solid phaseenzyme-linked immunosorbent assay using the spectroscopic detection of a colored reagent.However, the enzyme labeled on the antibody is limited which limit the sensitivity of the ELISAmethod. Electrochemical immunosensor, based on specificity of antigen-antibody interactions withelectrochemical transducers, has attracted considerable interest because of its intrinsic advantagessuch as low cost, high sensitivity, simple instrumentation, and excellent compatibility withminiaturization technologies. And the key to successful construct electrochemical immunosensorare follows:（1） Exploring simple, rapid, effective methods to immobilize proteins on the electrode;（2） Developing new signal strengthen technology. Therefore, this research focuses on thepreparation of composite magnetic nanoparticles, immobilization of proteins, and enhancement ofthe signal of immunoassay, especially on the enhancement of the signal of electrochemicalimmunoassay. The detail contents are as follows:1. The preparation of Fe₃O₄/Au composite magnetic nanoparticles and the HG-AFSdetermination of AFP based on the As-Fe₃O₄/Au. We demonstrate the possibility of HG-AFS as agood endpoint detector for the determination of biological molecules by measuring the elementattached to an immunoreagent. Firstly, Fe₃O₄/Au composite magnetic nanoparticle was preparedby self-assemble gold nanoparticles on the Fe₃O₄nanoparticles which was prepared by co-precipitation method. Considering Fe₃O₄has a good absorption of arsenic and Fe₃O₄/Au exhibitedgood binding capacity to antibody molecules, we use As-Fe₃O₄/Au to label anti-AFP （Ab2） assecondary antibody. After the sandwich immunoreaction, As can be dissociated from the labeledantibody in the sandwich-type immunocomplex and detected by HG-AFS. This method showsgreat promise to be applied to the clinical application.2. A new dual-amplification strategy of electrochemical signaling was proposed via porousnano-structure gold film as antibody （Ab1） immobilization matrix and Fe₃O₄/Au magneticcomposite nanoparticles as the nanocarrier for the immobilization of horseradish peroxidase （HRP）and secondary antibody （Ab2）. Greatly amplified sensitivity is achieved by using the bioconjugatesfeaturing HRP and Ab2linked to Fe₃O₄/Au at a high HRP/Ab2ratio to replace singly labeled secondary antibodies. The analytical procedure consists of the immunoreaction of the antigen （AFP）with Ab1, followed by binding Fe₃O₄/Au-HRP-Ab2. Electrochemical detection was performed inthe presence of hydrogen peroxide （H2O2） and hydroquinone （HQ）. Results demonstrated that theimmunosensor based on this amplification strategy has good dynamic range from0.005¹0ng/mLand the detection limit is3pg/mL for AFP. This simple, economic and sensitive immunosensorcould find wide potential application in clinical analysis.3. An ultrasensitive electrochemical ELISA method for the detection of alpha-fetoprotein wasconstructed by using core-shell Fe₃O₄/Au nanoparticles as labels. The analytical procedure consistsof the immunoreaction of the antigen （AFP） with anti-AFP coated on the96-well microtiter platesfollowed by binding Fe₃O₄/Au-HRP-anti-AFP. The formed HRP-labeled immunoconjugateshowed good enzymatic activity for the oxidation of OPD by H2O2and AFP can be quantitativedetected by the voltammetric response of the electroactive product. The linear range was from0.01²00ng/mL and the detection limit is5pg/mL. The usage of Fe₃O₄/Au-HRP-anti-AFP assignal antibody with a high HRP/secondary antibody molar ratio can greatly enhance thesensitivity. In addition, this strategy combines the merits of electrochemical method and traditionalELISA method, and smoothes away the cumbersome electrode modification process inelectrochemical method and low sensitivity in ELISA, which provided a promising potential inclinical diagnosis.