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Studies on Fabrication of the Biosensors for the Detection of α-fetoprotein

Author: WangZhi
Tutor: TuYiFeng
School: Suzhou University
Course: Analytical Chemistry
Keywords: α-fetoprotein immunosensor phenylboronic acid Ferroceneboronic acid Fe3O4 magnetic nanoparticles SiO2 nanoparticles
CLC: TP212.3
Type: Master's thesis
Year: 2009
Downloads: 103
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Abstract


α-fetoprotein (AFP) is a kind of acid glycoprotein. The concentration of AFP in the mother’s blood can be measured for certain fetal defects of the brain and spinal cord. For normal adults, the elevated serum AFP to abnormally high values occurs in several malignant diseases, usually including nonseminomatous testicular cancer and primary hepatocellular carcinoma. So as the one of the universally accepted marker of tumor, it is significant to detect AFP in clinical diagnoses.Recently, electrochemical biosensor has been greatly developed and widely used in a great amount of fields like environment, clinic diagnosis and biology. These biorecognition elements in combination with various transducers have created the rapidly expanding field of biosensors. Electrochemical detection for immunoassay ,being safe, economical, simple and easy to handle, and high sensitive, has found wide interest in the recently years.In this thesis, we have fabricated three kind of electrochemical immunobiosensor about AFP, which were based on the phenylboronic acid monolayer, Fe3O4 magnetic nanoparticles and SiO2 nanoparticles. And the rapid detection about AFP has benn realized.First, a novel reusable electrochemical immunosensor forα-fetoprotein (AFP) based on phenylboronic acid monolayer on gold was proposed. The sensor was fabricated by immobilizing of 3-aminophenylboronic acid (APBA) conjugated thiol-mixed monolayer on gold through 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3- tetramethyluronium tetra?uoroborate (TNTU) as linkage. AFP and enzyme-conjugated antibody were further trapped to the modified electrode surface through sugar–boronic acid and immunoaffinity interactions, respectively. The attached enzyme-conjugated antibody on the electrode surface could catalyze the reduction of hydrogen peroxide in the presence of thionine, which can be used to detect AFP in human serumby a competitivemechanism. Cyclic voltammetric, electrochemical impedance studies and photometric activity assayswere used to probe the assembly and regeneration process of the immunosensor. The in?uences of the competitive ratio of antigen and antibody, pH value of the measuring solution, incubation temperature and time were explored for optimizing the analytical performance. The whole assay process including incubation, detection and regeneration of the electrode could be completed in 35min. The reusable immunosensor for AFP with an RSD of 2.5% for five cycles. The detection of AFP in five serumsamples provided fromclinically diagnosed patients with liver cancer showed acceptable accuracy.Fe3O4 magnetic nanoparticles was used to prepare the immunobiosensor for no reagents. Ferroceneboronic acid was combined with antibody for preparing labeled antibody. Fe3O4 magnetic nanoparticles was silanized by APTES. Then the detected antigen was reacted with the antibody which was modified on the above nanoparticles. Finally, the nanoparicles was with sandwich structure by reaction with the mentioned labeled antibody. In the solution of detection, the final nanoparticles was absorbed on the magnetic glassy carbon electrode for cyclic voltammetry, differential pulse voltammetry detection. There is no need to add anything in the detection solution, therefore simply the complexity of the solution.SiO2 nanoparticles after silanizing was successively reaction with antibodies and ferroceneboronic acid. And then nanoparticles was combined with AFP antibody to prepare the probe molecule. L-cysteine formed self-assembled layer on the gold electrode, then AFP antibody was bind on the modified electrode. After that we incubated antigen on the above electrode. Then the labeled antibody was also incubated on the electrode by specific immunoreaction. Cyclic voltammetry (CV), electrochemical impedance (EIS), square wave pulse (SWQ) proved the formation of immunocomplex. At the certain experimental conditions, AFP antigen detection linear range was 10～100 ng/mL. This sensor has high sensitivity, wide detection range and the antibody was labeled without damange. SiO2 nanoparticles was also to enlarge the signal.

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