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Study of Immunosensors Based on Various Nanocomposites and Nanopaticles for Detection of α-fetoprotein

Author: GongJunHua
Tutor: ZhengJunSong
School: Third Military Medical University
Course: Clinical Laboratory Science
Keywords: α-fetoprotein amperometric immunosensor nanocomposites layer-by-layer electro-deposition layer-by-layer self-assembly
CLC: R318.08
Type: Master's thesis
Year: 2013
Downloads: 12
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Abstract

BackgroundTumor markers are associated with certain tumors and the level of tumor markers inhuman serum can give information regarding the stage of the disease.Therefore, risinglevels of tumor markers in human serum are associated with certain tumors. A number ofmethods have been reported for the determination of tumor markers, includingradioimmunoassay, chemiluminescence and enzyme-linked immunoassay. However, thesemethods have some limitations such as the radiation hazards, long analysis time, thecomplicated wash procedure, and expensive and cumbersome instruments. Biosensor,especially amperometric immunosensor has attracted much research interest because oftheir convenience, simple preparation, fast analysis time, high sensitivity anduncomplicated instrumentation. Therefore, the development of new determination methodsbased on amperometric immunosensor for rapid and sensitive detection of tumor markers isessential in clinical diagnostics.PurposeThe aim of this study was to construct a simple, rapid and sensitive electrochemicalimmunosensor to detect tumor markers. Using α-fetoprotein (AFP) as a model system, thecharacteristics of the immunosensor were determined.Materials and Methods1. Indium tin oxide (ITO) semiconductor glasses were cut into3mm×20mm slidesand cleaned by sonication with acetone, methylene chloride and distilled water, respectively.A3mm×3mm bare front end of the pretreated ITO slides was served as the working surface.2. The immunosensor was fabricated by two steps: first, nanogold particles (nano-Au)and prussian blue (PB) and nano-Au was alternately electrochemically deposited onto theITO surface. Then, the alpha-Fetoprotein antibody (anti-AFP) was adsorbed onto the surface of the nano-Au layer and bovine serum albumin (BSA) was used to block possibleremaining active sites of the nano-Au monolayer.3. The experimental parameters that could affect the performance of the immunosensor,including the pH of the detection solution, incubation temperature and incubation time,were optimized using cyclic voltammetry (CV) measurements.4. Under optimal condition, the performance of the electrochemical immunosensorincluding calibration curves, specificity, stability, reproducibility and applicability werestudied.5. Combination of MWNTs-PB-CHIT nanocomposites excellent conductivity, largespecific surface area, strong adsorption ability, and good biocompatibility with the protein A(PA) oriented immobilization of antibody, a sensitive amperometric immunosensor withgood stability has been prepared via layer-by-layer (LBL) assembly.6. The experimental parameters including the pH of the detection solution, incubationtemperature and incubation time, were optimized using CV measurements. Under optimalcondition, the performance of the electrochemical immunosensor including calibrationcurves, specificity, stability, reproducibility and applicability were studied.Results1. Electrochemical characterization of the electrode surface through CV is an effectiveand convenient method for probing the features of the modified electrode surface. The CVresults indicated that the BSA/anti-AFP/nano-Au/PB/nano-Au/ITO immunosensor wassuccessfully constructed.2. The CV measurements results of the BSA/anti-AFP/nano-Au/PB/nano-Au/ITOimmunosensor indicated that the optimal detection solution pH was5.5, incubationtemperature was25.0℃, incubation timewas8minutes.3. Under the optimal experimental conditions, the working range of the BSA/anti-AFP/nano-Au/PB/nano-Au/ITO immunosensor was from0.25to300ng/mL with a detectionlimit of0.04ng/mL. The studied results indicated that nano-Au/PB modified electrochemicalimmunosensor there were good specificity, reproducibility and stability, and the intra-assaycurrent response were-17.47±0.85μA and coefficient of variance were4.86%. After the100cycles CV measurements in PBS buffer, the immunosensor decreased2.49%and the initialcurrent response was-19.64μA. At the same time, after a long time store (30d), just7.97% decrease of the response was observed.4. Electrochemical characterization of the electrode surface through CV is an effectiveand convenient method for probing the features of the modified electrode surface. The CVresults indicated that the BSA/anti-AFP/PA/MWNTs-PB-CHIT/nano-Au/ITO immunosensorwas successfully constructed.5. The results indicated that the optimal the detection solution pH was6.5, incubationtemperature was25.0℃, incubation timewas6minutes.6. Under the optimal experimental conditions, the working range of theBSA/anti-AFP/PA/MWNTs-PB-CHIT/nano-Au/ITO immunosensor was from1.0to250ng/mL with a detection limit of0.5ng/mL. For MWNTs-PB-CHIT modified electrochemic-al immunosensor, there were good specificity,reproducibility and stability, and the intra-assaycurrent response were-20.52±0.51μA and coefficient of variance were2.48%. After the100cycles of CV measurements in PBS buffer, the immunosensor decreased2.24%andthe initial current response was-24.98μA.. At the same time, for the long time stability witha period of30d, just3.56%decrease of the response was observed.Conclusions1. Two amperometric immunosensors BSA/anti-AFP/nano-Au/PB/nano-Au/TIO andBSA/anti-AFP/PA/MWNTs-PB-CHIT/nano-Au/ITO have been studied in this work for thedetermination of AFP, which were developed based on LBL electro-deposition and LBLself-assembly technology. The studied immunosensor exhibits high sensitivity, fastanalytical time, good selectivity, reproducibility and stability,.2. Compared with the BSA/anti-AFP/nano-Au/PB/nano-Au/TIO immunosensor, theBSA/anti-AFP/PA/MWNTs-PB-CHIT/nano-Au/ITO immunosensor exhibited higher respo-nse current signal and stability, the preparation process of immunosensor was more simple.This is mainly attributed to the MWNTs-PB-CHIT nanocomposites with excellent conductiv-ity, large specific surface area, strong adsorption ability and biocompatibility.3. The developed immunosensor model provided a new idea for other tumor markersdetection, which would be a potential clinical diagnostic method.

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