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Prepararion of the Label-Free Electrochemical Immunosensor

Author: WeiZhiJing
Tutor: LinJieHua
School: Qingdao University of Science and Technology
Course: Analytical Chemistry
Keywords: Immunosensor Label-free Tumor markers MultianalyteMesoporous silicon Nano-materials
CLC: TP212.2
Type: Master's thesis
Year: 2012
Downloads: 46
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Abstract

In this paper, the finished major works are as follows,1. A label-free electrochemical immunosensor for the detection of a-fetoprotein (AFP) is proposed based on controlled fabrication of gold nanoparticles (GNPs) and monoclonal antibodies of AFP (anti-AFP) inside the pores of mesoporous silica (MPS). The MPS precursor was mixed with trimethylchlorosilane (TMCS) firstly to block the external surface silanol groups, afterwards the internal pore walls silanol groups were grafted by aminopropyltriethoxyl silane (APTS). Then the obtained modified mesoporous silicon was named as TMCS-MPS for short. The GNPs and anti-AFP were confined inside the mesopores of TMCS-MPS by the covalent linking with the amino groups, thus formed the label free immunoprobe (anti-AFP/GNPs/TMCS-MPS). The prepared immunoprobe were used to modify glassy carbon electrode (GCE) to construct a label-free electrochemical immunosensor. After incubating the sample AFP with the immunosensor, the immunoconjugates were formed on the surface of GCE and the spatial block increased. Thus, the peak current decreased with increasing concentration of AFP. The GNPs inside the mesopores could promote the electron transportation through the pore channel. Under the optimal experimental conditions, the fabricated immunosensor could detect AFP in a linear range from1.0to90ng mL-1with a detection limit of0.2ng mL-1(3a).2. A novel sensitive label-free electrochemical immunosensor was fabricated for the detection of AFP. The single wall carbon nanoparticle tubes (CNTs) were pretreated to reduce the length and modification with carboxyl functional groups at the ports. Then the label free immunoprobe anti-AFP/CNTs/TMCS-MPS was prepared by encapsulating the obtained CNTs and anti-AFP into the channel pores of modified MPS (TMCS-MPS) in sequence. The sensitive label free electrochemical immunosensor was fabricated by coating the graphene sheet solution (GS) and immunoprobe on the GCE surface layer by layer. After incubated in the sample AFP solution, immunoconjugates were formed inside the mesopores and resulted in the increment of spatial blocking and impedance. Hence, the current signals decreased with the increasing of antigen concentration. With the existence of CNTs in the mesopore and the bottom GS, the electron transport effectiveness through the electrode surface was greatly improved. Under the optimized conditions, the manufactured immunosensor could detect AFP in a linear range from0.1to100ng mL-1with a detection limit of0.06ng mL3. A label-free multiplexed immunoassay strategy was proposed for the simultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) and AFP. Monoclonal antibody of CEA was co-immobilized with ferrocenecarboxylic acid (FCA) inside the channels of modified MPS to prepare the label-free probe for detection of CEA. Monoclonal antibody of AFP was co-immobilized with horseradish peroxidase (HRP) inside the channels of MPS to prepare the label-free probe for detection of AFP by using o-phenylenediamine (OPD) and H2O2as the electrochemical substrates. Thus, the multianalyte immunosensor was constructed by coating the probes of CEA and AFP respectively onto the different areas of indium-tin oxide (ITO) electrode. When the immunosensor was incubated with sample antigens, CEA and AFP antigens were introduced into the mesopores of MPS after the immunoassay reaction. The nonconductive immunoconjugates blocked the electron transfer and the peak responses changed on the corresponding surface respectively. Then, the simultaneous detection of CEA and AFP achieved. The determination linear ranges for CEA and AFP were0.5-45ng mL-1and1-90ng mL-1, and the detection limits were0.2and0.5ng mL-1(S/N=3), respectively.4. A label free electrogenerated chemiluminescence (ECL) immunosensor for the determination of tumor marker was constructed based on the TMCS-MPS encapsulated CdS quantum dots (QDs) modified electrode. By applying TMCS-MPS, the binding of CdS QDs was confined in the MPS mesopores. Therefore, the nanohybrids of CdS/TMCS-MPS were obtained. By selecting AFP as the detection target, the anti-AFP were co-immobilized in the CdS/TMCS-MPS, and then the label free immunoprobe of anti-AFP/CdS/TMCS-MPS was prepared. By coating the immunoprobe on the surface of GCE electrode with poly (diallyldimethylammonium chloride)(PDDA), the label-free ECL immunosensor was constructed. When incubated with AFP antigen, immuno-complexes were formed inside the channel pores of MPS, which increased the spatial blocking and impedance. Thus, the ECL intensity decreased with the increment of AFP concentration. Under the optimum operation conditions, AFP could be detected in a linear range from0.5to100ng mL-1with a detection limit of0.39ng mL-1(3σ).

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