Study On Electrochemical Sensors For The Detection Of β-agonists With Nanomaterials And Competitive Immunoassay

In this paper, nanomaterials and competitive immunoassay were used to develop the electrochemical sensors forβ-agonists detection, and the development conditions were optimized, such as fixed antigen/antibody concentration, incubation time and substrate concentration. At the same time, sensitivity, selectivity, reproducibility and stability of developed sensors were evaluated. These sensors were used for the detection ofβ-agonists in real samples.The paper consists the following parts:1 . An electrochemical immunosensor was designed for the detection of clenbuterol (CLB) based on glass carbon electrode modified by the sequential electrodeposition of the gold nanoparticles and the adsorption of CLB-multi-wall carbon nanotubes (MWCNTs) conjugates. Under optimal conditions, the proposed immunosensor displayed a broad linear response and low detection limit to CLB, the working range being 0.3 to 1000 ng/mL with a detection limit of 0.15 ng/mL. The developed immunosensor exhibited other advantages such as simple fabrication, fast response, excellent reproducibility and long stability. Furthermore, a negligible matrix effect and good recoveries were obtained in the determination of spiked animal feeds.2. A new electrochemical immunosensor based on gold nanoparticles (AuNPs), polyaniline (PANI), multi-wall carbon nanotubes (MWCNTs) and horseradish peroxidase (HRP) labeled anti-clenbuterol antibody (AAb)-AuNPs bioconjugates layers was successfully prepared for the detection of clenbuterol (CLB) in this work. The active surface area of MWCNTs/PANI/AuNPs glassy carbon electrode was larger than that of a bare flat one. The presence of AuNPs enhanced the immobilized amount of HRP labeled AAb, which improved the sensitivity of the immunosensor. As a result of these two combined effects, the sensitivity of the immunoassay for the determination of CLB was increased significantly. The proposed immunosensor was proven to be specific to detect CLB between 0.24 ng/mL and 1000.00 ng/mL with a detection limit of 0.12 ng/mL. Good recoveries were obtained in the detection of real sample by using the developing technique.3. A new electrochemical immunoassay with the amplifications of porous Cu₂O-SiO₂ nanoparticles and biotion-avidin conjunction was developed in this work. The Cu₂O-SiO₂ nanoparticles immobilized on immunosensor surface acted as an amplification strategy to enhance the sensitivity of immunoassay. This is because Cu₂O-SiO₂ nanoparticles have good redox electrochemical activity, large surface-to-volume ratio, film-forming ability and high stability. In addition, owing to four identical binding sites of avidin to biotin, large amounts of biotinylated horseradish peroxidases (HRP) were attached, which improved the catalytic performance of the immunosenor. With the two amplification factors mentioned above, a wider linear range from 0.12 to 1000 ng/mL was acquired with a relatively low detection limit of 0.8 ng/mL for ractopamine. The fabricated immunosensor was successfully applied to the detection of ractopamine in real samples.4. An ultrasensitive electrochemical immunosensor based on nanogold particles (nano-Au), Prussian Blue (PB), polyaniline/poly (acrylic acid) (PANI (PAA)) and Au-hybrid graphene nanocomposite (AuGN) has been developed for the detection of salbutamol (SAL). Nano-Au, PB and PANI (PAA)-incorporated film was used to enhance the electroactivity, stability and catalytic activity for hydrogen reduction of the electrode. AuGN was used to immobilise chitosan, nano-Au and horseradish peroxidase-anti-SAL antibody (HRP-AAb). The resulting nanostructure (AuGN-HRP-AAb) was used as the label for the immunosensor. This is attributed to the high surface-to-volume ratio of graphene that allows the immobilisation of a high level of chitosan, nano-Au and HRP-AAb and its good electrical conductivity which can improve the electron transfer among HRP, H2O₂ and electrode. Under the optimized conditions, a calibration plot for SAL was obtained with a linear range between 0.08 ng/mL and 1000 ng/mL (r=0.9955). The detection limit was 0.04 ng/mL. The immunosensor was examined in real feed and pork samples for the analysis of SAL.5. An ultrasensitive electrochemical immunosensor based on chitosan-iron oxide-poly (amino-amine) dendrimers-gold nanoparticles (CS-Fe₃O₄-PAMAM-GNPs) nanocomposites and horseradish peroxidase-multiwall carbon nanotubes-antibody (HRP-MWCNTs-Ab) bioconjugates was developed for the detection of salbutamol (SAL). CS-Fe₃O₄-PAMAM-GNPs nanocomposites as immobilization matrix were used to enhance the electroactivity and stability of the electrode. HRP-MWCNTs-Ab bioconjugates as label were used to improve catalytic activity for hydrogen reduction of the electrode. Under the optimized conditions, a calibration plot for SAL was obtained with a linear range between 0.11 ng/mL and 1061 ng/mL (r=0.9984). The detection limit was 0.06 ng/mL. The immunosensor was examined in real samples for the analysis of SAL.