This work has explored the development of impedimetric immunosensors based on a conducting copolypyrrole film to which a histidine tag antibody has been bound using a complex NTA chelator coordinated with copper as metal. The electrical properties of the copolymer film have been characterized by Electrochemical Impedance Spectroscopy (EIS). The experimental study of EIS was performed over a range of dc voltages from 0 to -1.4 V (vs saturated calomel electrode SCE) where the polypyrrole layers transit from their conducting to insulating state and to demonstrate the contribution of the copper complex as redox probe. The electrical behavior of the biomaterial at these various potentials was studied according to an equivalent circuit fitting the experimental parameters. The results indicated that better immunosensor characteristics were obtained at an applied potential of -1400 mV where the reduction of copper occurs. The impedance obtained at this potential shows a linear variation of charge transfer resistance (R ct ) with the specific antigen concentration in the range of 0.1 ng mL -1 to 10 ng mL -1 with a detection limit of 0.1 ng mL -1 . Moreover, this study demonstrates that these immunosensors exhibit high sensitivity, stability and reproducibility.
This work has explored the development of impedimetric immunosensors based on magnetic iron nanoparticles (IrNP) functionalized with streptavidin to which a biotinylated FAB part of the antibody has been bound using a biotin-streptavidin interaction. SPR analysis shows a deviation on the measured (angle) during antigen-antibody recognition whereas label free detection using by EIS allows us to monitor variation of polarization resistance. Before detection, layers were analyzed by FTIR and AFM. Compared to immobilization of antibody on bare gold surface using aminodecanethiol SAM, antibody immobilization on nanoparticles permitted to reach lower detection limit: 500 pg/ml instead of 1 ng/ml to in the case of EIS and 300 ng/ml instead of 4.5 μg/ml in the case of SPR. Thus, it permitted to improve the sensitivity: from 257.3 Ω · cm 2 · μg −1 · ml to 1871 Ω · cm 2 · μg −1 · ml in the case of EIS and from 0.003• μg −1 · ml to 0.094 • μg −1 · ml in the case of SPR.
In this work, we describe an approach of detecting biomarkers by Surface Plasmon Resonance imaging (SPRi) technique in real samples. Two C-Reactive Protein (CRP)-antibody immobilization methods were used: The first method was based on direct physisorption of CRP-antibody onto gold surface; the second one was based on oriented CRP-antibody with protein G intermediate layer.The two developed immunosensors were tested against CRP antigen in phosphate buffer saline solution with the SPRi technique. The response of the developed immunosensors was reproducible and stable. The detection limit of 10 pg•mL −1 and 50 pg•mL −1 CRP-antigen was observed with and without protein G respectively with this technique. Moreover, the developed SPRi immunosensor was used for CRP-antigen detection in human plasma. A detection limit of 5 ng•mL −1 and 10 ng•mL −1 was obtained with and without protein G respectively. These obtained results were compared to those obtained with QCM (Quartz Crystal Microbalance) and Enzyme-Linked Immunosorbent Assay (ELISA) techniques.
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