Comparison of electrochemical immunosensors based on gold nano materials and immunoblot techniques for detection of histidine-tagged proteins in culture medium.

In this work, the direct electrochemical determination of poly-histidine tagged proteins using immunosensor based on anti-His (C-term) antibody immobilized on gold electrodes modified with 1,6-hexanedithiol, gold colloid particles or gold nanorods is described. The recombinant histidine-tagged silk proteinase inhibitor protein (rSPI2-His(6)) expressed in Pichia system selected as antigen for this immonosensor. An electrochemical impedance spectroscopy was used as label free detection technique for immune conjugation. The gold nanorods modified electrode layer showed better analytical response than gold nano particles. The linear calibration range was observed between 10 pg/ml and 1 ng/ml with limit of detection 5 pg/ml (S/N=3). Up to four successive assay cycles with retentive sensitivity were achieved for the immunosensors regenerated with 0.2M glycine-HCl buffer, pH 2.8. The performance of this immnosensor were compared with immuoblotting techniques.

Electrochemical impedance spectroscopy for the study of juvenile hormones-recombinant protein interactions.

The interactions of recombinant juvenile hormone binding protein (His8-rJHBP) with juvenile hormones (JHs), methoprene and farnesol have been studied with electrochemical impedance spectroscopy (EIS). The protein was immobilized on the dodecanethiol (DDT) modified gold electrodes. Each step of electrode modification has been confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The conformation changes of His8-rJHBP upon JHs and methoprene binding have been presented. The EIS determined association constants in the JHs analogs-immobilized His8-rJHBP system indicate that lack of the epoxide moiety in methoprene molecule is not critical for observed high affinity of this compound to the binding region of the His8-rJHBP protein.