Abstract
A label free real time method was developed for the detection as well as quantification of botulinum neurotoxin A (BoNT/A) using surface plasmon resonance (SPR). In the present work, antibody against rBoNT/A-HCC fragment and synaptic vesicles (SV) were immobilized on carboxymethyldextran modified gold chip. The immobilization of BoNT/A antibody and interaction of BoNT/A with immobilized antibody were in-situ characterized by SPR and electrochemical impedance spectroscopy. A sample solution containing BoNT/A antigen in the concentration ranging from 0.225 fM to 4.5 fM and 0.045 fM to 5.62 fM was interacted with immobilized antibody and immobilized SV, respectively. By using kinetic evaluation software, KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) values were calculated and found to be 0.53 fM and 38.23 mo for immobilized antibody and 0.22 fM and 116.0 mo for immobilized SV, respectively. Moreover, thermodynamic parameters such as change in Gibb’s free energy (ΔG), change in enthalpy (ΔH) and change in entropy (ΔS) were determined and the values revealed that the interaction between BoNT/A antigen and BoNT/A antibody as spontaneous, endothermic and entropy driven one. In order to optimize the detection method, temperature and pH variation studies were also performed.
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