The interaction between 3-thiol-4-(2,4-dichlorobenzylideneamino)-5-methyl-4H-1,2,4-triazole (CBTZ) and bovine serum albumin (BSA) under physiological conditions was investigated by fluorescence,UV-vis absorption and c...The interaction between 3-thiol-4-(2,4-dichlorobenzylideneamino)-5-methyl-4H-1,2,4-triazole (CBTZ) and bovine serum albumin (BSA) under physiological conditions was investigated by fluorescence,UV-vis absorption and circular dichroism (CD) spectroscopy as well as molecular modeling methods. The result of fluorescence experiment indicates the static quenching as a result of the formation of the CBTZ-BSA complex. The binding constants (Ka) at different temperatures were calculated according to the modified Stern-Volmer equation. The enthalpy change (-H) and entropy change (-S) were determined based on the van′t Hoff equation. Both negative-H and-S indicated that van der Waals and hydrogen-bonding forces were the dominant intermolecular forces to stabilize the CBTZ-BSA complex. Site marker competitive replacement experiments demonstrated that binding of CBTZ to BSA primarily took place in sub-domain IIA (Sudlow's site I). The binding distance (r = 7.2 nm) between CBTZ and the tryptophan residue of BSA was estimated according to the theory of fluorescence resonance energy transfer (FRET). The conformational studies by circular dichroism (CD) and three-dimensional fluorescence spectroscopy showed that the presence of CBTZ induced minor changes of the secondary structure of BSA. Molecular modeling study further confirmed the binding mode obtained experimentally.展开更多
Phage contamination is a very serious and unavoidable problem in modem fermentation industry. It is necessary to develop sensitive and rapid phage detection methods for the early detection of phage contamination. In t...Phage contamination is a very serious and unavoidable problem in modem fermentation industry. It is necessary to develop sensitive and rapid phage detection methods for the early detection of phage contamination. In the present work, a real-time, rapid, specific and quantitative phage T4 detection method based on surface plasmon resonance （SPR） technique has been in- troduced. Escherichia coli was immobilized onto the preformed MPA self-assembled monolayer （SAM） through the widely used EDC/NHS cross-linking reaction as the recognition element. The bacteria immobilization was verified efficiently through the electrochemical measurements and fluorescence microscopy observations. The specific adsorption was much stronger than the non-specific adsorption of phage T4 binding to the biosensor surface modified by E. coli, and the latter could be neglected. The detection sensitivity reached 1×10^7 PFU/mL within 10 min. Within the experimental phage concentrations, the linear cor- relation between the SPR response and the phage concentration was good. The results suggest that the SPR technique is a po- tentially powerful tool for the phage or other virus detections, as a label-free, real-time, and rapid method.展开更多
基金supported by the National Natural Science Foundation of China (20873096, 20921062 and 20621502)Fundamental Research Funds for Central Universities (1101007)
文摘The interaction between 3-thiol-4-(2,4-dichlorobenzylideneamino)-5-methyl-4H-1,2,4-triazole (CBTZ) and bovine serum albumin (BSA) under physiological conditions was investigated by fluorescence,UV-vis absorption and circular dichroism (CD) spectroscopy as well as molecular modeling methods. The result of fluorescence experiment indicates the static quenching as a result of the formation of the CBTZ-BSA complex. The binding constants (Ka) at different temperatures were calculated according to the modified Stern-Volmer equation. The enthalpy change (-H) and entropy change (-S) were determined based on the van′t Hoff equation. Both negative-H and-S indicated that van der Waals and hydrogen-bonding forces were the dominant intermolecular forces to stabilize the CBTZ-BSA complex. Site marker competitive replacement experiments demonstrated that binding of CBTZ to BSA primarily took place in sub-domain IIA (Sudlow's site I). The binding distance (r = 7.2 nm) between CBTZ and the tryptophan residue of BSA was estimated according to the theory of fluorescence resonance energy transfer (FRET). The conformational studies by circular dichroism (CD) and three-dimensional fluorescence spectroscopy showed that the presence of CBTZ induced minor changes of the secondary structure of BSA. Molecular modeling study further confirmed the binding mode obtained experimentally.
基金support from the National Basic Research Program of China (2011CB933600)the National Natural Science Foundation of China (21077081,20921062)+1 种基金the Natural Science Foundation of Hubei Province (2010CDB01302)the Fundamental Research Funds for Central Universities (1103005 and 1101007)
文摘Phage contamination is a very serious and unavoidable problem in modem fermentation industry. It is necessary to develop sensitive and rapid phage detection methods for the early detection of phage contamination. In the present work, a real-time, rapid, specific and quantitative phage T4 detection method based on surface plasmon resonance （SPR） technique has been in- troduced. Escherichia coli was immobilized onto the preformed MPA self-assembled monolayer （SAM） through the widely used EDC/NHS cross-linking reaction as the recognition element. The bacteria immobilization was verified efficiently through the electrochemical measurements and fluorescence microscopy observations. The specific adsorption was much stronger than the non-specific adsorption of phage T4 binding to the biosensor surface modified by E. coli, and the latter could be neglected. The detection sensitivity reached 1×10^7 PFU/mL within 10 min. Within the experimental phage concentrations, the linear cor- relation between the SPR response and the phage concentration was good. The results suggest that the SPR technique is a po- tentially powerful tool for the phage or other virus detections, as a label-free, real-time, and rapid method.
