| Based on the significance of determination of antibiotic, the characteristic and application of self-ordered ring (SOR) fluorescent microscopic imaging technique, in this paper we developed the methods for the determination of sparfloxacin (SPFX) and chlortetracycline (CTC), and employed the developed methods to detect the content of above drugs in serum, urine, muscle, liver, manure, kidney and dung respectively. Besides, The interactions of rolitetracycline (RTC), fleroxacin (FLRX), demeclocycline (DMCTC), methacyline (METC), and human serum albumin (HSA were studied by fluorescence spectra, UV-vis absorption spectra and FT-IR spectra and molecular modeling methods.Based on the self-ordered ring (SOR) fluorescence microscopic imaging technique on a hydrophobic glass slide with Zn2+and cetyltrimethyl ammonium bromide (CTMAB) as sensitizer, and poly (vinyl alcohol)-124(PVA-124) and NH3-NH4Cl (pH10.00) as the medium, a method has been developed for determining sparfloxacin (SPFX) concentrations in chicken serum and residues in chicken tissues and manures. When the droplet volume was0.20μL, SPFX was determined in the range of1.38×10-13-2.03×10-12mol-ring-1(or6.9×10-7-1.02×10-5mol.L-1), and the limit of detection (LOD) was14fmol-ring-1(or6.9×10-8mol·L-1). The recoveries of SPFX at all different spiked levels are in the range of90.74%-106.61%when the methanol or acetonitrile were used as extracting agent, respectively, and the relative standard deviations (RSD) are less than3.0%. This study expands the applied fields of SOR technique in drug concentrations and residues determination.With Zn2+and SDBS as sensitizer, PVA-124and Piperidine (0.06mol·L-1) as the medium, a simple and sensitive SOR technique was successfully developed to determine chlortetracycline (CTC). When the droplet volume is0.2μL, CTC in the range of1.40×10-13～1.62×10-12mol·ring-1(7.00×10-7~8.10×10-6mol·L-1) can be detected, and the detection limit can reach14.0fmol (7.0×10-8mol·L-1). This developed methods was applied to determine CTC concentrations in rabbit serum, the recoveries of CTC at all different spiked levels are in the range of92.5～105.6%, respectively, and the relative standard deviations (RSD) are less than6.1%. Furthermore, the milk, beef, cow liver, cow kidney, bovine urine and cow dung of Xilinguole were determined by using SOR methods, it was proved to be a viable method for analysis of CTC in those samples with the recoveries92.3～101.7% and RSDs less than3.0%.The binding of rolitetracycline (RTC) to human serum albumin (HSA) was analyzed by means of multi-spectroscopy and molecular modeling. The inner filter effect was eliminated to get accurate binding parameters. Fluorescence and UV-vis results revealed the presence of static quenching mechanism in the binding of RTC to HSA. Binding constants (Ksv) and the number of binding sites (n) were calculated using Stern-Volmer equations. The thermodynamic parameters (enthalpy change,△H and entropy change,△S) were calculated to be -95.86KJ mol-1and-216.48J mol-1K-1via the van’t Hoff equation, which indicated that the interaction of RTC with HSA was driven mainly by hydrogen bond and van der Waals force. RTC binds to the Sudlow’s drug binding site I in HSA, and the binding distance between them was calculated as2.58nm based on fluorescence resonance energy transfer. The three dimensional fluorescence showed that the conformation of HSA was changed after its complexation with RTC, and the alternations of protein secondary structure were quantitatively calculated from FT-IR with reduction of α-helices content about8.4%, with increases of β-sheet from30.3%to31.4%and β-turn from15.6%to16.1%. Furthermore, the binding details between RTC and HSA were further confirmed by molecular docking studies, which revealed that RTC was bound at subdomain ⅡA through multiple interactions, such as hydrogen bond, hydrophobic, polar forces, etc.The interaction of Fleroxacin (FLRX) and human serum albumin (HSA) has been studied by multi-spectroscopy and molecular modeling methods. The results showed that the fluorescence of HSA was quenched by FLRX in a static quenching mechanism. The thermodynamic parameters (enthalpy change, AH and entropy change,△S) were calculated to be-107.99KJ·mol-1and-240.99J·mol-1·K-1via the van’t Hoff equation, which indicated that the interaction of FLRX with HSA was driven mainly by hydrogen bond and van der Waals force. The calculated binding distance (r=2.58nm) indicated that the non-radioactive energy transfer coming into being in the interaction between FLRX and Trp-214 of HSA. Synchronous fluorescence and three-dimensional fluorescence spectra indicated the conformation of HSA changed after adding FLRX, and the alternations of protein secondary structure were quantitatively calculated from FT-IR with reduction of a-helices content about18.3%, with increases of β-sheet from30.3%to20.7%and β-turn from15.6%to30.3%. Docking experiments uncovered the binding mode of FLRX in HSA molecule.This study was designed to examine the interaction of demeclocycline (DMCTC) with human serum albumin (HSA) by multi-spectroscopic and molecular docking methods. The inner filter effect was corrected before we calculated the binding parameters. Fluorescence and UV-vis spectroscopy revealed that DMCTC induced the fluorescence quenching of HSA though a static quenching procedure. Thermodynamic analysis by Van Hoff equation found enthalpy change (△H) and entropy change (△S) were-53.01KJ·mol-1and-65.13J·mol-1·K-1, respectively, which indicated hydrogen bond and van der Waals force were the predominant force in the binding process. According to fluorescence resonance energy transfer (FRET), the specific binding distances between Trp-214(donor) and DMCTC (acceptor) were3.18nm. Through site marker competitive experiments, subdomain ⅡA of HSA has been assigned to possess the high-affinity binding site of DMCTC. The three dimensional fluorescence showed that the conformation of HSA was changed after its complexation with DMCTC, and the alternations of protein secondary structure were quantitatively calculated from FT-IR with reduction of a-helices content about4.8%,β-sheet from30.3%to21.6%and with increases of β-turn from15.6%to22.2%. Furthermore, the binding details between DMCTC and HSA were further confirmed by molecular docking studies, which revealed that DMCTC was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, polar forces,π-π interactions, etc. Moreover, the coexist metal ions such as Al3+, Fe3+, Cu2+, Cr3+and Cd2+can decrease the binding constants of DMCTC-HSA.Multi-spectroscopy and molecular modeling methods were used to study the interaction of methacyline (METC) with human serum albumin (HSA) under simulative physiological conditions. The quenching mechanism was suggested to be static quenching by monitoring the fluorescence and ultraviolet-visible (UV-vis) spectroscopy. According to the Vant’Hoff equation, the values of enthalpy (△H) and entropy change (△S) were calculated to be-95.29KJ-mol-1and-218.13J·mol-1·K-1, indicating that the main driving force of the interaction between HSA and METC were hydrogen bond and van der Waals force. By performing displacement measurements, the specific binding of METC in the vicinity of Sudlow’s site I of HSA had been clarified. An apparent distance of3.05nm between the Trp-214and METC was obtained via fluorescence resonance energy transfer (FRET) method. Furthermore, the binding details between METC and HSA were further confirmed by molecular docking studies, which revealed that METC was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, polar forces, hydrogen bonding, etc. The results of three-dimensional fluorescence and fourier transform infrared (FT-IR) spectroscopy showed that METC caused conformational and some microenvironmental changes of HSA and deduced the a-helix significantly reduce in the range of52.3%-40.4%in HSA secondary structure. Moreover, the coexist metal ions such as Ca2+, Al3+, Fe3+, Zn2+, Cu2+, Cr3+and Cd2+can decrease the binding constants of METC-HSA. |
PDF Full Text Request