Development Of Group-specific Receptors For Screening Of Multiple Quinolones Residues

The fast screening of mulitiple drug residues in food stuffs is now one of the key problems in the field of food analysis.In this study deoxyribonucleic acid(DNA) and broad-selective antibodies were developed,characterized and validated as group-sepcific receptors for quinolones which are widely used in veterinary as antibiotics.The important results were summerized as following:(1) The ability of DNA to extract qinolones from model solutions and real probes of food was demonstrated and investigated quantitatively.An interaction between quinolones and different types of DNA was studied by equilibrium dialysis.The first application of this direct approach allowed us to determine binding constants and binding stoichiometries in different conditions. The binding of enrofloxacin to heating-denatured DNA(d-DNA) from herring sperm is pH and magnesium dependent;the highest fraction of bound drugs was found at pH 7 and magnesium concentration of 0.5-1 mM.Results for three types of DNA:d-DNA,double stranded DNA(ds-DNA) and single stranded DNA(ss-DNA) were compared.The unwound DNA showed almost doubled binding constants(Ka) and stoichiometries thus indicating on preferable interaction of enrofloxacin with single strand regions of DNA.The binding of other fluoroquinolones(lomefloxacin,ciprofloxacin,norfloxacin,danofloxacin and sarafloxacin) with d-DNA are very similar to that of enrofloxacin:the binding constants are in the range from 0.94×10⁵ to 2.40×10⁵ M^-1,and the stoichiometries are from 4.1 to 6.9 of quinolone molecule per 100 DNA bases.The binding properties were quantitatively the same for extraction of quinolones from model aqueous solutions or from liquid food(milk).The results indicate on the efficiency of DNA for selective extraction of quinolones from real samples for further analysis.This selective binding also allows us to consider DNA as a natural receptor for development of analytical techniques for quinolones.(2) A DNA-based surface plasmon resonance biosensor was developed.Heating denatured DNA immobilized on the gold-coated glass surface was exploited as the broad selective receptor.The immobilization was performed by a layer-by-layer co-deposition with a cationic polymer.The sensor performance was tested with real biological probes.Direct and simple determination of enrofloxacin in milk samples was demonstrated.The sensor response obeys Langmuir binding isotherm being almost linear until about 20μg ml^-1.The detection limit in milk samples was estimated to be 3μg ml^-1.(3) A new molecular modeling mode was developed and validated for both quanlitive and quantitive cgaracterization of quinolones haptens.The quanlitive modeling of 3-D conformations showed that 1-substitute and 7-substitute of quinolones molecules have significant effect on the conformation of haptens;the conformation difference among quinolones is caused mainly by the different substitutes at 1 and 7 positions.The 8-substitute also showed some effect by its inter-reaction with 1-substitute.Then a quantitive model was for the first time developed and validated with 4 quinoloes and respective antibodies.The significant positive correlation between calculated similarity of haptens and cross reactivity of antibodies proves that the developed model can fit well with real properties,which allowed us to suggest its application for screen of hapten as well as the precision of the specificity of respective antibodies.(4) Based on molecular modeling,peifloxacin and a synthesize derivate of ciprofloxacin were chosed as haptens for their high structural similarity with other quinolones.The respective polyclonal antibodies were developed and characterized,and both of them showed expected broad selectivity to quinolones investigated.Antibodies against peifloxacin showed cross-reactivities over 25%to 9 of 10 quinolones investigated,among which the cross reactivity was approximately 70-100%to 6 quinolones.(5)With prepared anti-peifloxacin antibodies both ELISA and a dot immunogold filtration assay(DIGFA) were developed for screening of multiple quinolones residues.Using eel muscles as food samples,at least 6 quinolones drugs can be simultaneously determined.For enrofloxacin,norfloxacin,ciprofloxacin,peifloxacin,ofloxacin and danofloxacin spiked at concentrations of 10μg kg^-1 and 20μg kg^-1,the average recovery of ELISA was approximately more than 70%with a RSD less than 15%.With DIGFA the detection limit for 6 quinolones mentioned above was estimated to be lower than 20μg kg^-1;the detection can be completed in 30 min and the results can be judged by naked eyes instead of instruments, which proves its potenciality for fast field screening of quinolones in food samples.