Graphene Materials-based Optical Biosensing Methods For Oxytetracycline And Human 8-oxo-dG Glycosylase Detection

Antibiotics have been widely used in human life due to its excellent antibacterial ability.However,70%-80%of these antibiotics will excreted into natural environment without being metabolized,leading to serious residue problems in environment and foods,accompanied by a series of toxicity effects.Therefore,for controlling of antibiotics pollution and monitoring water safety,it is of great significance to develop simple,rapid methods for determination of antibiotics and its toxicity effects.Compared to the conventional methods,the graphene materials-based optical biosensing methods show a great potential in rapid detection of antibiotics and its toxicity effects on account of its simple operation,fast response,high sensitivity and specificity.In this thesis,oxytetracycline(OTC)and hOGGl activity,a repair enzyme of the marker of gene damage of antibiotics(8-oxo-dG),were chosen as targets.Focused on the key issues of constructing graphene-based sensing materials with high reactivity and improving the intrinsic shortcomings of sensing probe,several graphene materials-based optical sensing methods have been developed for detection of antibiotics and its toxicity effects.The main contents are listed as follows:(1)Graphene/Au nanoparticles(AuNPs)was fabricated through one-step hydrothermal reduction.The AuNPs uniformly grew on graphene surface and their interface exhibited peroxidase-like catalytic activity.Because of the ?-? interaction between ring structure in nucleobases and hexagonal cells of graphene,single-stranded DNA(ssDNA)could adsorbed onto graphene/AuNPs,inhibiting its catalytic activity.Accordingly,combined with the specific recognition of aptamer,a label-free colorimetric aptamer sensing method was develop for detection of OTC.Under the optimal experimental conditions,the absorbance of system was proportional to the OTC concentration in the linear range of 0.17-0.50 ?mol/L with a limit of quantitation(LOQ)of 0.17 ?mol/L and a limit of detection(LOD)of 0.09 ?mol/L.(2)3D Graphene/Fe3O4-AuNPs was fabricated through one-step hydrothermal reduction.Compared to planar graphene/AuNPs,the spatial structure was helpful to promote the mass transfer of catalytic reaction,and the bimetallic strategy decreased the Fermi level of catalyst and improved the rate of electron transfer,resulting in the improvement of the catalytic activity,the affinity to substrate,the rate of catalytic reaction.Through the kinetic analysis,the Michaelis constant(Km)of 3D graphene/Fe3O4-AuNPs as H2O2 substrate was 0.7‰ of that of graphene/AuNPs.Accordingly,combined with the specific recognition of aptamer,a label-free colorimetric aptamer sensing method was develop for detection of OTC.Under the optimal,experimental conditions,the absorbance of system was proportional to the OTC concentration in the linear range of 0.01-0.25 ?mol/L with a LOQ of 0.01 ?mol/L and a LOD of 0.008 ?mol/L.(3)An indirectly labeled OTC aptamer-based fluorescent probe was designed.In the presence of OTC,the OTC aptamer in the probe would combine to OTC molecule and released the fluorescent short-chain ssDNA which originally hybridized to the aptamer.Accordingly,combined with the fluorescence quenching effect of graphene,an indirectly fluorescence labeled aptamer sensing method was develop for highly sensitive detection of OTC.This strategy avoided the negative effects of intrinsic secondary structure of the long-chain aptamer to sensing performance.Under the optimal experimental conditions,the fluorescence intensity of system was proportional to the OTC concentration in the linear range of 0.01-0.2 ?mol/L with a LOQ of 0.01 ?mol/L.The LOD was 5.17 nmol/L,lowering that of the strategy based on direct fluorescence labeled OTC aptamer by 48.3%.The spiked recoveries for milk and tap water samples were respectively 94-96%and 97-105%.(4)Based on the flexibly regulated peroxidase-like catalytic activity of graphene/AuNPs,a label-free colorimetric sensing method was develop for detection of hOGGl activity with a detection time less than 60 min.The result of agarose gel electrophoresis proved hOGGl possessed DNA terminal protection effect,that is,it could specifically capture the ssDNA which contained the 8-oxo-dG damage site,protecting this ssDNA from hydrolysis.The protected ssDNA would adsorbed onto graphene/AuNPs and inhibited its catalytic activity.Under the optimal experimental conditions,the absorbance of system was proportional to the hOGGl activity in the linear range of 0.02-0.11 U/?L with a LOQ of 0.02 U/?L and a LOD of 0.0016 U/?L.The detection results in lysates of human breast cancer cell(MCF-7)and human normal liver cell(HL7702)based on this method were in accordance with reported research,and the spiked recoveries were 90-92%.In conclusion,combined with specific recognition of functional nucleic acids,several graphene materials-based optical biosensing methods have been developed for sensitive detection of OTC and hOGG1 activity.It shows guiding significance on the development of similar approaches for analyzing other antibiotics.And it also provided new orientation on evaluation of toxicity effect of antibiotics based on biosensing strategy.