Construction Of Cell Electrochemical Method Based On Graphene Quantum Dot Sensor And Study On Cytotoxicity Of PAHs

Polycyclic aromatic hydrocarbons?PAHs?,as a kind of persistent organic pollutants,are commonly found in air,water,and soil.PAHs could accumulate in organisms through the food chain,thereby endangering human health.At present,PAHs were listed as priority pollutants in many countries.Thus,it was very important to detect and evaluate the toxicity of PAHs in environment rapidly.At present,most of the in vitro cytotoxicity test methods at home and abroad have disadvantages such as complicated pretreatment,time-consuming,high cost,and limited detection range.Therefore,it was of significance to establish a new,efficient,rapid,simple,non-toxic and low-cost method for toxicity detection of environmental pollutants.The electrochemical method has become an important research tool for cell viability detection due to its simplicity,rapidity,high sensitivity,nontoxicity,low-cost and instant detection.Purine nucleotides in cells presented with metabolic abnormalities under the action of exogenous substances,which lead to the changes of purine in the relative content.Electrochemical method can be used to detect changes in the electrochemical signals of purine,and then to evaluate the effect of exogenous substances on cell activity.Therefore,the preparation of modified electrodes with high sensitivity and excellent electrocatalytic activity to purine was the key to the application of electrochemical methods in cytotoxicity detection.In this paper,based on the novel graphene quantum dots?GQDs?,graphene quantum dots/glassy carbon electrode?RGOQDs/GCE?and graphene oxide quantum dots/multi-wall carbon nanotubes/screen-printed electrode?GOQDs/MWCNTs/SPCE*?with high sensitivity,remarkable electrocatalytic activity,low consumption of reagents and wide window potential were prepared.The electrodes were characterized by X-ray diffraction analysis?XRD?,X-ray photoelectron spectroscopy?XPS?,scanning electron microscopy?SEM?,electrochemical impedance spectroscopy?EIS?and transmission electron microscopy?TEM?.The effects of accumulation time,accumulation potential and pH on purine detection were investigated to determine the optimal conditions.Human breast cancer cells?MCF-7?,mouse embryonic fibroblasts?BALB/c 3T3?and chinese hamster lung cells?V79?were used as model cells.Electrochemical methods were used to study the electrochemical behaviors of three cells,which were verified by high performance liquid chromatography?HPLC?.Additionally,the multi-signal cell electrochemical method was established,which was used to evaluate the effects of PAHs on cell activity and to investigate the cytotoxicity mechanism of PAHs.This study provided a new method for cytotoxicity evaluation of PAHs.The specific research results are as follows:?1?Reduced graphene oxide quantum dots?RGOQDs?were prepared by hydrazine reduction method,which were then modified on the surface of glassy carbon electrode?GCE?by electrodeposition to obtain RGOQDs/GCE.The characterization results showed that RGOQDs were still in the amorphous state.The electron conjugation of RGOQDs had changed due to more nitrogen atoms.RGOQDs increased the electrochemically active sites,enhanced the conductivity and improved the sensitivity of the GCE.The electrochemical behaviors of uric acid,xanthine and guanine were studied on RGOQDs/GCE by differential pulse voltammetry?DPV?.The detection limits of RGOQDs/GCE for uric acid,xanthine and guanine were 0.012?M,0.008?M and 0.005?M,respectively.The optimal electrochemical detection conditions were accumulation potential 0 V,accumulation time 360 s,and pH 7.4.Two electrochemical signals were detected at 0.28 V and 0.68 V in the suspension of MCF-7,BALB/c 3T3 and V79 cells,which were attributed to the oxidation of uric acid and xanthine/guanine.A double signal electrochemical system based on RGOQDs/GCE was established,which laid a foundation for cytotoxicity detection of PAHs.?2?The cell electrochemical method based on RGOQDs/GCE was used to evaluate the effects of fluorene on the activities of MCF-7,BALB/c 3T3 and V79 cells by measuring the changes of xanthine/guanine content in cells,and to study the time-effect and dose-effect relationships.The results showed that the optimal action time of fluorene on three types of cells was 30 h.The fluorene exposed to MCF-7 cells with the concentration below 0.050 mM could promote cell proliferation.The concentration of fluorene above 0.050 mM exhibited inhibitory effect on three kinds of cells.The mechanism of cytotoxicity induced by 3-ring fluorene in cells may be that the purine nucleotides presented abnormalities by inducing the expression of p53 protein,leading to the changes of purine content and cytotoxicity.The IC₅₀values of fluorene to MCF-7,BALB/c 3T3 and V79 cells were 2.14 mM,0.89 mM and 0.25mM,respectively.The order of response to the toxicity of fluorene was MCF-7<BALB/c3T3<V79.There were some differences in metabolism of purine nucleotides among different types of cells,thus the cytotoxicities were different.The results of electrochemical method were compared with those of the traditional MTT assay.The order of toxicity of fluorene to three kinds of cells was the same,and the IC₅₀ values detected by electrochemical method were lower than those of the MTT assay,indicating that the sensitivity of electrochemical method was higher than the MTT assay.Therefore,the electrochemical method based on RGOQDs/GCE could be used to detect the cytotoxicity of PAHs.?3?A miniature electrochemical detection system was constructed that integrated graphene oxide quantum dots and multiwall carbon nanotubes modified anodized screen printed carbon electrode?GOQDs/MWCNTs/SPCE*?with a mini reaction vessel,which reduced the consumption of sample from 500 to 80?L.The characterization results showed that GOQDs were incorporated into MWCNTs matrix to form a highly interactive structure,indicating that MWCNTs could be used as a conductive medium for electron transfer between GOQDs and SPCE.The existence of GOQDs/MWCNTs greatly improved the sensitivity of SPCE and expanded the detection range.The uric acid,xanthine,guanine,adenine and hypoxanthine were detected sensitively.The optimal electrochemical detection conditions were the accumulation potential of 0 V,the accumulation time of 240 s,and the pH of 7.4.The electrochemical behaviors of V79,MCF-7 and BALB/c 3T3 cells were studied by this miniature electrochemical detection system.Four electrochemical signals were detected at0.29,0.66,0.93,and 0.98 V,which were attributed to the oxidation of uric acid,xanthine/guanine,adenine and hypoxanthine,respectively.The signal of uric acid was significantly higher than that of RGOQDs/GCE.The contents of uric acid and purine in V79cells was the highest.Compared with MCF-7 cells,the BALB/c 3T3 cells showed lower level expression of purine and higher level expression of uric acid.The results of electrochemical method were consistent with those of HPLC analysis.The miniature electrochemical detection system had the advantages of easy preparation,low sample consumption,and expanded the detection range of cell electrochemistry,which provided a simple and convenient method for the simultaneous detection of purine and uric acid in cells.?4?The effects of six PAHs including fluorene,benzo[a]pyrene,naphthalene,fluoranthene,pyrene,and phenanthrene on the activity of V79 cells were studied by using the miniature electrochemical detection system based on GOQDs/MWCNTs/SPCE*.The results showed that the optimal action time of PAHs on V79 cells was 30 h.The IC₅₀ values and the toxicity order of PAHs on V79 cells were found to be different by using different metabolites as markers.The IC₅₀ values of the six PAHs measured with uric acid as the marker were the lowest,which indicated that the uric acid in cells was more sensitive to PAHs.The toxicity sequence to xanthine/guanine was the same with that of MTT assay,which was pyrene>phenanthrene>benzo[a]pyrene>fluoranthene>fluorene>naphthalene.The toxicity sequences of PAHs measured by different markers were different,which related to the PAHs with different structures might influenced the cellular aromatic hydrocarbon receptor?AhR?,intercellular junction communication?GJIC?and p53 protein.However,PAHs with different structures played different roles in the process of cell development and differentiation.Additionally,there was a significant positive correlation between the IC₅₀ values measured by electrochemical method and those of MTT assay.The IC₅₀ values detected by electrochemical method were lower than those of MTT assay,suggesting that the electrochemical method was more sensitive.The miniature electrochemical detection system based on GOQDs/MWCNTs/SPCE*enabled the simultaneous detection of four signals of cell metabolites,providing more information for multi-parameter evaluation and toxic mechanism study of PAHs and other environmental pollutants.