Development Of Cell Electrochemical Methods Based On Carbon Nanomaterials Modified Electrodes And Their Application In Toxicity Evaluation Of Pollutants

Water pollution has become one of the most widespread problems in the world.Some heavy metals and phenols have been listed as priority pollutants by the US Environmental Protection Agency?EPA?.As the main pollutants in the water environment,they have the characteristics of wide distribution and high toxicity.They not only cause water pollution but also harm organisms,which have attracted more and more attention of researchers and the public.It is thus vital to establish efficient biological techniques for the early warning and ecological risk assessment.However,most of the conventional in vitro toxicity testing methods were complex,time-consuming,toxic,and high-cost.It is of great importance to develop simple,rapid,non-toxic,and low-cost methods.The cell electrochemistry has emerged as a new research area in the recent decade.The cell electrochemical method can be used to reveal cellular structure-function relationship and evaluate the effect of exogenous molecules by analyzing the electrochemical behaviors of cells.Compared with other in vitro toxicity evaluation methods,it is label-free,non-toxic,simple,sensitive,and rapid.Hence it has become an important tool for the detection of cell viability.Currently,the researches in this area focus on analyzing the mechanism of electrochemical responses and improving the sensitivity with the use of new nanomaterialsmodified electrodes.The clear explanation from the perspective of nucleotide metabolism is that the mixed electrochemical signal is attributed to the oxidation of guanine and xanthine in cell cytoplasm.In contrast,the study on the modified electrodes is still at an early stage.The novel nano-materials are expected to be beneficial in enhancing the peak currents and finding new signals,which will promote the continuous development of cell electrochemistry.In this paper,several electrodes with high catalytic activities were fabricated based on two carbon nanomaterials including graphene and carbon nanotubes.The electrodes were characterized by the scanning electron microscopy?SEM?,X-ray photoelectron spectroscopy?XPS?,X-ray diffraction?XRD?,Raman spectroscopy,energy dispersive X-ray spectroscopy?EDS?,electrochemical impedance spectroscopy?EIS?,and cyclic voltammetry.The electrochemical behaviors of human cervical electricity?HeLa?cells and human hepatoma?HepG2?cells were investigated.The mechanism was further proposed using the high performance liquid chromatography.Cell growth curves were also described.The electrochemical methods based on different nanomaterials-modified electrodes were developed.The cytotoxicity of several heavy metals and phenols was studied and compared with that of the traditional MTT assay.This work provided novel methods for toxicity assessment of pollutants and safety evaluation of water environment.The main results are concluded as follows:1.The graphene/glassy carbon electrode?ERGO/GCE?was fabricated by the electrochemical reduction method,which avoided the complex operation,toxicity,and high experimental requirements of traditional chemical reduction process.The characterization results showed that the oxygenated functional groups of graphene oxide could be effectively removed,thus the electron transfer ability and electrocatalytic performance of ERGO/GCE were significantly enhanced.The electrochemical behaviors of HeLa cells were studied and an obvious oxidation peak ascribed to the oxidation of guanine and xanthine appeared at +0.68 V,which was further verified by the high phase liquid chromatography.The growth curve of HeLa cells was described and the result was consistent with the cell counting method.The electrochemical method was used to evaluate the toxicity of five heavy metals.Significant time-effect and dose-effect relationships were found.The cytotoxicity order was Cr6+ > Cd²⁺ > Cu²⁺ > Pb²⁺ > Zn²⁺,with the 30 h half inhibitory concentration?IC50?values of 5.56,19.68,36.56,57.50,121.00 ?M,respectively.Compared with the traditional MTT assay,the cell electrochemical method based on ERGO/GCE is simpler,faster,and more sensitive.2.The carbon nanotubes-ionic liquid/glassy carbon electrode?MWCNTs-IL/ GCE?was developed,which widened the electrochemical potential window from 0–+0.80 V to 0–+1.10 V.The IL reduced the entanglement of MWCNTs and promoted the electron transfer.The electrochemical impedance of MWCNTs-IL/GCE was almost reduced to zero and the electroactive surface area increased significantly.Two electrochemical signals attributed to guanine/xanthine and adenine/hypoxanthine were observed at +0.72 V and +1.03 V,respectively.The growth curve of HeLa cells was also studied.The cytotoxicity of three chlorophenols was investigated by the two-signal electrochemical method.The IC50 values of 2,4-dichlorophenol?2,4-DCP?,2,4,6-trichlorophenol?2,4,6-TCP?,and pentachlorophenol?PCP?were 451.76,172.51,and 75.92 ?M based on signal I,and 495.76,198.53,and 96.39 ?M based on signal II.The toxicity order was PCP > 2,4,6-TCP > 2,4-DCP,which was consistent with that of the MTT assay.3.The bromocresol purple/graphene/glassy carbon electrode?BCP/erGO/GCE?was fabricated with the electrochemical reduction and electropolymerization methods.The ?-? conjugated structure of graphene was restored,promoting the ?-? stacking with BCP molecules.The electroactive sites and the electron transfer ability were also improved.Two distinct oxidation peaks of HeLa cells were obtained at +0.62 V and +0.83 V,respectively.The BCP/erGO/GCE was used to study the growth curve of HeLa cells.The cytotoxicity of five heavy metals was assessed.The IC50 value for Cr6+,Cd²⁺,Cu²⁺,Pb²⁺,Zn²⁺ were 4.53,15.45,26.36,52.20,and 101.86 ?M based on signal I,and 7.06,21.32,35.81,67.28,and 119.67 ?M based on signal II.The toxicity order was Cr6+ > Cd²⁺ > Cu²⁺ > Pb²⁺ > Zn²⁺.The electrochemical method based on BCP/erGO/GCE?signal I attributed to guanine/xanthine?was more sensitive than that of ERGO/GCE on toxicity assessment of heavy metals.Meanwhile,the stability of BCP/erGO/GCE was significantly improved compare with the MWCNTs-IL/GCE.4.A poly rhodamine B/graphene/multi-walled carbon nanotubes/glassy carbon electrode?PRhB/GO/MWCNTs/GCE?with synergistic effect was developed.The agglomeration of GO nanosheets and MWCNTs was effectively prevented and the electroactive surface area and catalytic activity of the three-dimensional network structure were improved.The electrochemical behaviors of HepG2 cells were studied.Two irreversible oxidation peaks owing to the oxidation of guanine/xanthine and adenine/xanthine were found at +0.58 V and +0.91 V,which were confirmed by the high phase chromatography chromatography.The growth curve of HepG2 cells was also investigated.The cytotoxicity of 2,4,6-TCP and PCP was evaluated with the IC50 values of 201.37 and 71.09 ?M based on signal I and 217.02 and 89.74 ?M based on signal II.The toxicity order was PCP > 2,4,6-TCP.Meantime,the PRhB/GO/MWCNTs/GCE could be used for concentration determination of 2,4,6-TCP and PCP,and revealed the detection limits of 0.8 nM and 0.5 nM.Thus the quantitative detection and toxicity assessment of 2,4,6-TCP and PCP were achieved.5.The graphene oxide quantum dots/carboxylated carbon nanotubes/pencil microelectrode?GOQDs/CMWCNTs/PGE?was developed,which could be used in 96-well plates and saved the amount of cells from 1 mL to 100 ?L.The electrode exhibited excellent electron transfer ability and catalytic activity,thus the mixed signal of adenine/hypoxanthine was separated.Three electrochemical signals attributed to guanine/xanthine,adenine,and hypoxanthine were detected at +0.67 V,+0.94 V,and +1.00 V,respectively.The electrochemical behaviors and cell growth curve of HepG2 cells were also studied.The toxicity of six priority pollutants was evaluated.The IC50 based on the electrochemical signals I,II and III were 6.25,5.62 and 7.99 ?M for Cd²⁺,9.65,8.80 and 9.82 ?M for Hg²⁺,47.95,44.18 and 60.69 ?M for Pb²⁺,446.07,418.21,487.87 ?M for 2,4-dinitrophenol?2,4-DNP?,195.89,178.65 and 202.21 ?M for 2,4,6-trichlorophenol?2,4,6-TCP?,and 68.87,61.24 and 78.71 ?M for pentachlorophenol?PCP?,respectively.The cytotoxic tendency is Cd²⁺ > Hg²⁺ > Pb²⁺ > PCP > 2,4,6-TCP > 2,4-DNP,which agreed with that of the MTT assay.This research was beneficial in providing a low-cost and highly sensitive platform for the study of cellular metabolism,as well as high-throughput analysis of environmental pollutants.