Electrochemical Biosensors For Detection Of Intracellular Purine Metabolism Based On Graphene Electrode

Objective This thesis aims at in situ detection of four purines including guanine(G), xanthine(X), adenine(A) and hypoxanthine(HX) by electrochemical biosensors with high selectivity, sensitivity, and stability and establish an electrochemical method, which can be used to estimate cell activity and monitor cell cancerization by studying the electrochemical relation between normal and carcinogenesis cell.Methods This thesis uses RGI-GCE that is based on ion liquid(IL) and reduced oxidized graphene(RG) to achieve simultaneous detection of cellular adenine and hypoxanthine in human breast cancer(MCF-7) or mouse embryonic fibroblast(BALB/3T3) cell by linear sweep voltammetry(LSV) detection method. Meanwhile, we take POM@3DGF-GCE that is based on polyoxometalate modified three-dimensional graphene foam to achieve simultaneous detection for guanine and xanthine in MCF-7 cell by cyclic voltammetry(CV). Furthermore, we characterize the surface topography and intrinsic parameter of combination electrode by scanning electron microscope(SEM), transmission electron microscope(TEM), Raman spectrum, and X-ray powder diffraction apparatus. Traditionally high-performance liquid chromatography(HPLC) was utilized to verify the reliability of the proposed electrochemical method.Results It was confirmed that IL and POMs are successfully anchored on RG and 3DGF, and also proved the successful integration of IL and RG or POM and 3DGF by the results of electrochemical behavior. Low-level expression of purine bases in MCF-7 and noncancerous BALB/3T3 cells was first monitored by graphene-based sensor through three electrochemical signals at 0.7, 0.94 and 1.0 V, attributing to the signals of G/X, A and HX, respectively. In comparison with the negative control group, the peak currents increased significantly with growth rates of 300% for A and 317% for HX when it was treated with cancerogenic substance. It should be noted that the three electrochemical signals were first used to monitor the real process of cell canceration. POM with an average diameter of 3.07 nm was obviously observed on the surface of graphene, indicative of nano-property of POM molecules. When it was carried out CV, two oxidation signals ascribed to X and G bases were observed at about 1.05 and 1.13 V respectively, which could not be observed separately by previous reported 1D carbon nanotube or 2D graphene modified GCE. Furthermore, POM@3DGF modified GCE can be used to detect X and G bases in MCF-7 cell lysates, which extends the detection species of purines in cells by electrochemical approach. The results of HPLC verified the reliability of electrochemical method.Conclusions This project introduces nano-materials and advanced carbon material-based hybrids to successfully build perfect biosensors for detection of four purine bases in MCF-7 and BALB/3T3 cells. Moreover, this electrochemical biosensor, with high selectivity, sensitivity, and stability, monitors the real process of cell canceration. As a result, our study opens a promising way to accurate electrochemical assay of cellular purines by IL and POM-based carbon-materials, which is significant for the prediction or diagnosis of disease related to purine metabolism disorder.