Study On The Cholesterol Electrochemical Biosensing Based On Graphene Nanocomposites

The determination of cholesterol has vital significance in clinical diagnosis and food detection fields. Modern electrochemical biosensing technology perfectly combines the nanomaterials with electrochemical analytical methods, which produced a series of electrochemical cholesterol biosensor with excellent performance.In recent years, it has also been found that graphene could act as effective supports to immobilize electroactive enzymes on electrode, which not only could maintain the native bioactivity of immobilized enzymes, but also exhibited the ability to promote electron transfer reaction between the redox center of proteins or enzymes and the surface of electrode when used as electrode material to fabricate mediator free electrochemical biosensors. However, pure graphene is hydrophobic and tends to form agglomerates due to the van der Waals and strong ?-? stacking interactions, thus limit its applications, especially in the field of electrochemical biosenors. It is known that functioned graphene-based nanomaterials can effectively improve the dispersity of graphene in aqueous solution. Furthermore, some nanocomposites can also exhibit synergistic effect of the composed components. Based on that, the performance of the as fabricated electrochemical biosensor can be greatly improved.The nanomaterials based on functionalized graphene have been widely used in the application of electrochemical biosensors, but rarely reported in cholesterol biosensor. For developing highly performance electrochemical biosensors based on graphene, the polyelectrolyte and ionic liquid polymer, DNA, gold nanoparticles and ceramic liposomes, which could improve the dispersion in water and biocompatibility of graphene, were selected to prepare several graphene-based nanocomposites and fabricate distinctive cholesterol biosensors. The applicable performance of the as-prepared biosensors was studied systematically and deeply. The main contents of the thesis are summarized as follows:1. The polyethylenimine functioned graphene (PEI-GP) nanocomposites with good dispersion in water and positively charged were prepared by the modification of PEI onto graphene. The cholesterol biosensor was fabricated by layer-by-layer (LBL) self-assembling cholesterol oxidase (ChOx) on PEI-GP modified glassy carbon electrode (GCE). And the electrochemical behaviors of ChOx in the self-assembled (ChOx/PEI-GP)n film were investigated. Graphene as an conductive nanomaterial greatly improved the direct electron transfer between ChOx and the electrode. The layer by layer self-assembly technology greatly increased the amount of ChOx in the (ChOx/PEI-GP)n film. The surface concentration of ChOx was 1.2×10-10 mol·cm-2. And the ultrathin (ChOx/PEI-GP)n film on the electrode surface provided a favorable microenvironment to keep the bioactivity of ChOx. In addition, PEI used as an out-layer was adsorbed on the top of the (ChOx/PEI-GP)n film greatly improved the stability of the enzyme modified electrode. Accordingly, the developed PEI/(ChOx/PEI-GP)n/GCE biosensor has good reproducibility, stability and anti-interference ability.2. A novel graphene nanocomposite, functionalized by polymeric ionic liquids (PILs) with anion of poly(sodium-p-styrenesulfonate) (PSS) was prepared and exhibited excellent advantages of good conductivity, favorable biocompatibility and intensive film-forming properties as electrode material. Owing to the surface modification for graphene, PSS/PILs-GP can not only be well dispersed in aqueous solution, but also possess a strong charge property. Based on the electrostatic interaction, ChOx can be immobilized onto the surface of PSS/PILs-GP to fabricate ChOx/PSS/PILs-GP/GCE. The experiment results demonstrated that PSS/PILs-GP provided a biocompatible microenvironment for the immobilized redox enzyme, as well as supplied a necessary pathway for its direct electrochemistry. Furthermore, ChOx/PSS/PILs-GP/GCE also displayed a wide linear range of 10.5×10-6-10.4×10-3 mol/L, a low detection limit of 3.5 ?mol/L and good anti-interference ability towards the detection of cholestero. Meanwhile, the biosensor has good performance for application in the determination of cholesterol in serum, and the recovery rate is between 93%-104%.3. ssDNA-GP nanocomposite was prepared by non covalent interaction. The nanocomposite was endowed with excellent properties of the two independent components, such as biocompatibility of the ssDNA and the outstanding electric properties of graphene. Moreover, the existence of ssDNA on graphene could make it negatively charged, effectively prevented the stacking between graphene sheets assisting the dispersion of graphene in aqueous solution due to the electrostatic repulsion and approved the biocompatibility of graphene. The positive charged Au nanoparticles (AuNPs) were achieved by utilizing phase transfer of nanoparticles from organic to aqueous media, which could produce high concentrations of AuNPs with improved monodispersity relative to those prepared in water. Then, AuNPs/ssDNA-GP nanocomposites were prepared via electrostatic self-assembly process and were used to fabricate ChOx/AuNPs/ssDNA-GP/GCE. The modified electrode exhibited good electrochemical performance and commendably realized direct electron transfer of ChOx. Moreover, the modified electrode showed the catalytic linear range of 7.5-280.5 ?mol/L to cholesterol and good stability and reproducibility.4. Cerasomes are a novel organic-inorganic hybrid material composed of spherical lipid bilayer vesicles with an internal aqueous compartment and an inorganic silicate framework covering the vesicular surface. The obtained enzyme-modified electrode by immobilized ChOx on a cerasome modified GCE not only exhibited effective direct electron transfer between the enzyme and electrode surface, but also excellent electrochemical catalytic activity with a wide linear range of 5.0×10-6-3.0×10-3 mol/L towards the oxidation of cholesterol. The excellent catalytic performance of the modified electrode is attributed to the good biocompatibility of the cerasome providing a morphologically stable and soft interface that is suitable for enzymatic immobilization, allowing the enzyme to retain its catalytic activity, along with their specific affinity for water-insoluble cholesterol owing to the lipid bilayer structure of cerasome. The constructed biosensors have good application performance in the detection of real samples, and the recovery rate is between 96%-106%. The results indicate that cerasome are useful as a platform for electrochemical sensing of cholesterol and have the applicable prospects for immobilizing enzyme in biosensors.For improving the conductivity of ceraosome based on electrode, the biocompatible and conductive graphene quantum dots (GQDs), which was assembled onto the negatively charged cerasome modified electrode by alternatively assembling a cationic poly(ethylenimine) (PEI) layer and a ChOx layer through the electrostatic interaction to fabricate ChOx/PEI/GQDs/PEI/cerasome/GCE. The GQDs with negative charge were prepared and characterized by TEM, UV-Vis, fluorescence spectra (FL) and zeta potential measurements. Then, the electrochemical behaviours of the as prepared ChOx/PEI/GQDs/PEI/cerasome/GCE between redox enzyme (ChOx) and the electrode were investigated. Based on the direct electrochemistry of the immobilized ChOx, the modified electrode exhibited excellent catalytic performance for the detection of cholesterol with a linear range of 10.5×10-6-10.4×10-3. Compared with ChOx/cerasome/GCE, the ChOx/PEI/GQDs/PEI/cerasome/GCE exhibited higher electrochemical response and wider detection range for the detection of cholesterol.