Design And Specific Cell-cell Important For The Detection Of Small Molecules New Nano-catalysts

Numerous bioactive small moleculer species are ubiquitous in living cells, such as H2O2, O2et al., which show various physicological functions and play the key roles in life course. However, the mechanism of these bioactive species on the physiological events is closely related to their cellular level. At a low concentration, they act as a messenger in cellular signal transduction and signal amplification, play an important role as the regulator for a number of oxidative stress-related states, control the proliferation, differentiation and apoptosis of cells. Whereas at a high concentration, they can damage major cellular constituents and harm to living organisms, causing a number of pathological events ranging from aging, diabetes, neurodegeneration, even to cancer. Therefore, the quantitative determination of cellular bioactive species concentration could lead to a better understanding of the clinical consequences of the enhancement in their concentration as well assisting in studies designed to elucidate the biological effect of them in cells. Electrochemical detection method takes advantage of fast response, high specifity, and ease operation. More important, electrochemical detection not only can realize real-time determination of concentration of bioactive species with a great precision, but also can provide the dynamic information on the release process of these bioactive species from cells. Elucidating the detailed electron transfer (ET) mechanism of the redox molecules at the surface of novel electrocatalysts not only helps to understand the major factors that control the ET processes, but also make better design of the new synthesized electrocatalysts, which will significantly improve the detection sensitivity and specificity.In this paper, we synthesized a series of nanocatalysts, and applied them into electrochemical measurement of the concentration of cellular H2O2(the most important representative of small and bioactive moleculer species in cells) and monitor its dynamic release process from the cells. We also chose the nitrogen-doped grapheme and graphyne as study model, studied the mechanism of these bioactive small molecule (H2O2, O2) on their electrocatalytic reduction reations at the surface of nanocatalysts. The main results are as follows:1. Developed a new method for reliable determination of cellular ROS (reactive oxygen species) concentration could lead to a better understanding of the clinical consequences of the enhancement in ROS concentration, elucidating the relationship between ROS and environmental stresses and lipid peroxidation, and assisting in studies of the biological effect of ROS in cells. This work developed and validated an electrochemical approach for measuring the concentration of H2O2in RAW264.7macrophage cells. This approach is based on the electrocatalytic reduction of the releasing H2O2at the biosensor of HRP-Attapulgite/GC, which was fabricated by depositing the horseradish peroxidase-attapulgite nanohybrids on the glassy carbon (GC) electrode. The biosensor exhibited a rapid response (less than2s), a wide linear range (0.2to150μmol/L), a high sensitivity (55.8±2.9μA L/mmol cm2), a low detection limit (0.05±0.01μmol/L), as well as good stability and repeatability due to using the natural mineral (attapulgite) as the enzyme immobilization substrate. In addition, some common coexisting ROS and compounds in biological system such as hypochlorite (OC1-), nitric oxide (NO·), peroxynitrite (ONOO-), and ascorbic acid (AA) etc., did not cause any interference due to the use of a low operating potential (-400mV, versus SCE). Moreover, the developed approach can also be used for studying the effects of the stimulator loading on the generation of H2O2in cells. Therefore, this work has demonstrated a simple and effective sensing platform for detection of concentration of cellular H2O2in cells such as RAW264.7cells, which has potential utility to cellular biology and pathophysiology.2. Modulating the electronic characteristics of graphene is of great technological importance for improving and expanding its applications. Chemical doping with other elements is a promising way to achieve this goal. This work reports a facile synthesis of the nitrogen-doped graphene (N-graphene) at low temperature. This method, which involves the steps of graphite oxidation, exfoliation, and chemical reduction with the use of hydrazine as a reducing agent, can simultaneously realize the reduction of graphene oxide and doping graphene with nitrogen atoms. The spectroscopic results demonstrate that the N-graphene with N/C atomic ratio up to～4.5%can be prepared, and the doping N atoms consist of the pyridinic, pyrrolic, graphitic, and oxidized nitrogen structures with the surface atomic compositions of～28%,49%,19%, and4%, respectively. The prepared N-graphene exhibits the superior electrocatalytic activity toward H2O2reduction, and the contribution of the doped N atoms to the enhanced electrocatalytic activity is explained in details based on density functional theory (DFT) calculation. Moreover, the N-graphene is further used for studying the dynamic process of H2O2(a common representative of reactive oxygen species, ROS, in the living cells) releasing from living cells such as neutrophil, RAW264.7macrophage, and MCF-7cells. The results presented here has opened a new way for synthesizing the N-graphene, and also developed a new platform for a reliable collection of kinetic information on cellular ROS release. The approach established in this work could be potentially useful in study of downstream biological effects of various stimuli in physiology and pathology.3. Elucidating the detailed electron transfer (ET) mechanism of the redox molecules at the surface of novel materials with different microstructural features will helps to understand the mechanism on electrocatalytic reaction. We report here a density functional theory (DFT) study on the microscopically understanding the detailed effects of the bonding configuration of nitrogen-doped graphene (N-graphene) within the carbon lattice such as including pyridinic, pyrrolic, and graphitic N on the activity and mechanistic processes of the H2O2reduction. We simulated the adsorption process of H2O2, analyzed the mechanistic processes and calculated the reversible potential of each reaction step of H2O2reduction reaction at N-graphene. The simulation results indicate that the adsorption of H2O2on the pristine and N-graphene surface occurs via a physisorption without the formation of chemical bond. When H+is introduced into the system, a series of reactions can occur including the breakage of the O-O bond, formation of an O-C chemical bond between oxygen and graphene, and the creation of water molecules. The results also indicate a decrease in the energy of the system and a positive value of the reversible potential for each reaction step. The calculated results on the relative energy of the each reaction step and the value of the onset potential for H2O2reduction suggested the reactivity of pristine and N-graphene has an order of pyridinic N-graphene> pyrrolic N-graphene> graphitic N-graphene> pristine graphene. We also proposed an explanation for this dependence of reactivity order on the doping bond configuration in N-graphene based on the electrostatic potential calculation. The results presented here should help to understand microscopically the dependence of the reactivity N-graphene on its microstructure, inspire the study of heteroatom-doped graphenes to improve their catalytic efficiency, and provide a theoretical framework to analyze their catalytic activities.4. Graphyne, a new two-dimensional periodic carbon allotrope with a one-atom-thick sheet of carbon built from triple-and double-bonded units of two sp-and sp2-hybridized carbon atoms, has been shown in recent studies to have the potential for high-density hydrogen and lithium storage. We report here a density functional theory (DFT) study of an oxygen reduction reaction (ORR) involving graphyne and demonstrate that graphyne is a good, metal-free electrocatalyst for ORRs in acidic fuel cells. We optimized the geometrical structure, calculated the charge densities on each carbon atom in the graphyne, and simulated the each step of the ORR reaction involving graphyne. The simulation results indicate that the distribution of the charge density at each carbon atom on the graphyne plane is not uniform and that a large number of positively charged carbon atoms, which are beneficial to the adsorption of O2and OOH+molecules, can behave as catalytic sites to facilitate ORRs. When H+is introduced into the system, a series of reactions can occur including the formation of an O-C chemical bond between oxygen and graphyne, breakage of O-O bond, and the creation of water molecules. The results also indicate a decrease in the energy of the system and a positive value of the reversible potential for each reaction step on the graphyne surface. In addition, a spontaneous electron transformation process occurs during the ORR along a four-electron pathway. The results presented here should lead to an improvement in the catalytic efficiency of carbon nanomaterials and provide a theoretical framework for the analysis of their catalytic activity. This paper highlights the urgent need for new experimental syntheses for graphyne.5. The aptamer (S2.2)-guided Ag-Au nanostructures (aptamer-Ag-Au) have been synthesized by photoreduction and validated by UV-vis spectra and transmission electron microscopy (TEM) images. Differential interference contrast (DIC), fluorescence, and TEM images indicated that the aptamer-Ag-Au nanostructures can be targeted to the surface of human breast cancer cells (MCF-7) with high affinity and specificity. This targeting is completed via the specific interaction between S2.2aptamer (a25-base oligonucleotide) and MUC1mucin (a large transmembrane glycoprotein, whose expression increased at least10-fold at MCF-7cells in primary and metastatic breast cancers). However, the nanostructure cannot be targeted with HepG2(human liver cancer cells) or MCF-10A cells (human normal breast epithelial cells) due to these cells are MUCl-negative expressed. Moreover, the synthesized nanostructures exhibited a high SERS activity. Based on these results, a new assay has been proposed for high specifically detecting MCF-7cells, even down to a single cell. This assay can also discriminate MCF-7cells from MCF-10A cells and different cancer cell lines such as HepG2cells. In addition, the aptamer-Ag-Au nanostructures have high capability of adsorption NIR irradiation and are able to perform photothermal therapy of MCF-7cells at a very low irradiation power density (0.25W/cm2) without causing the destroy of the healthy cells and the surrounding normal tissues. Therefore, the proposed assay is significant for diagnosis of the tumors in its nascent stage. The synthesized nanostructures could offer a protocol to specifically recognize and sensitively detect the cancer cells, and would have a great potential useful in photothermal therapy of the cancers.