| As the elementary unit of organism morphological structure and life activity, cell generates numerous biointeresting molecules, such as reactive nitrogen species(RNS) and reactive oxygen species(ROS), which are a class of radical and non-radical nitrogen or oxygen-containing molecules that display high reactivity with lipids, proteins and nucleic acids. Depending on concentration, location and context, RNS and ROS can be either ??friends?? or ??foes??. Under normal in vivo conditions, RNS and ROS play essential roles in bio-regulatory and cellular signal transduction, cell proliferation and regulating oxidative stress-related states. However, excessive RNS and ROS generation cause damage to brain, proteins, DNA, tissues and lead to cell death, neurodegeneration, atherosclerosis, diabetes, cancer and so on. Therefore, quantitative in-situ monitoring of RNS and ROS released from living cells are very critical to understand mechanisms of cellular functions and pathology while offering important applications in diseases diagnosis and drug discovery. However, in-situ detection of RNS and ROS remains a huge challenge due to the low concentration of RNS and ROS released by cells. In addition, RNS and ROS both possess high activity and short half-life and can easily react with other surrounding molecules. Thus, construction of a sensitive sensing platform with good biocompatibility for cell growth is of extreme importance for the in-situ detection of RNS and ROS. Electrochemical methods exhibits its own advantages, for example, simplicity, relatively low cost, high selectivity and sensitivity, which is widely applied in real time detection of biointeresting molecules. Carbon materials, such as carbon nanotubes and graphene, have been reported in fabrication of electrochemical platform and cell-related investigation.In my Ph D research project, we design and synthesize various functional nanomaterials as synergetic biomimetic enzyme to greatly enhance the stability and selectivity of proposed sensors. Besides, we construct smart sensing interfaces which not only behave good biocompatibility but also provide excellent catalytic activity for real time detection of biointeresting molecules released from living cells. The catalytic mechanism is also deeply explored. In addition, we demonstrates living cells directly growing 2D and 3D free-standing sensing films with good cell adhesion property and smart electrochemical performance, which significantly increases the detection sensitivity of ROS. The thesis is divided into the following parts:1. NO is an important signal molecule released by most cancer cells under drug stimulation or/and disease development. However, it is extremely challenging to real-time sensitively detect NO due to its large diffusivity, low concentrations and fast decay. Herein, shape-controlled reduced graphene oxide nanocomposing with ceria(r GO-Ce O2) was synthesized via hydrothermal reaction to construct a highly sensitive real-time sensing platform for NO detection. The crystal shape of Ce O2 nanoparticles in r GO-Ce O2 composites significantly affects the performance of r GO-Ce O2 sensor, of which the regular hexagonal nanocrystal Ce O2 achieves the highest sensitivity as 1676.06 m A/(cm2?mol/L), a wide dynamic range(18.0 nmol/L to 5.6 μmol/L) and a low detection limit(9.6 nmol/L). This attributes to a synergical effect from high catalytic activity of the specifically shaped Ce O2 nanocrystal and good conductivity/high surface area of r GO. This work demonstrates a great potential to sensitively real-time detect NO released from living cells for studies of complicated biological process while vividly offers a universal approach to rationally compose individual merit components while well control the nanostructure for a superior synagistic effect on a sensing platform.2. O2?-, one of ROS released by cells, is an important bio-regular moleculer which can mediate cellular signal transduction under normal in vivo conditions. cell proliferation anding oxidative stress-related states. However, excessive O2?- generation can deregulate the redox homeostasis which will lead to oxidative stress that causes cell damages and is implicated in various pathological conditions. Thus, in situ quantitative detection of O2?- is very essential for precaution and diagnose of disease. In this work, manganese phosphate spindle-shaped materials(DNA@Mn3(PO4)2) are synthesized and selected as biomimic enzymes for O2?- to functionally modify graphene oxide(GO). The obtained specific biomimetic DNA@Mn3(PO4)2/GO sensor performs high sensitivity(1556.97 m A/(cm2?mol/L)), wide linear range(6.5 nmol/L to 8.85 μmol/L) and low detection limit(2.1 nmol/L). Furthermore, it realizes in situ sensing of O2?-released by human skin cancer cell and normal cell under drug effect, demonstrating perfect real time quantitative detection capability. Results show that O2?- released by cancer cells is 5 times of that released by normal cells.3. It is important to detect ROS in situ for investigation of various critical biological processes, which is however very challenging with limited sensitivity or/and selectivity of the existing methods that are mainly based on sensing the ROS released by cells with short life and low concentration in a culture medium. Herein we report a new approach to directly grow living cells on DNA/Mn3(PO4)2-immobilized and vertically-aligned CNT(VACNT) array nanostructure as a smart free-standing biofilm, of which the DNA/Mn3(PO4)2 and VACNT provides high electroactivity and excellent electron transport, respectively while the directly grown cell on the nanostructure offers short diffusion distance to reaction sites, thus constructing a highly sensitive in-situ detection of cancer cells released ROS under drug stimulations. Compared to the measured ROS release by cells in a culture medium, the detection sensitivity with this constructed biofilm increases by more than 6 times, which implies that ROS molecules(O2?-) secreted from living cells are immediately captured by this smart structure without diffusion process or with extremely short diffusion distance. This design considerably reduces the time from release to detection of the target molecules, minimizing the potential molecular decay due to the short life-time or high reactivity.4. As is known to all, physiologically-biologically relevant cell culture environments are based on three dimensional(3D) growth of human cells. However, most of the reported cell culture platforms are generally performed on two dimensional(2D) platforms, which deviate from the real in vivo environment. Herein we create a 3D culture environment for human Melanoma cells using Pt nanoparticles decorated 3D graphene foam(Pt@GF) through facial and yet green Substrate-Enhanced Electroless Deposition method, which behaves as a specific physical-chemical smart multifunctional bio-scaffold for in-situ detection of ROS released from living cells under drug stimulations. Fluorescence microscope and scanning electron microscope investigations show that cells cultured on 3D Pt@GF composites deliver improved morphology, expression, localization and metabolic activity than that on 2D Pt@GS composites. Compared to the measured ROS released by cells grow on 2D Pt@graphene sheets(Pt@GS), the detection sensitivity with this constructed bio-scaffold increases by 1.4 times. This may owing to electrons can directly transfer along the graphene skeleton between O2?- and GCE surface which functions as a electron transport shortcuts on 3D GF, significantly speed up the charge transfer rate. However, electrons in GS film have to travel much longer distance to reach the broken edge of graphene sheet to cross graphene layers and arrive at GCE surface. Thus, the 3D Pt@GF composite holds great promise for live-cell related investigation such as cellular functions, drug discovery, tissue engineering and regenerative medicine. |
PDF Full Text Request