Research On High Performance Electrochemical Biosensor And In Situ Analysis System For Cellular ATP Release Monitoring

Adenosine triphosphate(ATP)acts as a high-energy phosphate compound of an organism,can also be released as an important neurotransmitter from cells,and plays a significant role in physiological processes,such as the regulation of neural information,immune response,systemic damage,tissue damage and death,normal body development and homeostasis regulation,apoptosis and tumor cell clearance and so on.However,the mechanism of cellular ATP release and regulation is still unclear.Revealing the mechanism is crucial for understanding the pathogenesis,developing targeted drug therapies and unraveling the mystery of cell signal transduction.Therefore,it is of great significance to realize in situ monitoring of cellular ATP release from living cells.The electrochemical ATP biosensor based on glucose oxidase/hexokinase has been used for in vitro or in vivo ATP release detection,due to its unique high sensitivity,fast response,continuous monitoring,high temporal and spatial resolution.Considering ATP release with low concentration and short time,a high performance electrochemical ATP biosensor with high sensitivity,low detection limit,fast response,high selectivity,long-term stability and a high resolution,fast,dynamic in situ analysis system are required.However,there still remain some problems,such as complicated preparation process of microelectrode,too small electrode effective area,low enzyme activity and poor long-term stability.In order to resolve these problems,researches on screen-printed electrode H2O2 biosensors,performance optimization of screen-printed electrode ATP biosensors,platinum nanorods decorated planar microelectrode array ATP biosensors,3D gold-platinum nanoflowers decorated needle-type microelectrode ATP biosensors,in situ analysis system for cellular ATP release have been carried out.The system has been successfully applied for in-situ monitoring of ATP release from PC 12 cells induced by high potassium solution.The main contents and innovations of this work lies in:1.Propose a one-step electrodeposition method to immobilize pristine graphene and obtain an enzyme-free hydrogen peroxide biosensor based on layer-by-layer chitosan-decorated pristine graphene modified screen-printed electrodes,with enhanced sensitivity,wide linear range and low limit of detection.The film was characterized by scanning electron microscopy,Fourier infrared spectroscopy,Raman spectroscopy,X-ray photoelectron spectroscopy and electrochemical cyclic voltammetry.The H2O2 biosensor is the basis of ATP biosensor and its mechanism study lays the foundation for ATP biosensors.The proposed method can be extended to the immobilization of other nanomaterials and biomolecules.2.Realize a dual enzyme ATP biosensor based on screen-printed electrode.The effects of platinum deposition voltage and deposition time,glutaraldehyde dosage and cross-linking time,enzyme layer structure and Mg2+ concentration in PBS on biosensors sensitivity were explored and optimized respectively.The sensitivity after optimization was 20.1μA mM-1 cm-2.The key parameters for ATP biosensor fabrication were explored,contributing to microelectrode ATP biosensor fabrication.3.Realize ATP biosensors based on Pt nanorods modified planar microelectrode array,contributing to ATP biosensors for cellular ATP release monitoring.Optimize the preparation conditions of Au-Pt composite structure and realize a ultramicroelectrode ATP biosensor.Optical characterization was used to verify the successful fabrication of microelectrode array.The sensitivity was increased by 27 times and the detection limit was as low as 20 μM after Pt nanorods modification.4.Propose a simple strategy for microelectrode packaging,realize template-free electrodeposition of 3D bimetallic Au-Pt nanoflowers and construct a needle-type microelectrode ATP biosensor,expected to be used in cellular ATP release monitoring.The nanoflowers were characterized by scanning electron microscopy,energy scattering spectroscopy and electrochemical impedance spectroscopy,and the effects of HAuCl4 concentration and Au deposition time on the sensitivity were also explored.The optimized ATP sensitivity was 2.28±0.19 nA μM-1 mm-2,with a range of 2.5 μM～447 μM and a detection limit of 2.5 μM(S/N=3).After two weeks stored in PBS at 4 ℃,the ATP sensitivity remained 79.39 ±9.15%and the glucose sensitivity remained 95.44±6.97%.The excellent performance of ATP biosensor was due to the excellent catalytic activity and large electroactive surface area provided by the nanoflowers,which helps maintain enzyme activity and long-term stability.5.Design and implement a monitoring and analysis system for ATP release from cells,and successfully apply it for in situ monitoring of PC 12 cells induced by high potassium solution.Design and complete the system integration,select target cell and stimulus solution,consider the validity and comparison experiments.The decreased current was approximately 21.6±3.4 nA(N=6),corresponding to an ATP concentration of 12.2±2.8 μM,right in the micromolar range and consistent with the suggested levels.The system can be used for the monitoring of ATP concentration during glycolysis and apoptosis,and can also be extended for in situ monitoring studies of other neurotransmitters and cell secretions.