The Built Of Fluorescence-photoelectric Integrated Equipment For Simultaneously Sensing Of Intracellular And Extracellular Chemicals In Vivo

As the most important part of the central nervous system,the function of the brain is closely related to the homeostasis of different chemicals.The development of efficient in vivo brain analysis methods is of great significance to understand the physiological function and pathological process of the brain.Numerous methods have been reported for in vivo brain analysis,which are mainly used to collect different chemical signals in extracellular or intracellular.However,isolated implementation of extracellular or intracellular chemical signal analysis has great limitations in understanding the molecular mechanisms of the integral brain function.Therefore,it is urgent to develop methods for simultaneous analysis of extracellular and intracellular chemical signals to analyze the molecular mechanisms of brain-related physiological and pathological processes.In view of the key scientific issues in simultaneous analysis of intracellular and extracellular chemical signals in vivo,this thesis focuses on the following two aspects.（1）To address the problem that the in vivo electrochemical analysis technique for extracellular chemical analysis requires the application of additional voltage,an optical fiber-based photoelectrochemical sensor was designed and constructed to achieve highly accurate quantitative analysis of extracellular dopamine（DA）in vivo under near-infrared light（785 nm）excitation.The photoelectrochemical sensor was constructed by assembling layers of optical fiber（OF）,indium tin oxide（ITO）,titanium dioxide nanotubes（Ti O₂）,silver sulfide nanoparticles（Ag₂S）,and ferrocene-labeled DNA aptamer（DNA-Fc）.And OF was a carrier for excitation light transmission and photocurrent signal collection,ITO was used to increase the electrical conductivity of the optical fiber,Ti O₂ and Ag₂S were the main materials for the signal conversion of light-to-electric,DNA-Fc was both an electron donor and a recognition ligand for DA.The sensor showed a significant photocurrent response to DA,with good linearity between photocurrent density and DA concentration in the range of 1.00-500 n M,and detection limit down to 0.30±0.018 n M.By the sensor,it was found that DA concentrations in the cerebral cortex（S1BF）,striatum（CPU）,hippocampal area（CA1）,nucleus ambiguus（NAC）,and ventral tegmental area（VTA）were lower in depressed mice than in normal mice,with the largest decrease（～56.1%）in the CA1 brain region.（2）To address the challenge that the current in vivo brain analysis methods cannot achieve simultaneous analysis of intracellular and extracellular chemicals,a fiber optic sensing platform for simultaneous collection of intracellular fluorescent signals and extracellular photoelectric signals was designed and built,which consists of a laser,a spatial optical path,an electrochemical workstation,and a photoelectric detector with a fiber optic electrode as the core.Based on the sensing platform,the developed photoelectrochemical sensor and DNA fluorescent nanoprobe were further combined for simultaneous quantitative analysis of extracellular DA,intracellular p H and Ca²⁺.By the platform,simultaneous quantitative analysis of extracellular dopamine,intracellular p H and Ca²⁺concentrations in five brain regions（S1BF,CPU,CA1,NAC,VTA）of depressed mice was achieved.It was found that intracellular p H and Ca²⁺in depressed mouse brain cells could regulate dopamine secretion by neurons through acid-sensitive ion channel 1a（ASIC1a）.In addition,ASIC1a channel inhibitors can achieve depression treatment by increasing brain extracellular DA content as well as decreasing intracellular Ca²⁺concentration and increasing p H.