In Vivo Electrochemical Analysis Under Oxidative Stress

Oxidative stress refers to the imbalance between oxidants and anti-oxidants,in favor to oxidants.Excessive reactive oxygen species?ROS?cause cytotoxicity,which in turn leads to organism damage.Brain,with the highest oxygen demand,is more susceptible to oxidative stress than other organs,which in turn triggers a variety of brain diseases such as epilepsy,ischemia and so on.When oxidative stress occurs,various ions such as H+,Ca²⁺,K⁺,neurotransmitters such as glutamate,?-aminobutyric acid?GABA?etc.are involved.Therefore,insight to the chemical nature of oxidative stress is of great significance for the understanding of physiological and pathological process of oxidative stress related brain diseases.Electrochemical methods are widely used in brain analysis because of their excellent chemical specificity,high temporal and spatial resolutions,and real-time monitoring in vivo features.However,due to the complexity of brain microenvironment and the numerous interrelated neurochemicals,it is still a great challenge in analytical chemistry to establish new approaches for in vivo analysis with high selectivity,high sensitivity,high accuracy and simultaneous detection of multiple neurochemicals.Based on extensive literature research,this paper aims to solve the key scientific problems existing in the in vivo electrochemical analysis under oxidative stress.Novel electrochemical methods were designed and developed,and were summarized as follows:?1?In vivo monitoring of local pH values in a live rat brain based on the design of a specific electroactive molecule for H+.In this work,we designed and synthesized N-?6-aminopyridin-2-yl?ferrocene?Fc-Py?,with a specific recognition element for p H–pyridine?Py?,and an electroactive element–ferrocene?Fc?,as an electrochemical probe for determination of p H.The mid-point potential E1/2 of Fc-Py positively shifted with decreasing p H due to the increase in the electron-withdrawing power of the pyridine ring upon protonation of the pyridyl nitrogen atom,resulting in a p H-sensitive electrode with a linear range from 5.9 to 8.0.Meanwhile,6-?ferrocenyl?hexanethiol?Fc HT?with a different redox potential,inert to p H,served as an inner reference channel to provide a built-in correction for avoiding the complicated brain environment,thus resulting in a two-channel ratiometric electrochemical p H sensor.In addition,the current signal was amplified by ?6-fold through the electrodeposited gold bamboo shoot-like nanostructures on the carbon fiber microelectrode?CFME?with a diameter of 10 ?m.Finally,the developed biosensor for p H with high analytical performance was successfully applied for real-time monitoring of p H values in the live rat brain upon ischemia.We found that the p H values in the striatum and hippocampus of rat brain were reduced by ?0.53,and ?0.51,respectively,while negligible p H change was found in the cortex.?2?Simultaneous determination of glutamate and Ca²⁺ in the rat brain during spreading depression?SD?and ischemia processes.In this work,we developed a novel method to monitor glutamate and Ca²⁺ simultaneously with an integrated dual function microelectrode?DFME?biosensor.A glutamate sensing microelectrode was fabricated by co-modification of glutamate oxidase?Glu Ox?and Pt nanoparticles?Pt NPs?on the surface of Pt-microelectrode?Pt-ME?.Ion-selective membrane-modified carbon fiber microelectrode?CFME?was used for detection of Ca²⁺.DFME biosensor was integrated by efficiently combining the two kinds of microelectrodes together for the simultaneous monitoring of glutamate and Ca²⁺.In addition,the sensitivity of the glutamate biosensor was magnified via the electrodeposition of Pt NPs on Pt-ME.Allsolid-state Ca-ion selective microelectrode?Ca-ISME?allowed the study of deep brain areas in vivo.Finally,the integrated DFME for glutamate and Ca²⁺ with good analytical performance was successfully applied for the real-time monitoring of glutamate and Ca²⁺ changes in live rat brain during SD and ischemia processes.The methodology can be extended to design and develop biosensors for simultaneous detection of multiple species,and provide reliable approaches for brain chemistry studying.?3?An electrochemophysiology for real-time mapping and simultaneous quantification of multi-ions in the brain of freely moving rat.In this work,a novel electrochemophysiology was created for directly and simultaneously mapping and biosensing the individual chemical signal of K⁺,Ca²⁺,Na⁺ and p H using multiple ionselective microelectrode array?M-ISMEA?,in which open-circuit potential method was employed to measure the equilibrium concentration of ions,as well as for recording electrical signals,in the brain of freely moving rat.Metal ions such as K⁺,Na⁺,Ca²⁺,and p H were chosen as model molecules.A specific recognition probe for K⁺,triazacryptand?TAC?was designed and synthesized.Calcium ionophore I?ETH 1001?,hydrogen ionophore I,and sodium ionophore X were optimized for determination of Ca²⁺,H+,and Na⁺ respectively.Furthermore,an inner-reference electrode was used for providing a built-in correction to avoid the effects from the complicated brain system.Then,a novel 5 channel ion selective microelectrode array?M-ISMEA?consisting of K-SEME,Ca-ISME,Na-ISME,and H-ISME,and inner-reference element,as well as 8-channel ISMEAs?K-ISMEA,or Ca-ISMEA,or Na-ISMEA,or H-ISMEA?were assembled for large-scale mapping and accurate biosensing of K⁺,Ca²⁺,Na⁺,and p H with high temporal and spatial resolutions.In addition,the developed electrochemphysiology for monitoring of chemical signals can be combined with electrophysiology for recording electrical signals for neuronal activity at the same time without cross-talk for the first time,due to the employed current-free potentiometry.The notable analytical performance such as high selectivity and accuracy,together with the unique properties of ISMEAs,established an approach for reliable determination of K⁺,Ca²⁺,Na⁺,p H in a live rat brain followed by cerebral ischemia.Moreover,due to the high antifouling property and long-term stability,our developed biosensor was successfully contributed to real-time tracking the dynamic changes of K⁺,Ca²⁺,Na⁺,and p H in the live brain of freely moving rat under seizure status.Using this powerful tool,ISMEAs opened up a new vista in fundamental research on epilepsy as well as preclinical development of pharmaceutical candidates through dynamic monitoring of K⁺,Ca²⁺,Na⁺,p H at network level in the brain upon seizure treatment.