Electrochemical Analysis Of Calcium Ion In Living Brain

Calcium ion is one of the most important biologically active ions in vivo,and it is mainly distributed in the tissue fluid,various cells and plasma in the body.Calcium ions are involved in various physiological activities such as nerve signal transduction and energy metabolism in the brain.Especially in the pathogenesis and treatment of common brain diseases such as stroke,the changes of extracellular calcium ions in different brain regions directly predict severe pathological features such as cell membrane depolarization,abnormal activation of ion channels,and destructive increase of ROS.Therefore,the development of a new method for real-time monitoring of calcium ions in the living brain is of great significance for the rapid and accurate tracking of the pathogenesis of brain energy metabolism diseases caused by acute and chronic injury and the molecular mechanism of drug treatment.Existing research methods such as micro dialysis and small-molecule fluorescent probe methods are difficult to monitor the dynamic changes of calcium ions in vivo in real-time and reversibly in situ for a long time.The electrochemical microelectrode method provides a reliable method for continuous monitoring of chemical signals in vivo,in which the implanted functional microelectrodes exhibit excellent spatiotemporal resolution.However,current in vivo electrochemical detection methods still face challenges such as poor selectivity in complex biological environments,susceptibility to biological contamination of implanted electrodes,poor electrode reversibility,and poor biocompatibility.In order to solve the key scientific problems of current in vivo electrochemical research,this paper designs and develops the following new in vivo electrochemical analysis methods:（1）Graphene oxide transition layer-mediated ion-selective electrode for high-sensitivity and high-selectivity detection of Ca²⁺.In this work,a novel strategy for high-sensitivity Ca²⁺detection was constructed.First,graphene oxide microstrip（GO）was uniformly electrodeposited on the surface of carbon fiber electrode（CFE）as an ion-electron transfer layer to realize the electrical signal conversion after ion enrichment on the surface of micro-scale electrode.Second,gold microleaf（Au NL）was in situ electrodeposited on the surface of GO-modified CFE,which increased the electrode area by 4.4 times compared to CFE and provided functionalized molecular modification sites.On this basis,a highly selective Ca²⁺-recognizing ligand was designed and synthesized,and the ligand was modified on the electrode surface through Au≡C bonds,and a new high-performance Ca²⁺-selective microelectrode was constructed.The microelectrode exhibits excellent electrochemical performance,with a good linear response to Ca²⁺in the concentration range from 10μM to 10 m M,and the detection limit is as low as 5.91±0.46μM.Benefiting from the specific recognition ability of Ca²⁺ligands,the microelectrode has no obvious response to common interfering substances,showing excellent selectivity for Ca²⁺.In addition,the GO layer with high double-layer capacitance properties provides more carrier sites for Ca²⁺adsorption and electron exchange,resulting in a 2.4-fold increase in the sensitivity of the electrode.Finally,the microelectrode was applied to monitor the changes of Ca²⁺in blood after cerebral ischemia in rats.The study found that the concentration of Ca²⁺in blood samples decreased gradually with the prolongation of cerebral ischemia time,and the concentration of Ca²⁺decreased from the initial 1.3 after 120 minutes of cerebral ischemia.m M down to 0.95 m M.The results were compared with those measured by a blood gas analyzer,and the difference was less than 3.7%,demonstrating the high accuracy of the microelectrode in blood monitoring.（2）Long-term tracking and dynamic quantities of reversible changes in extracellular Ca²⁺in multiple brain regions of freely moving animals.In this work,an anti-biological contamination multifiber microarray detection platform was constructed to realize real-time monitoring of reversible changes in extracellular Ca²⁺and neuronal activity in multiple brain regions of the head-fixed and freely moving mouse brains.The sensitivity of this microfiber array for Ca²⁺detection remained above 92%after 60days of continuous measurement in live mouse brains.On the other hand,in this work,three molecules with different affinities for Ca²⁺were designed and synthesized.By comparing their selectivity and reversibility,it was found that the low binding strength of the Ca²⁺ligand-METH was favorable for the reversible capture and dissociation of Ca²⁺,and in the It can still maintain high selectivity in the face of other common interferences such as metal ions,ROS,amino acids,etc.Therefore,the METH-modified microelectrodes can achieve highly reversible detection of Ca²⁺.This is the first reported microelectrode array with excellent reversibility and selectivity for real-time monitoring of extracellular Ca²⁺levels in living brains for 60 consecutive days.Using this microarray,we found for the first time that the concentration of extracellular Ca²⁺gradually decreased from the superficial brain region to the deep brain region and the rate of change gradually decreased during ischemia,and gradually recovered from the deep brain region to the superficial brain region with a decreasing rate during reperfusion..This suggests that our microarrays provide a useful and powerful tool for studying complex pathological processes involving large-scale network dynamics across multiple brain regions.In addition,it was also found that the influx of extracellular Ca²⁺was effectively reduced with increasing doses of reduced glutathione（GSH）injected in advance with the ROS scavenger.Therefore,glutathione may act as a potent inhibitor to protect neuronal activity.Finally,it was found that the Ca²⁺channel blocker（flunarizine）could not only gradually restore the extracellular Ca²⁺concentration.It can also promote the recovery of neurons in the treatment of cerebral hemorrhage.This further confirmed the direct link between Ca²⁺influx and cerebral hemorrhage injury.（3）Highly biocompatible hydrogel-based Ca²⁺-selective microelectrodes.In this work,a highly biocompatible Ca²⁺-selective microelectrode sensor with rigid-flexible switching properties was constructed.A new strategy for the preparation of hydrogel microelectrodes doped with highly conductive hydroxypropyl cellulose（HPC）and hydrophilic graphene oxide（GO）in polyvinyl alcohol（PVA）hydrogels is proposed.Among them,the interaction of PVA and GO makes the compressive elastic modulus of the electrode as high as 175 MPa in the dry state,and the elastic modulus decreases to 1.5 KPa after infiltration,which makes the electrode exhibit high flexibility after implantation in the living body.Compared with metal electrodes of the same size,the expression of glial fibrillary acidic protein,which is used to indicate brain nervous system damage,decreased by 78.9%after implantation of the hydrogel electrode in vivo,thus confirming its excellent biocompatibility.The doping of HPC greatly improves the conductivity of the microelectrodes,so high-selectivity Ca²⁺selective recognition ligands can be doped into PVA/HPC/GO hydrogels to prepare a new type of hydrogel-based Ca²⁺selective Microelectrode PVA/HPC/GO/METH（PHGM）.Benefiting from the high electric double-layer capacitance of the hydrogel material and the porous structure with high permeability,the PHGM exhibits a sensitivity of 52.3mv/lg Ca²⁺,which is 1.77 times that of conventional ion-selective electrodes,thus enabling the monitoring of tiny fluctuations in Ca²⁺.Has better resolution.The highly selective Ca²⁺-recognition ligand makes the electrode insensitive to common interferents,and thus has the potential to accurately recognize Ca²⁺in complex living environments.This novel highly biocompatible hydrogel Ca²⁺-selective microelectrode provides a reliable research tool for investigating the relationship of Ca²⁺in the brains of freely moving animals with various behavioral and pathological models.