Miniature Electrochemical Sensor Analyzes Small Molecules In Mouse Brain In Situ

For many years,the brain has been an important area for scientists to study relentlessly.Intracerebral neurotransmitters,neurotransmitters and other neurochemicals are closely related to the normal work of the brain,and the chemical substances that affect the nervous system are responsible for the transmission of information between neuronal synapses.Brain chemistry is a complex research system.and the small changes of neurochemical substances(such as,NO,O2,pH,5-HT and etc.)could have a significant impact on the brain function.Therefore,it is very important to analyze the release and ingestion of neurochemicals in neurons of extracellular neurons in the brain,which is conducive to the further study of the molecular basis of the brain function.NO is key free-radical messenger and neuronal signal which is closely related to many brain diseases.It is still a challenge to develop a sensitive and reliable biosensor for in vivo monitoring NO in brain.Here,a facile ratiometric electrochemical biosensor for NO monitoring in rat brain followed by cerebral ischemia was developed on the basis of carbon nanotube fiber(CNF)modified by hemin and Nafion.in which the CNF was not only taken as a platform to confine hemin molecule,and greatly facilitated the electron transfer of hemin on the electrode surface.Additionally,hemin was designed to play dual roles:a stable catalyst for reduction of NO,making selective detection of NO at-0.68 V vs.Ag/AgCl,and an inner reference element to provide a built-in correction,avoiding the interference from complicated brain environment.The developed ratiometric biosensor can detect NO with a linear range from 25 to 1000 nm,with a low limit of detection of 10 nM,which can fulfill the requirements for in vivo measurement of NO.The remarkable analytical performance of the present biosensor,as well as the long-term stability and good reproducibility ascribed to the integration of microelectrode technique and carbon nanotubes,provided a reliable platform for in vivo monitoring NO in hippocampus of rat brains followed by cerebral ischemia.The average level of NO was 61±23 nM(n=3).But the concentration of NO increased to 141 ± 18 nM(n=3)after cerebral ischemia for 15 min.Oxygen(O2)and pH are closely correlated with physiological and pathological events of brain,however the detailed mechanism is still unclear,mainly owing to a lack of accurate analytical methods for determination in live brain.Herein,we create a single electrochemical biosensor for simultaneous determination and ratiometric quantification of O2 and pH in rat brain upon ischemia,by designing and synthesizing of Hemin-aminoferrocene(Hemin-Fc).The reduction peaks current of Hemin group increases with rising concentration of O2,and shows a good linearity in the range of 1.3 to 200.6 μM.Meanwhile,this peak potential positively shifted with decreasing pH from 8.0 to 5.5,resulting in simultaneous determination of O2 and pH by monitoring both current and potential signals output.Interestingly,Fc group can serve as an inner reference to construct a ratiometric biosensor,because both current and potential signals of Fc stay almost constant with variations of O2 and pH.The developed single biosensor with high temporal and spatial resolutions,as well as remarkable selectivity and accuracy,was successfully applied in real-time quantification of O2 and pH in rat brain upon cerebral ischemia.The accurate values of O2 and pH in different regions of normal rat brain and that followed by ischemia are also reported in detail.5-Hydroxytryptamine(5-HT).or serotonin,is an electroactive indole that acts as an important neurotransmitter in the brain.5-HT is one of the major neurotransmitters of brain and is known to play a central role in wide variety of pharmacological,biological and psychopathological functions including depression,eating disorders,alcoholism,obsessive-compulsive disorders and anxiety.There are few methods of determination for 5-HT can meet the requirements of sensitivity and lowest detectable limit as well as significant selectivity in vivo according the previous reports.Additionally,there are fewer electrochemical methods for direct in situ monitoring of 5-HT in rat brain up to now.But differential pulse voltammetry(DPV),a demonstrated analytical method for the vivo detection of ascorbic acid,is extended to the detection of in.vitro and in vivo 5-hydroxytlyptamine(5-HT).Because the DPV has almost no background current and relatively high sensitivity and selectivity,as well as very fast response speed.Besides,we choose 3,3-Dithiodipropionic Acid Di(N-succinimidyl ester)(DSP)molecule to recognize the 5-HT in complex environment and react with it quickly at room temperature in aqueous solution,connecting it to the electrode.Based on oxidation peak current of 5-HT on electrode,we can monitor 5-HT in vivo and vitro.Of cause,not only can gold nanoparticles on carbon fiber microelectrode(CFME)amplify the electrochemical signal but also it can increase the electron transfer.More importantly,co-modified MB with DSP molecules on microelectrode act as inner-built reference to increase the accuracy of measurement results when microsensor in vivo.