In Vivo Analysis Of Oxidative Stress Related Small Molecules

Oxidative stress refers to the imbalance between oxidation and antioxidation in the body,which leads to the overproduction of highly active molecular-active free radicals in the body.The body cannot remove it in time,resulting in a large number of oxidation intermediates,thereby causing damage to the body and inducing many diseases,especially brain diseases such as ischemia.Numerous molecules involved in the oxidative stress process and have complex molecular mechanisms.When oxidative stress occurs,oxidative stress-related molecules such as reactive oxygen species,neuromodulators such as cysteine,energy molecule such as glucose,and pH are present at abnormal concentrations.Therefore,the real-time,in-situ measurement of the concentration of each related molecule in the oxidative stress process in vivo is of great significance to the analysis of oxidative stress and physiological diseases caused by oxidative stress.Electrochemical methods are widely used for in vivo detection due to their advantages of miniaturization,in situ,real-time,and dynamic detection.However,due to the complexity of the brain's microenvironment,the large number of interferences,the large differences between the internal and external environments,and the close links between molecules,the establishment of electrochemical analysis methods with high selectivity,high accuracy,and simultaneous analysis of multiple substances is still a huge challenge!Based on a large number of literature surveys,in order to solve the key scientific issues in the in vivo electrochemical analysis of oxidative stress-related molecules in rat brain under ischemic conditions,the following electrochemical analysis methods were designed and constructed,and successfully used for the analysis of small molecules in rat brain.The specific contents are as follows:?1?An electrochemical biosensor for ratiometric monitoring the levels of cysteine in rat brain.In this work,single-walled carbon nanotubes?SWNT?modified carbon fiber electrodes?CFME?were used as electrode substrate,acrylate naphthalene?1-NA?as cysteine-specific recognition ligand,and ferrocene?Fc?was also introduced as an internal reference.The reaction mechanism can be explained as:cysteine can undergo a nucleophilic addition reaction with acrylic groups in the 1-NA molecule to generate naphthol,and the naphthol can undergo a one electron-one proton electrochemical reaction on the surface of the electrode to generate an electrochemical signal,and use this signal to quantify cysteine.The linear range of cysteine is 1-50?M.And the biosensor also possess high selectivity,good stability and fast response time.Finally,the detection of cysteine in the brain of ischemic rats was successfully achieved.The concentration of cysteine was significantly increased after 30 min of ischemia,reaching about 4 times of the normal level.?2?An electrochemical biosensor with dual signal outputs for ratiometric monitoring the levels of H₂O₂ and pH in the microdialysates from a rat brain.In this work a current-potential signal outputs ratiometric electrochemical probe Cat+Fc/SWNT/CFME was designed for simultaneous quantitative analysis of pH and H₂O₂ in rat brain dialysate.The electrochemical probe is mainly composed of three parts:?1?for the first time,catalase was selected as a specific recognition unit,and the dual signal output of current and potential through a single redox peak was used for simultaneous analysis and detection of H₂O₂ and pH;?2?The introduction of electrochemically stable ferrocene?Fc?as a reference molecule to eliminate the measurement error caused by the difference of in vivo and in vitro environment;?3?The carbon nanotube electrode?CFME?modified with single-walled carbon nanotubes?SWNT?was used as an enzyme modifying substrate,which greatly improved the direct electrochemical peak of Cat.The reduction peak of Cat increased with the addition of H₂O₂,which can realize the detection of H₂O₂.The linear range of H₂O₂ is 1-230?M.At the same time,the peak potential of Cat gradually shifted positively with the decrease of pH,and pH detection can be realized.The linear range of pH is 5.91-7.81.And the electrochemical peak of the reference molecule Fc remains unchanged.The ratiometric electrochemical sensor has high selectivity,good sensitivity,stability and repeatability,and also uses a cubic spline interpolation method to study the relationship between pH and H₂O₂.Finally,the simultaneous detection of pH and H₂O₂ in rat brain dialysate was successfully achieved.It was found that the concentration of H₂O₂ increased significantly?5-7 times?and the p H level decreased significantly?0.5 pH?under ischemic conditions.More importantly,this method can also be extended to the design and construction of biosensors for other biomolecules in the rat brain and provide feasibility for further study of physiological and pathological processes.?3?Single biosensor for simultaneous quantification of glucose and pH in a rat brain of diabetic model using both current and potential outputs.In this work a dual signal outputs ratiometric electrochemical biosensor GOD+ABTS/SWNT/CFME was developed for simultaneous quantitative analysis of pH and glucose in rat brain of diabetics.The electrochemical probe firstly selects glucose oxidase as a specific recognition unit,and uses the current and potential signal output for simultaneous detectionofglucoseandpH;electrochemicallystable2'-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid?ABTS?that does not respond to both glucose and pH is used as an internal reference molecule to eliminate measurements error caused by differences of in vitro and in vivo environmental,and improve the detection accuracy in living complex micro-environment more effectively;finally,the carbon nanotube electrode?CFME?modified with single-walled carbon nanotubes?SWNT?was used as an enzyme-modified substrate,which greatly improved the direct electrochemical peak of glucose oxidase.The oxidation peak of GOD increase with the increase of glucose concentration,which can realize the detection of glucose.The linear range of glucose is 0.3-8.2 mM.At the same time,with the decrease of pH values,the peak potential of GOD gradually shifts positively,and the pH can be detected.The pH linear range is 5.67-7.65.The electrochemical peak of the reference molecule ABTS remains unchanged.The sensor has high selectivity,good stability and repeatability,and high accuracy.The data of the relationship between glucose and pH were processed using cubic spline interpolation to improve the accuracy of detection.Finally,the ratiometric dual detection sensor was successfully applied to detect pH and glucose levels in the brain of diabetic model.It was found that the concentration of glucose in the brain of diabetic model was significantly increased by 2-4 times and the p H was decreased by 0.2-0.3 pH.The glucose level in each brain area of normal rats is reduced to about half of the normal level under ischemic conditions.