Design Of Hydroxyl Radicals Electrochemcial Sensors And Their Application In Cell Biology Studies

Reactive oxygen species?ROS?are a group of very unstable oxygenates produced during metabolic process of aerobic organisms.The normal concentration level of ROS in organisms plays an important physiological role in cell differentiation,proliferation,apoptosis and transcription factors.However,it will lead to cellular damage and many diseases when the ROS generates excess in vivo.It has been reported that hydroxyl radical?·OH?is one of the most toxic and harmful free radical in ROS.·OH can react with various molecules in the organism,including carbohydrates,nucleic acids,lipids,amino acids,etc.,by means of electron transfer,addition and dehydrogenation,resulting in various diseases..Thus,considering the serious toxicity and other adverse effects of·OH,it is urgent to develop a highly sensitive and selective detection method for·OH determination.Due to their low cost,rapid response,high specificity and sensitivity,easy operation and ease of miniaturization,electrochemical sensors have attracted much attention.Therefore,this paper developed three novel,sensitive and fast electrochemical sensors for tracking and detecting·OH released by living cells.The specific work is as follows:?1?Electrochemical sensor based on ferrocene hexyl mercaptan modified gold electrode for detection of·OHThe 6-?ferrocenyl?hexamercaptan?FcHT?with reversible redox characteristics was self-assembled on the surface of gold electrode?FcHT/GE?by mild and controllable self-assembly technology.The morphology of the prepared sensor was characterized by X-ray photoelectron spectroscopy?XPS?and contact angle,and the electrochemical properties were studied by cyclic voltammetry,electrochemical impedance spectroscopy and square wave voltammetry.In addition,the mechanism of redox peaks produced by FcHT in HClO₄ solution,the number of electrons transfer in FcHT redox process,diffusion coefficient on electrode surface,coverage and self-assembly mechanism of FcHT were explained by experimental data.Under the optimal experimental conditions,the linearity of·OH detection was established by double redox peaks of FcHT.?2?A novel electrochemical sensor for·OH determination by coupling nanoporous gold layer with self-assembled FcHTThe detection of·OH in live cells is significant to study its physiological and pathological roles,while it is full of challenge due to the extremely low concentration and short lifetime of·OH.Herein,we have developed a novel electrochemical sensor based on FcHT self-assembled nanoporous gold layer?NPGL?modifided GE?FcHT/NPGL/GE?,which can detect the release of·OH from living cells with high sensitivity and selectivity.The superior sensitivity could stem from the unique porous architecture of NPGL,which enlarged electrode surface area and expedited electron transportation during electrochemical reactions.Additionally,NPGL provides more active binding sites for capture agent?FcHT?of·OH,thus ensuring high selectivity.For comparison,FcHT/GE was applied to detect·OH,and the obtained sensitivity was 0.03118 mA nM^-1 and detection limit was 0.128 nM in the linear range of 0.45 nM to 80 nM.After modification of NPGL,the sensitivity of FcHT/NPGL/GE to the·OH response was increased to0.4248 mA nM^-1,the detection limit was reduced to 0.571 pM,and the linear range was extended from 2pM to 60 nM.It is worth mentioning that a plenty of extra merits has also been validated like reproducibility,repeatability and stability,enabling to direct?straight-forward?electrochemical detection of·OH in HepG2cells.?3?Double signal amplification through a functionalized nanoporous Au–Ag alloy microwire and Au nanoparticles:development of an electrochemical·OH sensor based on a self-assembled layer of FcHTDetermination of·OH with high sensitivity and accuracy in live cells is a challenge for evaluating the role that·OH plays in the physiological and pathological processes.Herein,electrochemical sensors for the analysis of·OH released from live cells with enhanced sensitivity have been developed.The enhanced sensitivity is achieved by functionalizing a self-assembled monolayer?SAM?of electroactive FcHT onto a3D nanoporous Au–Ag alloy microwire?NPAMW?or Au nanoparticles-linked NPAMW?AuNPs/NPAMW?.FcHT/NPAMW allows the first signal amplification to detect·OH corresponding to a sensitivity of 0.08624 mA nM^-1 in the linear range of 0.5 nM to 80 nM.FcHT/AuNPs/NPAMW allows for dual signal amplification,which yields an improved sensitivity of 0.1748 mA nM^-1 and extended linear range of 2 pM to 80 nM.Most impressively,the as-prepared sensors reveal other good merits including high selectivity,reusability and stability,enabling direct electrochemical detection of·OH released from live cells.?4?Electrochemical microfluidic chip based on FcHT Functionalized AuNPs and NPAMW applied for·OH detectionIn this work,a novel electrochemical detection platform was established by integrating self-assembly technology with microfluidic chip and applied for detection of·OH released from live cells.The chip foundation is acrylic panel with designed grooves.In the detection unit of the chip,Pt wire is used as the counter electrode,Ag/AgCl is prepared as the reference electrode,and the SAM of the electroactive FcHT is functionalized the AuNPs/NPAMW as working electrode.Detailed characterization of the chip and the working electrode was performed,and the properties were explored by square wave voltammetry and cyclic voltammetry.The electrochemical microfluidic platform detect·OH corresponding to a detection limit of 0.028 nM in the linear range of 0.1 nM to 150 nM.Low detection limit ensures trace measurement of·OH released from live cells.This simple and low-cost chip presented here has great potential serving as a promising assay device for POCT.Future development of the chip will be focused on extension of the target anlytes,from small chemical molecules to ions,biological macromolecules,and even microorganism like viruses and bacteria.Furthermore,other electrochemical methods will be involved,such as electrochemical impedance spectroscopy,linear sweep voltammetry and differential pulse voltammetry,in order to achieve better analytical performance.