Real-time Monitoring Of Nitric Oxide From Single Cells By High-performance Graphene-field Effect Transistor Biosensor

Nitric oxide(NO)is both a messenger molecule and a neurotransmitter,which can receive and convey information to regulate cell activity and direct the body to perform certain important functions such as neurotransmission,vasodilatation,immune response and angiogenesis.It is found that NO not only acts as a signaling molecule in the activities of immune regulation,neuronal communication,smooth muscle relaxation and so on,but also participates in the physiological processes such as plant growth and development,stress response,respiratory metabolism and aging.However,NO,as an active gas present in vivo at a nanomolar concentration,has a relatively short half-life(<10 s),and is easily oxidized by oxides or superoxide ions,making it difficult to accurately quantify the NO levels in physiological conditions.Hence,the development of a method that can efficiently,accurately and quantitatively obtain the information of NO in cell and in vivo is the basis of further exploring the mechanism of its action.Many techniques have been developed for NO detectionsuch as bioassay,electron magnetic resonance spectroscopy,luminol/hydrogen peroxide chemiluminescence,laser-inducedfluorescence,spectrophotometric assay,etc.However,most of these methods suffer from high costs or complicatedpretreatments or low sensitivity,which restricts real-timemeasurement.Electrochemical detection provides a usefultool to directly measure NO in real time with high sensitivityand ultrafast response time,which has been widely used for NOdetection in biological systems.Yet,electrochemical NOsensors are usually operated at a relatively high oxidationpotential(>0.6 V)to achieve the goal of NO detection.Such anattempt can disturb the intrinsic cellular response or inducesignificant signal interference from other electrochemicallyactive molecular species.Compared to amperometric electrodes,field-effect transistor(FET)nanobiosensors allow real-timemonitoring of physiological information with higher sensitivitydue to their intrinsic amplification capabilities and high signaltonoise ratio.More importantly,it is operated at arelatively lower detection potential(Vds = 0.1 V),which haslittle impact on cells.So it will be very meaningful to detect NOin physiological processes by using the FET biosensor.In recent years,several works have been reported for realtime monitoring of NO in physiological solution based onsemiconductor FETs.Wu et al.reported a GaAs-basedsemiconductor FET for detection of NO in physiologicalsolution,in which hemin molecules were self-assembled on theGaAs surface.Li et al.developed a graphene-based FETbiosensor for detecting NO gas,in which the sensitive channelswere composed by a palladium-decorated reduced grapheneoxide(RGO).However,the sensitivity of the recentlydeveloped sensors is still limited to the micromolar orsubmicromolar level,and these sensors are not suitable fornanomolar levels cell detection.Recently,Jiang et al.developed a NO sensor based on heminfunctionalized graphene FET for directly detecting NO in livingcell with subnanomolar sensitivity.Nevertheless,in order torealize realtime monitoring of NO release at single-cell level,sensitivity needs to be further improved.Cells are the basic unitof the organism,and knowledge at the single-cell level willenhance the understanding of diverse cellular processes such asintracellular and intercellular communication,cell differentiation,physiological effects of external stimuli and diseasestates,etc.Additionally,for diagnosis of cell-based diseases,it isimportant to develop methods of extremely high sensitivitydown to single-cell level to gain the best ability fordiagnosis.In view of the above situation and challenges,this workaimed at developing high-performanceFET biosensor to solve the bottleneck problems encountered in real-time monitoring of singlecells.In this work,we explore an excellent performance of FETbiosensor by combination of a highly specific molecularmetalloporphyrin and a highly electrical conductivity ofgraphene for ultrasensitive and highly specific monitoring ofNO in real time.Firstly,the base electrode was prepared by photolithography,and the graphene was deposited on the prefabricated FET sensor surface as a highly conductive bridge to facilitaterapid transport of electrons between source and drain by liquid phase separation method.Afterward,the composite ofFe(III)meso-tetra(4-carboxyphenyl)porphyrin(FeTCP)andRGO,named FGPCs,is then deposited onto the RGO layer.Not only does the formation of the compound(FGPCs)showsuperb nature in selectivity and sensitivity,but it also improveselectrical contact between FeTCP and graphene.Due tothe remarkable synergistic effects of RGO and FGPCs,thebiosensor thus enhanced superior conductivity and behavedwith good catalytic properties,enabling highly sensitive andspecific detection of NO.The detection limit was found to be 1pM in PBS and 10 pM in the cell medium(S/N ? 3),which isthe lowest compared to that reported in the literatures.Theprepared sensor also showed good cytocompatibility for cellculture.The ultralow detection limit and good biocompatibilitymade the sensor suitable for real-time monitoring of NO atsingle-cell level.This methodhas great potentials forapplications in clinical laboratory diagnostics.