N-doped Graphene Based Electrochemical Aptasensors For The Detection Of Microcystin-LR In Agro-Products

Microcystins?MCs?is one of the most common cyanobacterial toxins in watery environment,and among them the most widely distributed and acute toxic one is the subtype microcystin-LR?MC-LR?.MC-LR not only can be directly harmful to aquatic organisms,but also can migrate into the soil via the irrigation,overflow and fertilization,etc,which is further absorbed by crops,then seriously affects the quality safety of agricultural products and posts a potential threat to human health.Therefore,it is very necessary to establish a sensitive,reliable,simple and convenient,easy to realize,online detection and tracking methods for MC-LR in agricultural products.Compared with the traditional detection methods,electrochemical sensors,as a novel sensing technique,because of a series of advantages such as portability,low cost,high sensitivity and fast analysis time,have drawn many researchers'eyes in the agricultural product quality monitoring fields.In this dissertation,we aim at starting with the design and preparation of a series of N-doped graphene?NG?based functional nanomaterials,coupling with new electroanalytical techniques,including electrochemiluminescence?ECL?,photoelectrochemical?PEC?,and self-powered electrochemical sensing,establishing a series of electrochemical sensing methods and being applied for the determination of MC-LR and further analysis of MC-LR contents in agricultural products.The main research contents are as follows:?1?A highly sensitive and selective ECL sensor for MC-LR detection was built successfully with the as-prepared three-dimensional boron and nitrogen codoped graphene hydrogels?3D BN-GHs?via the self-assembled hydrothermal method as the luminescent molecule Ru?bpy?₃²⁺immobilization platform for further loading the designed MC-LR aptamer through electrostatic adsorption.This sensor has good analytical performance,including the linear range of 0.1¹000 pM and the detection limit of 0.03 pM,and can be used for the detection of MC-LR in water samples from the farmland.More importantly,with the aid of the biological dynamic experiments of electrochemical quartz crystal microbalance and the comparison results of sensing performances between two-dimensional and 3D nanomaterials,a novel sensing mechanism was proposed for the first time:avoiding using conventional double-stranded DNA configuration,3D BN-GHs nanomaterials directly amplify the steric hindrance effect between the single-strand DNA?aptamer?and MC-LR,so as to enlarge the quenching efficiency of the ECL signals to a detectable range,finally realizing the signal amplification.This detection methodology supplies a simple and versatile platform for specific and sensitive determination of a wide range of aptamer-related targets.?2?A selective and sensitive method for detecting MC-LR in fish samples by integrating the PEC technique and the specific recognition ability of aptamer was developed.Specifically,as an efficient PEC transducer,the NG-BiOBr electrode was utilized as the aptamer immobilization platform via the?–?stacking interaction.The target MC-LR was captured by aptamer of the photoelectrode surface and then oxidized by the photo-induced holes of the photoactive electrode,resulting in the enhancement of the PEC signals,which would be a biosensor enabling the convenient and exquisite PEC analysis.Subsequently,the PEC response of constructed aptasensor was only specific binding to MC-LR,Other isoforms did not interfere with the detection process,and thus,it could be applied for the highly selective determination of MC-LR level.Under the optimized condition,the PEC signal versus the logarithm of the MC-LR concentration was in good linear relationship ranging from 0.1 pM to 100 nM with detection limit about 0.03 pM.The constructed method was employed to analyze fish samples collected from the local supermarket.?3?A direct formatted PEC aptasensing platform was constructed for ultrasensitive MC-LR detection with NG-AgI composites as photocathode and aptamer as recognition element.The developed aptasensor can be used for MC-LR detection in fish samples.Furthermore,to investigate the sensing mechanism,the experiments with photoluminescence and time-correlated single-photon counting technique were implemented as supporting evidence.The experimental and theoretical efforts elucidate that the theory of flow directions of electron give reasonable explanation for the sensing process:prevailing electron-hole recombination constitutes a signal-off strategy,while electron transfer playing the leading role presents a signal-on performance.For the detection process,in the presence of MC-LR,the specific recognition of the targets with the sensing interface would trigger the formation of the aptamer/MC-LR complex,resulting in reduced electron transfer rate,increased charge recombination,and hence quenched photocurrent intensity of the NG-AgI with the detection limits of 0.017 pM.This work will help to complement the vital theoretical explanation about the underlying sensing mechanism,and future designing of PEC aptasensors.?4?According to the fundamental matching principle,the Fermi level of photoanode should be higher than that of the photocathode.By employing with photo-assisted fuel cell technique,we selected TiO₂ as photoanode nanomaterials and NG-BiOBr as photocathode nanomaterials for the MC-LR assay.In the self-powered sensing system,MC-LR is not only as fuel molecules,but also as target analytes.As the concentration of MC-LR increased,the electricity power output increased.The electrochemical impedance spectra results showed that the sensing process actually occurred in the photoanode interface and the detailed mechanism was that the MC-LR captured by the photoanode could promote the charge separation process and thus enhance the output signal.The constructed self-powered platform avoids using biological enzymes,reducing the production cost,but the open circuit potential can still reach to the level of 0.54 V.The maximum output power density versus the logarithm of the MC-LR concentration was in good linear relationship ranging from 2pM to 155 pM with detection limit of 0.67 pM.Such a self-powered conceptual sensor needs no additional power supply,and the detection device itself can supply energy for the detection process.Therefore,it is easy to be miniaturized and portable,and thus could be applied to monitor the MCs contents in water samples.?5?By introducing Ag nanoparticles with surface plasma resonance effect and NG into the photoanode TiO₂ and combining with MC-LR aptamer as specific recognition elements,a double photoelectrode assisted visible-light responsive self-powered aptasensor was developed for the selective detection of MC-LR.With the increase of MC-LR concentration,the output signal of the power density will decrease.Under the support of the experiment results of electrochemical impedance spectra,UV-vis absorption spectra and photoluminescence spectra,the detailed sensing mechanism was puts forward:the photoanode interface specifically recognizing and capturing MC-LR,produced steric hindrance effect,and reduced the absorption efficiency of light,thus promoting the recombination of the electron and hole pairs,finally resulting in a low power output signal.The self-powered sensing platform showed a good linear relationship with a wide detection range?1 pM³16nM?and can be used for the detection of MC-LR in vegetables.The constructed aptasensing system exhibited good selectivity and sensitivity and facilitate the real-time field detection due to no need of additional power supply.?6?Different electrochemical detection methods for MC-LR from this dissertation and previous reports were systematically evaluated in term of their respective advantages,disadvantages and applicability.Meanwhile,the performances of the constructed five MC-LR electrochemical sensors in this dissertation were comprehensively evaluated by comparing with existing electrochemical sensors for the MC-LR detection,and analyzing the advantages and disadvantages of these developed photo-,electro-and self-powered-electrochemical sensors so as to provide support and guidance for related research work in the future.