Study On Electrochemiluminescence Enzyme Biosensor For Detecting Lactate,Choline And Organophosphorus Pesticides Based On Novel Nanomaterials

The enzyme biosensors,which use the immobilized enzyme membrane as the biological recognition element and the modified electrode as the signal converter,achieve the detection by the changes of the electrochemical signal with the enzymatic reaction.Electrochemiluminescence?ECL?is a luminescence process,in which the emitter formated a highly excited state after the electrochemical and chemical luminescence on the surface of the electrode,and then decayed to the ground state.ECL enzyme biosensor is a new biosensor which combines ECL detection technology and enzymatic reaction.It not only has the characteristics of high specificity,molecular recognition and selective catalysis of enzyme biosensor,but also has the advantages including simple operation,high sensitivity,and low background signal of ECL technology.The enzymatic reaction based on the oxidoreductase generally involves the consumption of dissolved oxygen?O₂?and the generation of hydrogen peroxide?H₂O₂?.As is well-known,O₂ and H₂O₂ could serve as the coreactants of many emitters.Thus,the detection of substrates or enzyme inhibitor can be achieved based on the consumption of oxygen and the generation of H₂O₂.Conventional ECL biosensors for the detection of substrates and enzyme inhibitor were constructed based on the luminol-H₂O₂ system or CdTe QDs-O₂ system.In this thesis,single signal-based ECL biosensors based on luminol-H₂O₂ system and conjugated polymer dots-hydrogen peroxide?PFO-H₂O₂?system were constructed to determine lactate and choline,respectively.Additionally,PFO-H₂O₂ system was further combined with CdTe QDs-O₂ system to construct the ECL ratiometric biosensor for the detection of the enzyme inhibitor organophosphorus.The main research is as follows:Part 1 An electrogenerated chemiluminescence biosensor based on g-C₃N₄-hemin nanocomposite and hollow gold nanoparticles for the detection of lactateA novel electrochemiluminescence?ECL?biosensor based on g-C₃N₄-hemin nanocomposite and hollow gold nanoparticles?HGNPs?was constructed to detect lactate.Firstly,the g-C₃N₄ nanosheets were prepared through ultrasonication-assisted liquid exfoliation of bulk g-C₃N₄,which was obtained through polymerizing melamine under 600 °C.Then,the nanocomposites of g-C₃N₄ nanosheets and hemin were prepared to modify a glassy carbon electrode.Subsequently,HGNPs were self-assembled onto the electrode for adsorbing lactate oxidase to achieve a lactate biosensor.Due to the excellent catalytic effect of g-C₃N₄/hemin and HGNPs on the luminol/H₂O₂ ECL system,the as-prepared biosensor exhibited a good response performance to lactate with a linear range of 1.7×10-8⁵.0×10-4mol·L-1and a detection limit of 5.5×10-9 mol·L-1.In addition,the prepared ECL biosensor exhibited satisfying reproducibility and stability.The g-C₃N₄-hemin naocomposite might hold great potential application in luminol/H₂O₂ ECL system.Part 2 Anodic electrogenerated chemiluminescence behavior and the choline biosensing application of blue emitting conjugated polymer dotsThe anodic electrochemiluminescence?ECL?behavior of poly?9,9-dioctylfluorenyl-2,7-diyl??PFO?was studied and the possible ECL mechanisms were proposed.Based on a fact that H₂O₂ can serve as the coreactant for amplifying the ECL signal of PFO,with choline oxidase as a model enzyme,the application of PFO in oxidoreductase-based ECL sensing was investigated through integrating PFO and C60-polyamidoamine?C60-PAMAM?composite.Since both C60 and PAMAM could facilitate an increase in the ECL signal of PFO,our prepared biosensor exhibited a good response to choline with a linear range from 1.25×10-9 mol·L-1 to 9.45×10-5 mol·L-1 and a detection limit of 5.00×10-10 mol·L-1.Furthermore,the C60-PAMAM-PFO modified electrode as an enzymeless H₂O₂ sensor also can achieve the quantitative detection of H₂O₂ in a wide linear range from 2.30×10-9 mol·L-1 to 8.05×10-3 mol·L-1 and a low detection limit of 6.10×10-10 mol·L-1.The integration of C60,PAMAM and PFO would provide a new platform for the construction of ECL biosensors and biocatalysis.Part 3 Application of novel double-potential ratiometric ECL enzyme inhibition biosensor for organophosphorus detectionGenerally,electrochemiluminescence?ECL?ratiometric assays were constructed based on the energy transfer?ET?between an emitter and a metal nanomaterial or between two different emitters.The choice of suitable energy donor-acceptor pair andthe distance dependence of ET would greatly limit the practical application of ratiometric assays.In this work,a novel double-potential ECL ratiometry without the ET was explored and used to detection organophosphorus pesticides?OPs?.The opposite change trend of two ECL signals was achieved using the reactant and the product in enzymatic reactions as the coreactants of cathodic and anodic ECL emitters,respectively.The reduced graphene oxide-CdTe quantum dots?RGO-CdTe QDs?and carboxyl-conjugated polymer dots?PFO dots?were chosen as cathodic and anodic ECL emitters,respectively.Dissolved O₂ and the product?H₂O₂?in enzymatic reactions served as the coreactants of cathodic and anodic,respectively.With OPs as the target analyte model,in the presence of the substrate acetylthiocholine?ATCl?,with the occurrence of the enzymatic reactions caused by acetylcholinesterase?AChE?and choline oxidase?ChOx?,the dissolved oxygen was consumed,the cathodic ECL signal from RGO-CdTe QDs was quenched;meanwhile,the in situ generated H₂O₂ would enchance the anodic ECL signal from PFO dots,resulting a strong ECL signal of PFO dots.In the presence of OPs,due to the inhibition of OPs on the activity of AChE,the consumption of dissolved O₂ and the generation of H₂O₂ would be decreased,resulting the cathodic ECL signal increased while the anodic ECL signal declined correspondingly.The OPs were detected based on the ratio of two ECL signals.Such a ratiometric system not only overcome the defects of ET,but also achieved a high accuracy,reliability and sensitivity,so it exhibited a wide application prospect in the field of ECL ratiometric analysis.