Electrochemiluminescent Biosenor Based On New Luminescent Materials For The Detection Of Concanavalin A And Insulin

Electrogenerated chemiluminescence（ECL）refers to a special chemiluminescence phenomenon which is generated by electrochemical redox reaction.It is the ideal combination of electrochemical and spectroscopic methods.Because of its good sensitivity,wide dynamic range,easy operation and rapid detection,ECL has been widely used in many analytical environments,such as DNA detection,metal ion detection,immunoassays and biological molecule recognition.Concanavalin A（Con A）is a lectin which is usually selected as a protein model for clinical research because it mediates the pathology and physiology process.Insulin is the only hormone in human body that can lower the blood glucose levels,and its disorder can lead to diabetes.Therefore,it is clinically significant to achieve the sensitive,rapid and accurate quantitative detection of Con A and insulin.Based on these importance,in this paper,three ECL sensors were constructed for detecting Con A and insulin detection.Part 1 A sandwich electrochemiluminescent biosensor based on enzymatic reactionto in situ generate coreactant of conjugated polymer for Con A detectionA novel“signal-on”biosensor for the detection of concanavalin A（Con A）was designed utilizing an enzymatic reaction to in situ generate coreactant of poly（9,9-di-n-octylfluorenyl-2,7-diyl）（PFO）.Firstly,PFO polymers were wrapped with graphene oxide nanosheets to obtain GO@PFO nanocomposite,which was modified onto the surface of the electrode as a matrix for immobilizing phenoxy-derivatized dextran（Dexp）throughπ-πstacking interaction.Then Con A interacted with Dexp through the specific carbohydrate-protein interaction.Finally,glucose oxidase（GOx）and lactateoxidase（LOx）decoratedgoldnanoparticles@C₆₀-Polyamidoamine(Au@C₆₀-PAMAM)was bound onto Con A via a biospecific inter action between Con A and the internal residues of GOx,thus achieving a sandwich structure.In this construction strategy,LOx catalyzed the oxidation of lactate to in situ generate H₂O₂,which could act as the coreactant of PFO to enhance the ECL intensity.Due to the excellent ECL property of PFO-H₂O₂ system,our prepared biosensor showed a wide linear range from 0.001 ng/mL to 10 ng/mL,and a low detection limit down to 0.0003ng/mL.The integration of excellent ECL property of PFO-H₂O₂ system and excellent catalytic activity of Au@C₆₀-PAMAM would open a new and promising platform for fabricating ECL biosensors and biocatalysts.Part 2 A ratiometric electrochemiluminescent biosensor for the detection of Con A based on competition of oxygenA novel ratiometric electrochemiluminescent（ECL）biosenor was designed for the detection of concanavalin A（Con A）based on two ECL emitters competing for the coreactant dissolved oxygen（O₂）.In this strategy,CdTe quantum dots（QDs）were used as cathodic emitter and N-（aminobutyl）-N-（ethylisoluminol）（ABEI）as anodic emitter.With the dissolved O₂ as coreactant,CdTe QDs showed an ECL emission at-1.7 V,and ABEI showed an emission at+0.6 V.CdTe QDs was functionalized with graphene oxide（GO）to synthesize graphene-CdTe QDs（G-CdTe QDs）,which worked as a matrix for immobilizing phenoxy-derivatized dextran（Dexp）throughπ-πstacking interaction.Afterwards,Con A was further immobilized through the specific carbohydrate-protein interaction.Then Dexp,gold and platinum nanoparticles decorated ABEI（Dexp-Au-Pt-ABEI）was introduced into the modified glassy carbon electrode to interact with Con A through the biospecific interaction between Con A and Dexp.Thus,the sandwich immunoassay for Con A detecting was successfully obtained.Upon the proposed biosensor,with the increasing concentration of analyte Con A,the ECL signal of cathodic CdTe QDs decreased and the anodic response of ABEI increased,and the ratio for the two kind of intensity was logarithmically related to the concentration of Con A.This ratiometric ECL biosensor exhibited excellent stability,selectivity and reproducibility with a linear range of 1.0×10^-410 ng/mL,and a detection limit of3.0×10^-5 ng/mL.The ratio ECL biosensor can effectively overcome environmental and instrumental disturbances,improving analysis sensitivity and reliability,and also eliminate the need for additional coreactant,making the ratiometric detection more convenient.It shows a good application prospect in biological analysis and clinical application.Part 3 A novel electrochemiluminescent biosensor based on resonance energy transferbetweenpoly（9,9-di-n-octylfluorenyl-2,7-diyl）and3,4,9,10-perylenetetracar-boxylic acid for insulin detectionAn electrochemiluminescencent（ECL）biosensor was designed for the determination of insulin using a novel ECL resonance energy transfer（ECL-RET）strategy.In this strategy,carboxyl poly（9,9-dioctyfluorenyl-2,7-diyl）dots（PFO dots）were worked as ECL donor and 3,4,9,10-perylenetetracar-boxylic acid（PTCA）exploited as ECL acceptor,and hydrogen peroxide（H₂O₂）employed as the coreactant.The ECL donor and ECL acceptor were separately labeled with primary antibody（Ab₁）and secondary antibody（Ab₂）,forming a sensing interface to the analyte target,insulin.In this expected sandwich-type ECL biosensor,PFO dots acted as sensing platform and PTCA employed as labels to quench the ECL emission of PFO dots.During the determination process,ECL signal of PFO dots was decreased in a gradual way by the increase of insulin concentration,and the quenching mechanism was also investigated.Under the optimal experimental conditions,the constructed biosensor exhibited an excellent performance,including a wide linear range from 1.0×10^-5 ng/mL to 1.0×10²ng/mL,low detection limit of 3.0×10^-6 ng/mL,good stability and selectivity for the detection of insulin.This pair of PFO-PTCA,as a new donor-acceptor pair in ECL-RET system,would provide a promising platform for bioanalysis in ECL field.