Quantum Dots-based Electrochemiluminescent Immunosensors For Monitoring Okadaic Acid In Aquatic Products

As a main component of diarrhetic shellfish poison?DSP?,okadaic acid?OA?can accumulate in the body and cause deformities and even cancer.Pollution of OA has become a serious problem affecting the shellfish industry and food safety.Thus,it is significantly important to establish a rapid,effective,and sensitive detection method for monitoring OA in shellfish.Electrochemiluminescence?ECL?is an ideal analytical method that combines the simplicity and controllability of electrochemistry with the high sensitivity,low background and wide linear range of chemiluminescence.Thus ECL analysis technique presents promising potential for their applications in food safety and environmental monitoring.The performance of ECL sensor is greatly affected by the ECL luminescent reagent.Graphene quantum dots?GQDs?are a kind of carbon-based nanomaterials with a lateral dimension of less than 100 nm and thickness of less than 10 layers,which have excellent quantum confinement and edge effects.In this dissertation,we discovered new methods for the synthesis of P,S-GQDs and N,S-GQDs,and developed new ECL systems based on P,S-GQDs/K₂S₂O₈,Ru?bpy?₃²⁺doped silica nanoparticles?RuSiNPs?/N,S-GQDs,and RuSiNPs/copper nanoclusters?CuNCs?.The ECL probe is formed by coupling the nanocomposite with the antibody of OA?anti-OA-MAb?,and the ECL response and stability of the detection system are enhanced due to the multiple amplifications and the good biocompatibility of the nanomaterial.Finally,the ECL immunosensors were successfully applied to the convenient,fast,accurate and sensitive detection of OA in shellfish.This dissertation is mainly divided into the following parts:1.Electrochemical synthesis of P,S-GQDs as efficient ECL immunomarkers for monitoring OAIn this chapter,P,S-GQDs were prepared by the one-step electrolysis of a graphite rod in an alkaline solution containing sodium phytate and sodium sulfide.Compared with GQDs and mono-doped GQDs?P-GQDs and S-GQDs?,the P,S-GQDs dramatically improved the ECL performance due to high efficiency doping of P and S.Therefore,they were used as bright ECL signaling markers through covalent conjugation with a monoclonal antibody against okadaic acid anti-OA-Mab to form ECL immunoprobe?named P,S-GQDs-Ab?in the following tests.Moreover,the carboxylated multiwall carbon nanotubes-poly?diallyldimethylammonium?chloride-Au nanocluster?CMCNT-PDDA-AuNCs?composite was used to promote electron transfer and enlarge the surface area,which provided a good platform for immobilization of OA.A novel competitive indirect ECL immunoassay based on the competition of P,S-GQDs-Ab for free and immobilized OA was developed to detect free OA.Under the optimized conditions,the50%inhibitory concentration(IC₅₀)of the immunosensor was 0.25 ng mL^-1,and its linear range was 0.01-20 ng mL^-1 with a low detection limit of 0.005 ng mL^-1.In addition,the proposed ECL immunosensor was applied to detect OA in spiked Ostrea plicatula samples.The recoveries of the immunoassay were more favorable than those obtained from OA ELISA kits,suggesting the low matrix effect of the immunoassay and high potential in monitoring OA in real samples.2.Establishment of OA immunosensor based on synergistic effect on ECL enhancement between N,S-GQDs and RuSiNPsIn this chapter,a competitive ECL immunosensor for fast and highly sensitive quantitation of OA was exploited by using a novel self-enhanced solid-stated ECL marker.N,S-GQDs were first prepared by electrolysis of graphite carbon rod in an alkaline solution containing 3-?N-morpholine?propane sulfonic acid?MOPs?.It is interesting that the resulting N,S-GQDs can be used as co-reactants for they dramatically improve the anodic ECL performance of Ru?bpy?₃²⁺in phosphate buffer solution.After functionalized Ru?bpy?₃²⁺-doped silica nanoparticles?RuSiNPs?with PDDA,the N,S-GQDs were immobilized on the surface of RuSiNPs by electrostatic interaction.As the N,S-GQDs@RuSiNPs embeds the luminophor and co-reactant together,which improved their electron transfer rate as well as shorten their interaction distance and reduced the energy loss of light irradiation,the core-shell structural N,S-GQDs@RuSiNPs reveals advanced ECL property than RuSiNPs.Using the“bright”ECL beacon,the ECL immunosensor expressed a low IC₅₀ of 0.14 ng mL^-1,a wide linear range of 0.003-40 ng mL^-1 and a low detection limit of 1 pg mL^-1 for the determination of OA.Finally,the as-developed ECL immunosensor was successfully applied to monitor OA content in the certified reference mussel?CRM?and Solen gouldi samples.3.Development of a novel dual-signal ECL immunosensor based on the promotion of CuNCs on RuSiNPs bipolar ECL signalsIn this chapter,we found that the co-reactant CuNCs are capable to simultaneously promote both cathodic?-0.95 V?and anodic?+1.15 V?ECL signals of RuSiNPs,thus a new dual-signal ECL immunosensor was developed to detect OA.It was found that the ratio of the enhancement of ECL signals between the anode and cathode of RuSiNPs was a constant when the relative concentration ratio of the co-reactant CuNCs and the luminescent RuSiNPs was determined.Based on these observations,RuSiNPs@CuNCs were used as immunomarkers through effectively covalent conjugation with anti-OA-MAb to form probes with excellent ECL performance.Finally,dual ECL signals of the proposed immunosensor was fabricated and showed good linear relationships with OA concentrations in the range of 0.05-70 ng mL^-1,IC₅₀was 1.972 ng mL^-1,and the detection limit was 0.039 ng mL^-1.Moreover,from the constant ratio of the cathode and anode ECL peaks,the self-calibration of the detection signal is realized and the reliability of the experimental results is improved.Finally,the as-fabricated ECL sensor was successfully to be used in the detection of OA in spiked scallop samples.