Nanoelectrochemical Immunosensing Interface Fabrication And Their Application On The Detection Of Microcystins

Microcystins produced by cyanobacterical are a family of hepatotoxic cyclic heptapeptides which are known as the potent hepatotoxins and tumor promoters.So far,MCs has been found to reach more than 90 isomers,in which microcystin-leucine-arginine?MCLR?is the most common and toxic variant of the MCs.The traditional methods of detection MCs might be limited due to their shortcomings,such as expensive equipments,time-consuming sample pretreatment.Therefore,the developing of convenient,rapid and sensitive methods for detection MCs is not only urgent and but also important for ensuring water quality and protecting human health.The methods of electrochemical immunosensor are important for rapid detection of MCLR.In recent years,the combination of nano technology and sensor has become the research hotpot of immunosensor.In this paper,different types of electrochemical immunosensors were constructed for detection of MCLR based on various nano materials,such as Fe₃O₄ magnetic nanoparticles,Au nanoparticles?AuNPs?,graphene nanosheet,quantum dots?QDs?and molybdenum disulfide,etc.The main research contents are as follows:?1?The carboxyl-functionalized Fe₃O₄ magnetic nanoparticles?MNPs?were modified onto the surface of a home-made magnetic glassy carbon electrode?MGCE?to construct a voltammetric immunosensor for detection of MCLR.Direct competitive immunoassay format was adopted.In the presence of horseradish peroxidase-conjugated MCLR?MCLR-HRP?,MCLR in solution was determined by differential pulse voltammetry?DPV?.Under the optimum conditions,the linear range and the detection limit of the immunosensor to MCLR were investigated.The immunosensor was applied in the determination of MCLR in real samples.The carboxyl group was modified on the surface of Fe₃O₄ MNPs via the chemical reaction of succinic anhydride and dimethylsulfoxide at room temperature.Therefore,it was avoided that the magnetic properties of Fe₃O₄ nanoparticles were reduced by high temperature heating,which can enhance the adsorption between MNPs and MGCE.A composite in solution was prepared by the coupling reaction between carboxyl groups on the Fe₃O₄ MNPs and amino groups on the antibody of MCLR?anti-MCLR?.The composite was modified onto the MGCE surface.The coupling reaction was adopted to immobilize anti-MCLR onto the surface of Fe₃O₄ MNPs.Compared with traditional cross-linking reaction,the coupling reaction possesses better selectivity and can better maintain the biological activity of the antibody.The developed analytical method in this work owns these advantages:convenient,quick and be easy to implement field analysis.?2?The electrochemical reduction of AuCl₄^-was performed at a constant potential which can electrodeposited AuNPs as a matrix to immobilize the antibody of MCLR?anti-MCLR,Ab₁?by Au-S bonds on the surface of a glassy carbon electrode?GCE?.Take the thiohydracrylic acid as stabilizer,water-soluble CdS QDs was synthesized.Transmission electron microscope image?TEM?,photoluminescence spectra,ultraviolet-visible?UV-vis?absorption spectra were used to characterize the CdS QDs.The synthesized QDs CdS was used as a signal probe and was coupled with anti-MCLR?Ab₂?bycondensationreaction.Asandwich-type electrochemiluminescent?ECL?immunosensor for detection of MCLR based on CdS quantum dots?QDs?was constructed.Under the optimum conditions,the linear range and the detection limit of the ECL immunosensor were investigated.The data of determination of real samples obtained from the ECL immunosensor were compared with those of HPLC and satisfactory results were obtained.The immunosensor for MCLR constructed by this paper possesses some advantages,e.g.stable light emitting signal and high sensitivity etc.?3?The AuNPs sol used in this work were prepared by the sodium citrate reduction method and were characterized by TEM and UV-vis absorption spectroscopy.AuNPs were modified on the surface of gold electrode?GE?via the thiols on the two sides of ethanthiol?HDT?which not only increase the electroconductivity but also enlarge the specific surface area of GE.Therefore,the immobilized amount of the antibody in the immunosensor is increased.A novel electrochemical immunosensor for MCLR based on gold electrode modified with Au nanoparticles has been developed.MCLR and horseradish peroxidase conjugated MCLR?MCLR-HRP?competitively combined with immobilizing anti-MCLR.The determination of MCLR using the immunosensor was investigated by DPV and amperometry,respectively.Under the optimized conditions,the linear range and the detection limit of were investigated.The immunosensor was applied in the determination of MCLR in real samples.The immobilization method of anti-MCLR could preserve the biological activity well.The thin sensing film on the GE surface was benefit for diffusion of molecule and ion.The developed immunosensor in this work has the advantages of simple preparation method,good reproducibility and high selectivity.?4?Graphene nanosheet which owns the advantages of good eletroconductibility,great specific surface area and excellent biocompatibility was prepared by Hummers method and was characterized by scanning electron microscope?SEM?and TEM.A nonenzymatic electrochemical immunosensor based on AuNPs as a signal probe was developed for the detection of MCLR using grapheme sheet?GS?as a matrix to connect bovine serum albumin coupling MCLR?BSA-MCLR?.MCLR in solution and BSA-MCLR which modified on the immunosensor competitively combined with the AuNPs labeled anti-MCLR.After the immune reaction is complete,AuNPs were electrochemically oxidazed into AuCl₄^-at constant potential.Cathodic potential scan was carried out by DPV and AuCl₄^-ions were reduced into Au at the immunosensor.Peak current of DPV was used as the detecting signal to determine MCLR concentration.Under the optimal conditions,the linear range and the detection limit of the immunosensor were investigated.Compared with enzyme-labeled probe,AuNPs labeled probe made the determined process have the virtues,e.g.low cost,high stability,excellent detection result.?5?Positively charged poly?diallydimehyl-ammonium chloride??PDDA?was assembled on the surface of the GS which loaded large amounts of negatively charged CdSe QDs to prepare a composite?denoted as P-GS@QDs?.The composite was used to label antibodies and to amplify ECL signal.The new-type two-dimension nanomaterial layered Mo S₂ interacted with AuNPs on the surface in order to increase the conducting and loading amount of biomolecules.The ECL immunosensor was constructed using layered MoS₂-Au as the as ground substance to carry the BSA-MCLR biomolecules and graphene nanosheet as the scaffold to load large number of CdSe QDs.Under the optimal conditions,direct competitive immune model was adopted.The linear range and the detection limit of the immunosensor were investigated.The sensitivity of the ECL immunosensor constructed in this work using nanocomposite material was extremely enhanced.The immunosensor with the characteristic of high sensitivity and stability was applied in the determination of MCLR in real samples and satisfactory results were obtained.