Construction Of Microcystin Immunosensor Based On Nanomaterials

Microcystins?MCs?,produced by cyanobacteria,are widely distributed and severely damaged algae toxins and they have multiple organic toxicity,genetic toxicity and carcinogenicity.MCs can pollute drinking water and contaminate aquatic animals as well.MCs can also transfer through food chain that does harm to human health.The national standard drinking water handbook regulates the concentration of MC-LR no more than 1?g/L.Therefore,the detection and monitoring of MCs in source water is of great significance for ensuring the safety of water bodies.Traditional instrument methods have better analytical sensitivity,but the expensive instrument,complicated operation and the need of specialized technicians limit their wide applications.Hence,it is urgent for researchers to establish a simple,rapid and sensitive detection method to monitor the MCs in water.Immunosensors have the advantages of good selectivity,high sensitivity,fast analysis speed,low cost,continuous monitoring in complex systems.In recent years,with the rapid development of nanotechnology,various special physical and chemical properties of nanomaterials have been gradually explored,such as excellent electrical conductivity,chemical stability,large specific surface and good biocompatibility.Applications of nanomaterials are constantly expanding into all aspects of chemistry,biology,medicine and other fields.The use of nanotechnology can improve the sensitivity of immunosensors.In this paper,microcystin-LR?MC-LR?was chosen as object and several functionalized nanocomposites were synthesized.By using nanocomposites and the nucleic acid signal amplification techniques,sensitive immunosensors were constructed for selective and quick determination of MC-LR.More importantly,the biosensors showed good accuracy in real water sample.The main works are as follows:?1?The construction of electrochemical immunosensor based on Fe₃O₄@PDA/RGO substrate and RCA signal amplification strategyThe Fe₃O₄@PDA/RGO nanomaterial synthesized through hydrothermal process was served as substrate and gold nanorod was served as carrier to conjugate secondary antibody and primer.A multi-enzyme labeled electrochemical immunosensor was established based on Fe₃O₄@PDA/RGO nanomaterial and gold nanorod carrier.Abundant MC-LR antigen was immobilized onto ITO electrode due to the high specific surface of Fe₃O₄ and strong adhesive capacity of dopamine.After immunoreaction,the DNA primer could trigger rolling circle amplification?RCA?to get long single DNA strand.The DNA strand was served as template to link signal probe ST-HRP,which could enhance signal response and improve the detection sensitivity of immunosensor.The proposed immunosensor shows a linear range of 0.01-50?g/L and LOD of 0.007?g/L?S/N=3?for MC-LR detection.?2?The construction of dual-signal readout electrochemical immunoassay based on AuNP-CNT composite and HCR-induced copper nanoparticlesAn electrochemical immunoassay was proposed based on AuNP-CNT substrate and AgNP/AuNR nanocarrier.AuNP-CNT nanocomposite owned good biocompatibility and superior protein affinity that it was easy for MC-LR antigen to attach.The DNA primer conjugated by AgNP/AuNR nanocarrier could trigger hybridization chain reaction?HCR?.The HCR product could serve as template for the formation of copper nanoparticles.Both the silver nanoparticles that came from AgNP/AuNR nanocomposite and the copper nanoparticles were determinated through differential pulse stripping voltammetry?DPSV?measurements after dissolving by nitric acid.Two current peaks assigned to silver and copper were recorded on DPSV voltammogram.Such dual-signal readout strategy was available to improve detection sensitivity and decrease detection LOD and this immunoassay showed low LOD of 2.8 ng/L?S/N=3?for the determination of MC-LR.?3?The construction of fluorescent aptasensor based on DNA double strand induced copper nanoparticlesA simple and fast fluorescent aptasensor was established by using aptamer as recognition probe.Aptamer and its complementary strand could form double helix structure when they coexisted in solution.Fluorescent copper nanoparticles could grow into DNA double strand that high fluorescence intensity was achieved.If MC-LR was added into detection system,MC-LR could bind with aptamer specially,which decreased the fluorescence intensity.The concentration of MC-LR was reflected through the decreased fluorescent intensity.This aptasensor could quickly detect MC-LR with a linear range of 0.5-500?g/L and a LOD of 0.1?g/L.Though the LOD of this method was higher than electrochemical analysis method,its simple construction process provides some theoretical support for the miniaturization and portability of biosensors.