Sensitive Immunosensor For Microcystin-LR Detection Based On Nano-composite Biomaterials

Microcystins（MC）is a kind of hepatotoxin wildly in fresh water,and it is one of the routine testing items of urban drinking water.The conventional technique for MC detection is high-performance liquid chro matography（HPLC）,but it needs the bulky equipment and tedious sample pretreatment,causing time-consuming,high cost and difficultyof real-time monitoring in environment.Hence,it is important to establish a sensitive and simple technique for MC detection.Due to the specific molecular recognition between antibodyand antigen,the immunosensor is more specific than any other biosensors,and has been wildly investigated in the last decade s.Moreover,taking the advantages of the optical and electrochemical techniques,the immunosensor has become more and more sensitive,so it has been wildly used in many fields and developed towards more sensitive,specific and miniaturize.In this paper,three immunosensors based on electrochemical or optical detection signals were constructed to detect the most toxic microcystins（MC-LR）.The sensitivity of the immunosensor was further improved by nano-enhancement technique,enzyme-catalyzed amplification technology and DNA amplification technology.The main works and its results are as follows:（1）In-situ assembly of biocompatible core–shell hierarchicalnanostructures sensitized immunosensor for MC-LR detection.In this work,a sensitive biocompatible electrochemical immunosensor was constructed for MC-LR detection by synthesizing the three dimensional villiform-like carbon nanotube/cobalt silicate（CNT@Co silicate）core–shell nanocomposites as the substrate to immobilize the antigen of MC-LR（Ag）and Fe₃O₄nanoclusters/polydopamine/goldnanoparticles（Fe₃O₄@PDA–Au）core–shell magnetic nanocomposites as the label carrier of the immunosensor to conjugate the second antibody（Ab₂）and horse radish peroxidase（HRP）.Because of the high surface area and well biocompatibility of the as-prepared CNT@Co silicate and Fe₃O₄@PDA–Au,more immune molecules（Ag and Ab₂）and signal response molecules（HRP）can immobilize on their surface,which can further improve the sensitivity of the immunosensor.The as-prepared electrochemical immunosensor can successfully detect MC-LR in a range of0.005μg/L and 50μg/L with a detection limit of 0.005μg/L.（2）Multiple amplified electrochemical immunosensor based on DNAzyme functionalized mesoporous silica for MC-LR detection.In this work,the Au nanodendrites with huge surface area were electrodeposited on the ITO electrode as the substrate of immunosensor to capture more MC-LR antigen.Then,the monodisperse core-shell mesoporous silica nanoparticle（SiO₂@MSN）with electroactive material methylene blue（MB）embedded in its uniform pore channe l was synthesized as the label of the immunosensor to load Ab₂ for immunoreaction and DNA primer S₀ for signal improvement.Subsequently,two auxiliary DNA strands was selected for the in-situ propagation to form many double-helix DNAs on the surface of SiO₂@MSN through hybridization chain reaction（HCR）,and forming numerous DNAzymes（G-quadruplex/hemin）on their both sides after adding hemin.Finally,MB was inserted into the formed double-helix DNA to improve the detection signal.With the aid of MB,the formed DNAzymes can catalyze thereduction of H₂O₂to produce the electrochemical signal.The as-prepared electrochemical immunosensor can successfully detect MC-LR in a range of 0.5 ng/L and25μg/L with a detection limit of 0.3 ng/L,which was far below the MC-LR detection standard of WTO in drinking water（1μg/L）by taking advantages of nano-enhancement technique,enzyme-catalyzed amplification technology and DNA amplification technology.（3）Sensitive immunosensor for MC-LR visual detection based on nanozymes and DNAzyme integrated mimic enzyme.In this work,the silica/nickel silicate（SiO₂@Ni silicate）with large surface area was synthesized as the substrate of immunosensor to capture the MC-LR antigen.Then,a cage-like C u（OH）₂（Cu（OH）₂ SC）nanozyme with peroxidase activity was prepared as the label of the immunosensor to capture Ab₂ for immunoreaction and DNA primer S₀ for the formation of DNAzyme on its surface.Subsequently,two auxiliary DNA strands was selected for the in-situ propagation to form many double-helix DN As on the surface of C u（OH）₂ SC through hybridization chain reaction（HCR）,and forming numerous DNAzymes（G-quadruplex/hemin）on their both sides after adding hemin.The as-prepared nanozymes and DNAzyme integrated mimic enzyme（Cu（OH）₂ SC-G-quadruplex/hemin）with numerous catalyst units showed excellent peroxidase activity for the chromogenic reaction of ABTS,which can achieve the visual detection of MC-LR and provide the method for the commercialization of portable detection device of MC-LR.