Construction Of Fluorescent Biosensor And Its Application In Detecting The Heavy Metal Ions

The fluorescence sensor is an intelligent device that could convert the specific molecular recognition into the visual signal with the assistance of the signal conversion system.Fluorescence sensors show some excellent properties,such as simple devices,fast response,high sensitivity and stability.Heavy metal ions are non-degradable and bio-enrichable in living organisms,which threaten seriously the human health.For example,Cr（VI）is highly carcinogenic,causing the severe irreversible damage to the human respiratory system,gastrointestinal system and kidney system.Excessive lead ions(Pb²⁺)are particularly harmful to children,which will lead to a decline in their cognitive and memory level.In addition,Pb²⁺also affect the normal development of the fetus and even lead to deformities.Toluene,a toxic organic reagent,is widely used in various fields and seriously harms the human respiratory and nervous systems.Therefore,it is very important to construct a series of novel fluorescent sensors for the accurate detection of toxic Cr（VI）,Pb²⁺and toluene.In this paper,the novel multifunctional N/S co-doped fluorescent carbon dots were prepared for detecting Cr（VI）and toluene.In addition,with the aid of toehold-mediated nucleic acid signal amplification technology,different fluorescence sensing platforms were developed for detecting trace Pb²⁺in environmental samples.The relevant experimental contents were summarized as follows:Part 1 A novel multifunctional fluorescent sensor based on N/S co-doped carbon dots for detecting Cr（VI）and tolueneIn this work,multifunctional nitrogen and sulfur doped fluorescence carbon dots（N,S-CDs）were synthesized with citric acid monohydrate and thiosemicarbazide as raw materials.The fluorescence intensity of N,S-CDs could be quenched obviously by Cr（VI）due to the inner filter effect,achieving a low detection limit of 0.33 n M for Cr（VI）.While excessive Ba²⁺was added,the fluorescence intensity could be restored.Meanwhile,based on the fact that the fluorescence of the N,S-CDs could be enhanced by toluene,the detection of toluene was achieved and the detection limit was low as0.03μmol/L.Furthermore,the practical application of the N,S-CDs for detecting Cr（VI）was assessed in the river water.The proposed N,S-CDs could provide a promising fluorescence platform for detecting different toxic pollutions.Part 2 DNA branched junctions induced the enhanced fluorescence recovery of FAM-labeled probes on r GO for detecting Pb²⁺The reduced graphene oxide（r GO）could strongly adsorb and quench the fluorescence of dye-labeled single-stranded DNA（ss DNA）.Thus,it is widely applied in fluorescent sensors.However,these sensors may suffer from a limited sensitivity due to the low fluorescence recovery when adding the complementary DNA（c DNA）sequence.In this work,the powerful DNA branched junctions were constructed to improve the fluorescence recovery of FAM-labeled probe on r GO.In the presence of target Pb²⁺,the ribonucleotide（r A）in the substrate was cleaved specifically and the catalytic hairpin assembly of three metastable hairpins was further initiated,accompanied by the formation of DNA branched junctions.Then,the liberated Pb²⁺could be recyclable.Impressively,the DNA branched junctions not only hybridize with the FAM-labeled probes with a high efficiency,but also are significantly undesirable for the r GO.Thus,a high fluorescence recovery of FAM-labeled probe on r GO was expected.The integration of the high fluorescence recovery and dual-cycle signal amplification endows the sensing strategy with a good performance for detecting Pb²⁺,including low detection limit（0.17 n M）,good selectivity,and satisfactory practical applicability.The proposed DNA branched junctions offer a novel avenue to improve the fluorescence recovery of the dye-labeled probes on r GO for biological analysis.Part 3 The fluorescence sensor based on enzyme-free localized hairpin-DNA cascade reaction and magnetic beads for the ultra-sensitive detection of Pb²⁺In the traditional hairpin-DNA hybridization reaction,the hairpin reactants randomly find their target hairpin to hybridize in three-dimensional space,so the hybridization efficiency is limited.In the localized hairpin-DNA cascade reaction,DNA hairpin reactants are closely arranged in a compact space with a specific order.The sequential DNA cascade reactions can be easily performed among the hairpin reactants,which significantly reduce the hybridization time and improve the efficiency of hairpin-DNA hybridization.Therefore,the localized hairpin-DNA cascade reaction has become an important way to obtain a high hairpin-DNA hybridization efficiency.However,the reported localized hairpin-DNA cascade reactions usually need the participation of expensive proteases.The proteases could lose their biological activity in the harsh environments,which leads to a limited application of the localized hairpin-DNA cascade reaction.In this work,an enzyme-free localized hairpin-DNA cascade reaction and magnetic beads with the excellent loading capacity were integrated to effectively improve the hybridization speed and efficiency of the DNA cascade reaction.In the presence of the target Pb²⁺,the DNAzyme was activated.The released DNAzyme chain could trigger the sequential hybridization of hairpins on MBs.The fluorescence emission was obvious quenched.The proposed fluorescence sensor showed the satisfactory detection performances for Pb²⁺with the low detection limit of3.0 pmol/L and was successfully able to monitor Pb²⁺in natural river.The excellent signal amplification ability of the enzyme-free localized DNA cascade reaction provides a new method for constructing the highly sensitive fluorescent sensing platform.