Construction Of Host-Guest Interaction-based Electrochemical And Electroehemiluminescent Biosensors And Their Applications

Supramolecular chemistry is a highly interdisciplinary and fast-growing research field in chemistry,biochemistry,and materials science.Supramolecular intreaction is based on molecular recognition,namely host-guest recognition.Host-guest interaction is the process in which the host selectively combines the guest to form a supramolecular with specific function,including selective substrate–enzyme interactions,specific DNA–protein interactions,hydrogen bond guided interactions among DNA and RNA,antigen–antibody interactions.As an important research area in supramolecular chemistry,host-guest chemistry has found wide applications in the field of chemical sensing,especially in analytical chemistry,sensors,environment protection and biomedical research.In this thesis,we developed a series of host-guest reaction-based electrochemical and electrochemiluminescent biosensors with high sensitivity and good specificity,and applied them to sensitive and selective detection of uracil DNA glycosylase（UDG）and M.Sss I methyltransferases（M.Sss I MTase）.We develop a low-background electrochemical biosensor for one-step detection of uracil DNA glycosylase（UDG）based on the host-guest interaction and iron-embedded nitrogen-rich carbon nanotube（Fe–N–C）that mimics enzyme-mediated electrocatalysis to achieve signal amplification.In this work,Fe–N–C is initially immobilized on a glassy carbon electrode,followed by the immobilization ofβ–cyclodextrin（β–CD）.We construct the signal probes by assembling the methylene blue（MB）-labeled hairpin DNAs onto the surface of Au nanoparticles（Au NPs）to form the MB-hairpin/Au NPs probes.Due to the steric effect of Au NPs and the stem–loop structure of hairpin DNA,MB is prevented from entering the cavity ofβ-CD on the electrode.In contrast,UDG enables the removal of uracil from the U·A pairs in the stem of hairpin DNA probe to generate apurinic/apyrimidinic（AP）sites,leading to the assembly of MB-hairpin/Au NP probes on the electrode based on host–guest reaction betweenβ-CD and MB.Meanwhile,L-cysteine（RSH）is oxidized by O₂ to disulfide L-cystine and H₂O₂.In the presence of H₂O₂,Fe–N–C catalyzes the oxidation of MB to generate an amplified electrochemical signal.Notably,the Fe–N–C catalyzed oxidation of MB is mediated by the oxidation of RSH by O₂ instead of external H₂O₂,greatly simplifying the experimental procedures and improving the electrochemical signal.Due to the introduction of host–guest recognition,this electrochemical biosensor displays a low-background signal and high signal-to-noise ratio,enabling the one-step sensitive measurement of UDG with a detection limit of7.4×10^-5 U m L^-1.Moreover,this biosensor can measure UDG in crude cell extracts and screen the inhibitors,providing a new platform for biomedical research.We demonstrate for the first time that the host-guest recognition coupled with triple signal amplification endows the electrochemiluminescent（ECL）biosensor with enhanced sensitivity for uracil DNA glycosylase assay.We design a dually labeled hairpin DNA probe with ferrocene（Fc）as the guest molecule and the Fe MOF/Au NPs@luminol as the ECL tag.In the presence of UDG,it can remove uracil from the hairpin DNA probe stem to produce an apurinic/apyrimidinic site,resulting in the unfolding of hairpin structure and subsequently making the Fe MOF/Au NPs@luminol-modified single-stranded DNAs（ss DNAs）immobilize on the graphene oxide-modified electrode via host-guest between Fc andβ-CD.The reaction of acid and K₄Fe（CN）₆ with Fe³⁺in Fe MOF leads to the formation of prussian blue.Prussian blue can catalyze the deposition of H₂O₂ to produce OH^·which subsequently reacts with luminol radical to form 3-aminophthalate(AP^2-*)for the generation of an enhanced ECL signal.This assay involves triple signal amplification including:（1）electrochemically oxidized Fc（Fc⁺）catalyzes the decomposition of H₂O₂ to form AP^2-*;（2）the Fe MOF/Au NPs@luminol probe loads abundant luminol molecules to amplify the ECL signal;and（3）prussian blue catalyzes luminol radicals to form AP^2-*in the presence of H₂O₂.This biosensor exhibits good selectivity and extremely high sensitivity,superior to most of the reported biosensors.Furthermore,the proposed biosensor can be used to screen the UDG inhibitors and measure cellular UDG activity,holding great potential in clinical diagnosis and drug discovery.We develop for the first time a host-guest interaction-based and MOG-based biosensor with aggregation-induced electrochemiluminescence enhancement for M.Sss I Mtase assay.Metal-organic gels（MOGs）are new soft materials with the characteristics of high colloidal stability,superb luminescence properties,and facile synthesis.This biosensor employs a MOG as the luminophor and potassium persulfate as the coreactant,and the formation of the Ag-MOG from the aggregation of silver nanoclusters can induce significant ECL enhancement.Two complementary single-stranded DNAs（ss DNAs,i.e.,biotinylated DNA-1 and Fc-labeled DNA-2）that contain specific recognition sequence 5′-CCGG-3′can form a double-stranded DNA（ds DNA）probe.In the absence of M.Sss I MTase,the ds DNA probe will be digested by restriction endonuclease Hpa II,leading to the release of Fc from magnetic beads（MBs）.Theβ-CD can specifically recognize the released Fc through guest-host interaction,resulting in the quenching of an ECL signal.In contrast,the presence of M.Sss I MTase enables the formation of fully methylated ds DNA,which cannot be cleaved by Hpa II,making Fc remain on the MBs surface and consequently generating an improved ECL signal.This biosensor can specifically detect M.Sss I MTase with a linear range of 0.05-100 U m L^-1 and a limit of detection of 3.5×10^-3 U m L^-1,and it enables accurate detection of M.Sss I MTase in human serum.In addition,it can be used for the screening of inhibitors,with wide applications in drug discovery and disease diagnosis.