Study On AgNCs-based Fluorescence Biosensor Through Specific Target Recognition To Induce Conformation Switch Of DNA Structures

DNA-stabilised fluorescent silver nanoclusters（DNA/Ag NCs）are a new class of fluorescent nanostructures formed by a specific single-stranded DNA sequence stabilizing a small number of silver atom clusters.Due to their excellent photostability,good biocompatibility,high fluorescence quantum yield,and thus they have been widely used for the detection of small molecules,DNA,RNA and proteins,as well as for biomarking and bioimaging.Compared with traditional organic fluorescent dyes and fluorescent nanoparticles,DNA-Ag NCs are easier to synthesize and modulate their properties.Moreover,the fluorescent biosensors,developed from DNA-Ag NCs are characterized by high sensitivity and good selectivity,were widely applicated in the field of biochemical sensing.In this paper,three functionalized fluorescent biosensors were designed and assembled using DNA/Ag NCs as fluorescent signal probes,combined with target-specific recognition and DNA structural conversion.The specific works are as follows:1.Antibody-specific recognition regulated immunosensor for fluorescence signal conversion of d-Ag NCsImmunosensors have the advantages of simple operation,high sensitivity and high selectivity,which have great advantages in detecting antibodies,especially divalent antibodies（such as digoxin antibody,Dig A）.In this work,we reported a sensitive immunosensor for Dig A based on the switchable fluorescence of DNA-templated silver nanoclusters（Ag NCs）modulated by hemin/G-quadruplex（h Gq）.To explore the fluorescing performance of one h Gq quenching dual-Ag NCs（d-Ag NCs）,here we designed a functional hosting DNA strand（HGH）encoding with one centered G-rich segment for forming h Gq complex in the presence of hemin and K⁺,two terminated C-rich template sequences for the identical clustering of d-Ag NCs,and two flanked symmetric domains for specific recognizing and linking.Because of the photoexcited electron transfer process to the stacked h Gq,the originally presynthesized d-Ag NCs colocalized in two ends are non-emissive,which are retainable after complementarily hybridizing with two recognizable single strands that are both tagged with a digoxigenin hapten,a Fab fragment.Upon introducing Dig A,the steric strain associated with the affinity binding of two haptens results in the opening and stretch of h Gq in a coil spacer,thereby lighting-up the d-Ag NCs fluorescence.Based on a one-step signal switch stimulated by targeting antibodies,this label-free immunosensor strategy is of high specificity and sensitivity with a low picomolar detection limit,as well as simplification,rapidness and cost-effectiveness.2.Dual-channel fluorescence biosensor of DNA"Butterfly"structure loaded with two-color Ag NCs signal transformationIn this work,we used two specific sequences of COVID-19 as dual targets,T_R and T_O,and two-coloured silver nanoclusters as signal molecules,driven by chain substitution reactions,to achieve amplified detection of the dual target signals.Initially,the template sequences of g-Ag NCs and r-Ag NCs are locked at the diagonal positions of the"Butterfly"structure,and when the two specific sequences of the target,T_R and T_O,are present,they are first hybridised with the auxiliary hairpins H_R and H_O,resulting in a DNA double-stranded structure that is then rigidly assayed.The two diagonal strands of the"Butterfly"structure are then replaced by the two diagonal strands,exposing separate templates for the Ag NCs.Under complexation and reduction of Ag⁺and Na BH₄,a dual fluorescent signal is emitted,while sensitive detection of dual-target signal amplification is achieved by cyclic release.The method allows the synthesis of two silver clusters by one method,and the signal amplification response to the dual target is achieved by a chain substitution reaction.The synthesized g-Ag NCs and r-Ag NCs and structurally independent of each other,with good stability.By g-Ag NCs can achieve accurate and sensitive detection of Rd Rp-COVID in the concentration range of 1 pmol/L to 1μmol/L,with a detection limit of 0.52 pmol/L,and r-Ag NCs can achieve accurate detection of ORFlab-COVID in the concentration range of 0.1 pmol/L to 1μmol/L.The detection limit was 0.048 pmol/L.The method can simultaneously and sensitively detect two novel coronavirus-specific sequences,improving the specificity of COVID-19 detection and avoiding false positives,which is important for the expansion and construction of novel fluorescent sensors for rapid and accurate clinical identification and detection of COVID-19.The method has important implications for the expansion and construction of novel fluorescent sensors for the rapid and accurate clinical identification and detection of COVID-19,and is expected to be applied to the sensitive detection of other disease markers.3.Fluorescence biosensor study on signal transduction of Ag NCs induced by DNA triplex conformational transformationTriplex molecular beacons have good binding strength controllability and great potential for applications in biosensing.In this work,we rationalized the design of a DNA triplex fluorescent probe（TFP）to enable the detection of HIV-related sequences（T_H）.In the TFP,we use the protonated（C-G·C+）and（T-A·T）as the ring part,where Watson-Crick and Hoogsteen base pairing together to control the binding strength,which can be used to synthesize a DNA triplex structure,using the two separated C-rich templates of DNA/Ag NCs as the stem Initially,TFP is in a hairpin state and hybridizes with S_T to form a triplex structure,at which point the two sequences used to synthesize the silver cluster cannot be synthesized because they are kept away,and when the related sequence T_H of HIV is present,it breaks the triplex structure and brings the two sequences of the silver cluster template close together,synthesizing green fluorescent Based on this,the method adjusts the triplex state of TFP by affinity binding to HIV-related sequences to achieve a change in fluorescence signal and achieves sensitive detection of HIV in the concentration range of 0.1 pmol/L～1μmol/L with a detection limit of 0.041 pmol/L by detecting the fluorescence signal of TFP-g-Ag NCs.The rapid,specific and sensitive detection of HIV can be easily achieved,allowing early detection of potential HIV infections,effective control of virus transmission and diagnosis of the disease,which is of great significance for the prevention and control of virus replication and improvement of patient prognosis.