Construction Of Fluorescent Biosensor Based On Functional DNA Nanomachine And DNA Sequence-harbored Silver Nanoclusters

As a promsing fluorescent nanomaterial for biosensing and bioanalysis,DNA sequence-harbored silver nanoclusters（DNA/Ag NCs）can convert the specific interaction between the recognition and analyte into fluorescent signal for realizing quantitative detection of the analyte.Because of extremely attractive photophysical characteristics such as ultra-small size,convenient synthesis,excellent biocompatibility and long-term photostability,recently substantial research interests have been received for Ag NCs,which tend to have more extensive application in biosensing,imaging,disease diagnosis,and etc.In this paper,aiming at constructing more convenient,accurate,sensitive and efficient fluorescence platform of biosensors,we proposed several new fluorescent biosensors based on DNA-templated Ag NCs by combining with biomolecular sensing and signal amplification strategy.The specific works are as follows:1.Antibody-responsive signal-off fluorescence of DNA-harbored silver nanoclusters for direct,rapid and sensitive immunoassaySpecific DNA sequence-templated silver nanoclusters（Ag NCs）are extremely promising fluorescent emitters in biosensing and bioanalysis.Here,based on the targeted antibody-responsive fluorescent quenching of Ag NCs harbored in a unique DNA-based nanoassembly,we propose a specific,sensitive and one-step immunoassay of bivalent anti-digoxigenin（Dig）antibody（anti DA）as a model analyte.For proof-of-concept,this functional nanoassembly（CS1/RS/CS2）consists of a reporter strand tethering Ag-nucleable template（RS）and two capture strands（CS1 and CS2）labeled with hapten Dig,in which two tethered Dig haptens are closely positioned,and brightly emissive Ag NCs can be accommodated and stabilized in the unpaired Ag-nucleable scaffold to produce significant fluorescent emission（CS1/RS/CS2-Ag NCs）.In the presence of anti DA,the specific conjugation of anti DA with two Dig haptens results in the conformation stretch of CS1/RS/CS2,and simultaneously the harbored Ag NCs are destabilized to aggregate each other into larger non-fluorescent silver nanoparticles（Ag NPs）.The remarkable decreasing of Ag NCs fluorescence emission is linearly proportional to the anti DA concentration with a high sensitivity down to 4.5 pmol/L.With this regulatory antibody-dependent signal off scheme,the advantageous rapidness and simplification would be attractive and promising for highly specific detection of diverse bivalent antibodies or small molecules.2.Guanine-lighting-up fluorescence biosensing of silver nanoclusters populated in functional DNA constructs by a p H triggered switchDark or weak-emissive DNA-harbored silver nanoclusters（Ag NCs）can be remarkably lighted-up when approaching to guanine bases.The resultant bright Ag NCs acting as fluorescent reporter are fascinating in biosensing.To explore the applicable guanine-enhanced emission of Ag NCs for biosensing micro RNA-155（mi R-155）as model,here we designed a unique stem-loop hairpin beacon（HB）encoding with mi R-155-recognizable sequence and Ag NCs-nucleable template,as well as a hairpin helper tethering partially locked guanine-rich（15-nt）tail(G₁₅H),while two identical cytosine-rich segments were inserted in HB and G₁₅H to merge for folding/unfolding of i-motif at changed p Hs.Initially,the intact clusters populated in HB（HB/Ag NCs）were almost nonfluorescent in buffer（p H 7.0）.Then mi R-155was introduced to trigger a repeated hairpin assembly of HB and G₁₅H by competitive strand displacement reactions at decreased p H 5.0 within 10 min,consequently generating numerous duplex DNA constructs（DDCs）.With the resultant template of p H-responsive i-motifs incorporated in DDCs,their folding at p H 5.0 brought the proximity of unlocked G₁₅overhang to the clusters in a crowded environment,remarkably lighting-up the red-emitting fluorescence of HB/Ag NCs(λ_em=628±5 nm)for amplified signal readout.About 3.5-fold enhancement of quantum yield was achievable by using different variants of i-motif length and G₁₅ position.Simply by adding OH^-,the configuration fluctuation of i-motifs was implemented for switchable fluorescence biosensing to variable mi R-155.Based on one-step amplification and signaling scheme,this subtle strategy was rapid,low-cost and specific for mi R-155 with a high sensitivity down to 67 pmol/L.3.DNA tweezer-programmed ratiometric fluorescence of silver nanoclusters for precise and sensitive detection of ORFlab-COVIDAs a typical fluorescenct nanomaterials,DNA-scaffold silver nanoclusters（DNA/Ag NCs）have been widely applied in bioimaging and biosensing.However,the poor stability and accuracy performance disturbed by complex system still exist in the conventional fluorescent biosensors based on self-color Ag NCs.Additonally,DNA tweezers are used to construct functional nanomachines as usual,which owed to the ability in effectively and accurately regulating the spatial position between two molecules or within DNA structures.But,the lack of efficient signal amplification strategy in assembly progress limits the sensitivity of the methodology.Herein,based on the ratiometric fluorescence of double-molecule Ag NCs populating in both ends of DNA tweezer,we proposed an accurate and sensitive fluorescent strategy for biosensing the ORFlab-COVID,which a selected sequencen from the severe acute respiratory syndrome coronavirus 2 associated with the novel coronavirus disease).In short,the assembly of target-induced DNA tweezer can effectively realize the recycling and sensitive detection of target ORFlab-COVID.Morever,the dual-color Ag NCs of yellow-emissive Ag NCs and red-emissive Ag NCs（y-Ag NCs and r-Ag CNs）can be precisely programed by DNA tweezer in spacial.Simultaneously,the fluorescence of y-Ag NCs remained unchanged,while the fluorescence of r-Ag NCs was significantly enhanced when guanine-rich sequence proximity.By virtue of such ingenious design,this reported biosensor of dual-color Ag NCs harbored in DNA tweezer was accuracy and sensitive in response to ORFlab-COVID with a high sensitivity down to 0.27 pmol/L.