Fluorescent Biosensing Based On G-quadruplex And DNA-AgNCs Combined With Exo?-assisted Signal Amplification For MicroRNA And S1 Nuclease Analysis

Recently,researchers have discovered that many small molecules and proteins can be used as markers of cancer,such as nucleases,DNA,and miRNAs.Their abnormal performance is closely related to cancer and plays an important role in biological research and clinical diagnosis.Therefore,the highly sensitive and specific detection of related markers is of great significance for the early diagnosis and treatment of cancer.In the biosensing method,many isothermal signal amplification techniques assist in the detection of markers and improve the sensitivity of the method.Among them,the amplification technique based on the Exo III-assisted target cycle has been widely applied to various nucleic biosensing design because it does not require a specific sequence recognition site.DNA-AgNCs have excellent spectral properties,good biocompatibility and low toxicity,the G-quadruplex combines with small molecules to form a label-free probe that catalyzes the reaction and illuminates the fluorescence.Based on these biological characteristics,unlabeled probes composed of DNA-AgNCs and G-quadruplex have been widely used for the detection of biomarkers such as nucleic acids,proteins,small molecules,and metal ions.In this paper,a new method for miRNA and S1 nuclease analysis based on G-quadruplex and DNA-AgNCs fluorescent probes in combination with Exo III-assisted target multiplex cycles has been established.The specific research contents are as follows:Chapter one:we mainly introduce several commonly used nucleic acid signal amplification techniques in biosensing,as well as the properties of metal nanoparticles and G-quadruplex and their application of biosensing method.Chapter two:we build a novel method for rapid and simple detection of miRNAs based on G-quadruplex binding to small molecule emission fluorescence and Exo III-assisted target dual-cycle signal amplification.Two hairpins have been designed in the experiment,HP1 and HP2,which contain a G-quadruplex sequence and are resistant to Exo III cleavage,and the 3'end of the hairpin HP1 contains the recognition sequence of the target.The G-quadruplex sequence is blocked with in the hairpin in the absence of miRNA,G-quadruplex structure cannot form and resulting in a weak fluorescent signal.When miRNA is present,the miRNA hybridizes with HP1 to form a double-stranded miRNA-HP1 complex with a 3'-flat end,and after Exo III hydrolysis releases the X-sequence with the G-quadruplex and undergoes a miRNA cycle reaction;The released X-sequence specifically binds to HP2 to form a double-stranded X-HP2complex with a 3'-flat end,and Exo III continues hydrolysis the X-HP2 complex to release the Y-sequence with the G-quadruplex and perform X-sequence cycling reaction.The miRNA and X sequences undergo cyclic reaction to release more X and Y sequences.K⁺is added to the reaction system to fold the two sequences into a G-quadruplex structure,NMM can be inserted into the G-quadruplex to produce strong fluorescence.The method detects the linear range of miRNA is 0.08⁴ nmol/L,and the detection limit(LOD)is 6 pmol/L(S/N=3,n=11).In addition,the strategy has highly specific,and the target recovery is achieved in real human serum samples,the recovery rate are 98.0¹10.0%,which has a certain application value in the detection of real samples.Chapter three:we design a biosensing method for microRNA analysis based on DNA-AgNCs fluorescence quenching and Exo III-assisted target triple-cycle.This method designs three hairpins H1,H2 and H3 that can resist Exo III cutting,the 3'-end of hairpin H1contains target recognition sequence,the rings and stem parts of hairpin H3 are rich in cytosine(C)bases as silver nanocluster template chains.When there is no miRNA,the three hairpins can resist the cutting of Exo III and in a stable state,the strongly fluorescent DNA-AgNCs are synthesized by the addition of AgNO₃ under the reduction of NaBH₄;When miRNA is present,the miRNA first hybridizes with H1 to generate a 3'-flat end double-stranded miRNA-H1complex,and Exo III cleaves to release the X-sequence and perform miRNA cycle reaction.X-sequence hybridizes with hairpin H2 to form a 3'-flat end double-stranded X-H2 complex,and Exo III continues to cleave the X-H2 complex to release the Y-sequence and carry out the X-sequence cycle reaction;Y-sequence hybridizes with hairpin H3 to form a 3'-flat end double-stranded Y-H3 complex,and Exo III continues to cleave the Y-H3 complex to release the Z-sequence and perform a Y-sequence cycle reaction.Through the triple-cycle reaction of microRNAs and their substitutes X and Y-sequences,more hairpin H3 silver nanocluster templates were cut into single base nucleotides,which could not synthesize DNA-AgNCs leading to weak fluorescence signals.The method detects the linear range of miRNA is 0.05⁴nmol/L,and the detection limit(LOD)is 31.7 pmol/L(S/N=3,n=11).In addition,the bioassay can distinguish 100 times the concentration of other miRNA,and the target recovery is achieved in real human serum samples,the recovery rate is 99.5%¹20.7%,indicating that this method has a good application value in the detection of actual samples.Chapter four:we develop a biosensing technology for S1 nuclease detection based on the proximity-effects of DNA-AgNCs and the helper chain to enhance fluorescence.Two DNA probe P1 and P2 were designed,and P1 partly hybridize with P2.DNA fragment(5'-CACCGCT-3')of P1 as an auxiliary chain can enhance fluorescence of DNA-AgNCs.DNA fragment(5'-TGCCTTTTGGGGACGGATA-3')of P2 as a silvercluster template is used to synthesis DNA-AgNCs,which has no strong emission at 660 nm when excitation wavelength of600 nm.When there is no S1 nuclease,the hybridization of P1 and P2 leads to the adjacent between helper chain fragments and silvercluster template fragments.The addition of AgNO₃under the reduction of NaBH₄,resulting in a strong fluorescence emission at 660 nm.When S1nuclease is added,the S1 nuclease cleavage the P1 chain into small fragments,and the P2 chain can only be in a free state.The synthesized DNA-AgNCs can not emitting strong fluorescence at660 nm.The method detects the linear range of miRNA is 0.1¹ U/mL,and the detection limit(LOD)is 0.028 U/mL(S/N=3,n=11).In addition,the bioassay has good specificity and can distinguish 5 times concentration of other endonucleases and protein.The recovery rate of S1nuclease in real human serum samples is 97.0¹02.9%,indicating that this method has potential application in the detection of actual samples.