The Construction Of Self-assembled Multivalent Functional Nucleic Acid Nanoprobes And Their Biosensing Application

Sensitive detection of tumor markers has important clinical significance for early diagnosis,monitoring recurrence and judging prognosis of cancer.However,the content of tumor markers in clinical liquid samples(urine,plasma or tumor cells)is very low.How to develop probes with high output signal intensity is the bottleneck problem of tumor marker detection.Functional nucleic acid nanomaterials,as a new kind of biological nanomaterials,have attracted more and more attention in the field of biological analysis because of their programmability and the advantages of carrying a variety of functional units.Among them,the functional nucleic acid probes constructed based on the multivalent binding strategy can effectively enhance the affinity and signal output intensity of the detection system by means of the overall event of multiple monomer recombination.The construction of conventional polyvalent nucleic acid functional probes often requires the tedious design process and the assembly procedure,which limits its application in the field of life analysis.Therefore,how to develop simple multivalent functional nucleic acid nanoprobes to meet the demand of high signal output in complex biological matrices has become a common expectation of researchers.In order to develop simple and efficient multivalent DNA binding probes with high signal-to-noise ratio and satisfy the requirement of high signal output in clinical liquid samples,two kinds of multivalent signal output probes(rigid "Y-type" and "FRET network cluster")were constructed,which could be modularized and flexibly designed.In addition,the morphological characterization of multivalent signal model was performed.We also introduce magnetic nanoparticles to reduce background signal interference.It consists of the following two parts:1.Sensitive label-free fluorescence detection of p53 DNA based on KFP polymerase driven Y-type multivalent nucleic acid probeTaking p53 DNA as model,three nucleic acid hairpin probes were used as the multivalent assembly unit.With the help of KFP polymerase,the opening and polymerization extension of the three hairpin probes were achieved sequentially under the trigger of p53 DNA,forming a large number of rigid double-stranded "Y-type" multivalent signal output probes.After that,SYBR Green I(SG I)was embedded into "Y-type" double-stranded DNA to realize the high sensitivity of label-free detection of p53 DNA.At the same time,magnetic nanoparticles were introduced to reduce the background signal and improve the signal-to-noise ratio.The rigid double-stranded "Y-type" multivalentsignal output probe constructed is simple to synthesise,carries abundant SG I binding sites,and still shows a good ability to distinguish mismatched target sequences.The rigid "Y-type" structure is stable and has a satisfactory recovery rate in urine samples,which indicates that the constructed "Y-type" multivalent signal output probe has the potential to provide a powerful tool for biomedical research.2.Sensitive detection of p53 DNA by multivalent X-crosslinked "FRET network cluster" output probe based on strand displacement reactionIn this study,p53 DNA was used as a model.X-FAM and X-TAMRA self-assembled nucleic acids were designed as multivalent assembly primitive units.First,a large number of short single stranded DNA(ssDNA)were generated under the trigger of the recognition of p53 DNA,the dual drive of KFP DNA polymerase and Nb.Bbv CI endonuclease.With the help of base complementary pairing,ssDNA will bind a large number of X-FAM and X-TAMRA to form a polyvalent network cluster structure.With the help of complementary base pairing,ssDNA binds and aggregates a large number of X-FAM and X-TAMRA together to form a multivalent network cluster.The fluorescence was transferred from the donor X-FAM to the acceptor X-TAMRA,resulting in a significant FRET,thus realizing the fret fluorescence detection of p53 DNA.The constructed multivalent FRET network cluster probe can generate a large amount of FRET crosslinking agent by utilizing a polymerization cleavage reaction so as to bind a large amount of donor X-FAM and acceptor X-TAMRA.X-FAM and X-TAMRA were arranged in high density manner,which was beneficial to the high intensity output of fluorescence signal.In contrast,in the absence of p53 DNA,donor X-FAM and acceptor X-TAMRA failed to achieve FRET fluorescence transfer due to the lack of binding of FRET crosslinker.The proposed "FRET network cluster type" signal output probe has strong structural stability and has a satisfactory recovery rate for p53 DNA in cell lysis.In addition,the constructed multivalent signal output probe was successfully used for imaging detection of p53 DNA in fixed cells.These results indicate that the spatial configuration of the probe can effectively avoid being degraded in a complex cell environment by virtue of large steric hindrance effect,and the probe stability is enhanced,so that the probe can be expected to provide a powerful tool for biomedical research.