Fluorescent Biosensor Based On Functional Nanomaterials And Signal Amplification Technology

With the development of society,people pay more attention to their physical health.As a class of disease closely related to gene expression,the early diagnosis of cancer are of great significance.Tumor biomarkers refer to nucleic acids,proteins and other related substances that can play an indicative role in cancer.Therefore,the high sensitive detection of biomarkers has far-reaching significance for the early diagnosis,prevention and targeted treatment of cancer.As a mature detection method,fluorescent biosensor has the characteristics simpleness,celerity and stability,which shows the unique advantages in cancer detection and clinical medicine.In this paper,a variety of fluorescence biosensor systems were developed for sensitive detection of nucleic acids based on the unique identification ability,regulation of DNA and combining the enzymatic signal amplification strategy.These strategies consists of following parts:1.Detection of targets with double cycle amplification based on nano-gold and nanosilver clustersThis work achieved a double cycle amplification detection of target through enzymeassisted DNA chain replacement and rolling circle amplification reaction.When the carboxyfluorescein(FAM)modified report probe approch the gold nanoparticles(AuNPs),due to the fluorescence resonance energy transfer(FRET),the fluorescent signal of selfassembled molecular beacon was quenched.After the dual-cycle amplification-process,the product of cyclic reaction opened the hairpin structure of the report probe,resulting in the fluorescent groups of FAM away from AuNPs,thus realized the recovery of the fluorescent signal of the biological sensor.This method could achieved the simply,rapidly and sensitively detection of the target with the fluorescent biosensor.In addition,in the presence of target,the enzyme-assisted double circle amplification strategy also combined with the detection method of in situ synthesis of nanosilver clusters based on hybrid chain reaction.The product of the reaction triggered the hybridization chain reaction(HCR),opened the hairpin structure of HP1 and HP2,the rich-C sequence of HP1 and HP2 exposed.This method also achieved the fluorescence sensing of biological targets via in situ reduction to generate silver nanoclusters.2.A kinase detection method based on enzyme-assisted DNA walker and target detection of nanowire cascade reactionMany studies have shown that polynucleotide kinase(PNK)is related to the dysfunction of many cell activities,which eventually lead to various human diseases.Therefore,PNK plays a very important role in biochemical research.In this work,a DNA walker activated by T4 PNK was firstly constructed.In the presence of PNK,DNA swing arms were formed by the enzymatic hydrolysis of exonuclease,the endonuclease sheared the report probe which modified on the surface of AuNPs.The fluorescence group of FAM was dissociated from AuNPs into the solution,the fluorescence signal was recovered.In addition,the remaining sequence of reporting probe on the surface of the AuNPs was used as the primer for the next experiment,and the circle template was added to grow the DNA nanowire under the action of polymerase.The designed H1 and H2 were successively hybridized with DNA nanowires and formed the DNA nano-switches.In the presence of the target mRNA,the mRNA triggered cascade amplification reaction of the DNA nanoswitch.The fluorescence group of FAM in H1 was far away from the quenching group BHQ1,the fluorescence sensing signal is recovered.3.Hyperbranched isothermal amplification assisted multi-site HCR for ultra-sensitive detection of miRNAAs an oncogene,miRNA is overexpressed in many cancers.In this experiment,miRNA was combined with the rolling circle template,and the enzyme-assisted hyperbranched isothermal amplification was initiated to generate a large number of singlestrand DNA of different lengths.The generated DNA sequence provided multiple HCR sites,which were partially hybridized with the FAM modified hairpin HP1.The HP1 hairpin structure was opened and exposed its 5’ toehold.After the added of 5-carboxyl methyl rhodamine N-succinimide ester(TAMRA)modified HP2,HP2 replaced the HP1 from the product of hyperbranched hoop amplification,the formed fluorescent signal double-stranded DNA made the FAM close to TAMRA,thus occurred fluorescence resonance energy transfer(FRET)process,this method could avoid the false positive signals and the error of system.This simple fluorescent biosensor method is applied to the sensitive detection of miRNA.