Fluorescence Biosensing Based On Exo?-Aided Signal Amplification Technology And Nanomaterials For MicroRNA Assay

In recent years,nanomaterials have been widely used in biochemical analysis due to their unique photoelectric properties,size effect and surface effect,which has greatly promoted the rapid development of biosensors.In general,detection of biological samples based on nanomaterials depends on their excellent optical,electrochemical reading characteristics and strong fluorescence quenching ability.However,there are higher requirements for the water solubility and biocompatibility of nanomaterials for the detection of actual biological samples.Carbon nanomaterials have the advantages of good water solubility,high efficiency of fluorescence quenching and large specific surface area.DNA-templated fluorescence copper nanoparticles(DNA-CuNPs)with large Stokes shift,short synthesis time and high quantum yield.Combining the above advantages,carbon nanomaterials and DNA-CuNPs meet the development needs of fluorescent nanomaterials.In this paper,the use of graphene oxide as a fluorescent quencher and DNA-CuNPs were selected as a signal unit to construct a series of fluorescent biosensing methods for the detection of miRNA.This paper is divided into four parts;the brief contents are outlined as follows:Part one:An overview of miRNAs and their routine detection methods,the application of the biosensing platform based on graphene oxide and DNA-CuNPs.Part two:In this work,we constructed a fluorescence biosensing method to detect miRNA-122 using Exo? as a signal amplifier and GO as a fluorescence quencher.Exo? recognizes a 3'-flat or concave terminal double stranded nucleic acid substrate and catalyses the hydrolysis of 3,5-phosphodiester from the 3'-hydroxyl end of the polynucleotide chain,while it has low activity for 3'-protruding termini of dsDNA or single-stranded DNA.Two hairpin DNA probes,H₁ and H₂,are designed with a 3'-termini protrusion for resistance to degradation by Exo?,a FAM-containing ssDNA(P-DNA).After adding the target miRNA,the H₁ probe hybridizes with the target miRNA to form a DNA/RNA heteroduplex with a 3'-blunt terminus.The heteroduplex is the substrate for the Exo III cleavage,and then the target and trigger1 are released.The released target again hybridizes with H₁ to initiate the target recycling cycles to generate numerous trigger1(recycle 1).The trigger1 hybridizes with H₂ to form partially dsDNA,which is the same as H₁ of the target miRNA.Then Exo? cleaves them liberating a lot of trigger2,and trigger1 can hybridize with H₂ and start another recycling cycle(recycle 2).Meanwhile,trigger2 hybridizes with P-DNA to form dsDNA with a 3'-protruding terminus,which will not be adsorbed on the nanosheets due to the weaker interaction between the dsDNA and GO,thus retaining a strong fluorescence signal.In contrast,in the absence of the target miRNA,that will not happen with a series of hybridizations and enzymatic reactions,and the P-DNA will be adsorbed onto the surface of GO and the fluorescence is almost entirely quenched.The detection range of this method is 30 pmol/L to 3 nmol/L,and the detection limit was 21.4pmol/L according to the 3S/N rule.It can distinguish target well when the analog concentration was 30 times higher than the target concentration,this fluorescence strategy exhibits high selectivity.This method is capable of detecting miRNA sensitively in real complex samples,the recoveries are found to be in the range of 99.3%to 105.8%.Besides,this method is fast and the whole detection can be completed within 1 hour.Part three:Describe a method for the determination of microRNA-122 by using terminal deoxynucleotidyl transferase(TdT).It is based on the use of polythymine and exonuclease III-aided cycling amplification.A 3'-phosphorylated hairpin probe 1(H₁)and a hairpin probe 2(H₂)were designed.The H₁ and H₂ are not able to be opened and the Exo III can't digest without the target miRNA,which also means there will not occur the hybridizations and enzymatic reactions,two hairpin DNA probes will not be tailed by TdT and converted into long polythymine(polyT)sequences.In the presence of the microRNA,hybridization and enzymatic cleavage will occur and produce lots of 3'-hydroxylated ssDNA which can be tailed by TdT and converted into long polyT sequences.These can be used to synthesize copper nanoparticles(CuNPs)with a strong fluorescence signal.This method has the following advantages:Firstly,by taking advantage of the Exo?-mediated two-cycle amplification,both the primers released by two-cycles can be used for terminal transferase extension,which shows high sensitivity with a linear response in the 1.00×10² fmol/L and 1.00×10⁶ fmol/L miRNA concentration range and a44 fmol/L limit of detection.Secondly,it can distinguish target well when the analog concentration was 50 times higher than the target concentration,this strategy exhibits high selectivity.Furthermore,it has strong anti-interference capacity applied in detection real complex biological samples and the recoveries are 92.2%¹03.0%.Finally,the method is label-free,sensitive,low-toxic,easy to operate and versatile.It can be used to detect other DNA or miRNA by changing the target recognition sequence in H₁.Part four:The detection of miRNA is based on Exo?-assisted target cycle amplification and T₃₀ sequences to synthesize fluorescence copper nanoparticles(T₃₀-CuNPs)fluorescent copper nanoparticles.In this method,two DNA hairpin probes H₁ and H₂ are designed with a3'-termini protrusion for resistance to degradation by Exo?.The T₃₀ sequences are enclosed in hairpin H₂.In the presence of the microRNA,the T₃₀ sequences can't be released,and then the fluorescence intensity of system is very weak.In the presence of the miRNA,hybridization and enzymatic cleavage will occur and release T₃₀ sequences to synthesize CuNPs with a strong fluorescence signal.The detection limit is 9.2 pmol/L and the linear range from 0.01 nmol/L to1.5 nmol/L.Besides,this method has good selectivity,and it was successfully applied to determination of microRNA in spiked serum samples and the recovery rate is 96.0%¹09.3%.This method is economical,easy to operate simple operation and short detection time.It has potential application value in practical biological samples.