| With the development of science and technology,fluorescence resonance energy transfer?FRET?technology has been more and more widely used in biochemical analysis and clinical diagnosis.F?ster resonance energy transfer?FRET?,occurred between two photoluminescent?PL?chromophores with the absorption spectra of one chromophore,commonly named as acceptor,overlapping the emission spectra of another chromophore,commonly named as donor,through the interaction such as dipole-dipole,has found wide applications since it can supply long wavelength shift and low signal to noise ratio.FRET as a homogeneous analytical detection technique has the advantages of high sensitivity and selectivity,easy operation and so on,and thus was applied for analytical detection and biological imaging.Nucleic acid FRET?naFRET?probes,which signal the relative state changes of both the donor and the acceptor during the FRET process,are easily constructed since the oligonucleotides with the backbones labelled with one or several chromophores including donor fluorophores and acceptor fluorophores,have been used through varieties of hybridization reactions.However,most naFRET probes reported up to now consist of one-donor-one-acceptor assembly,limiting the energy transfer efficiency(EFRET).Probably owing to the weak dipole-dipole interactions that makes the energy of donors can't be transferred to the acceptors effectively,the as-resulting high background signal of the FRET probe of one-donor-one-acceptor assembly is not benefit to the sensitive detection of target.On the contrary,one-donor-multiple-acceptors?D–mA?naFRET probes can avoid this problem.Besides it is known that polypurine–polypyrimidine sequences can assemble into triple helical DNA structures under acidic conditions,forming T-A·T and C-G·C+nucleobase triplets upon protonation of cytosine bases through Watson-Crick and Hoogsteen interactions.The formation of triple-helix structure,supplies us a good chance to modify the oligonucleotides with several chromophores simultaneously,making it advantageous to the construction of D–mA naFRET probes.In this paper we successfully designed a D–mA naFRET strategy to detect biomarkers sensitively.And the main works are listed as follows:?1?A one-donor-four-acceptors?D–4A?FRET nucleic acid probe with lower background and higher sensitivity was designed through the formation of triple-helix DNA structure.the D–4A FRET probe consists of three ssDNA strands,two of which were target specific aptamer sequences flanked by two arm segments which are doubly labelled with acceptors?BHQ-1?at the 5-and 3'-end respectively,and the other is donor?FAM?-labelled hairpin structure.When the three sequences of ssDNA strands above were mixed,heated and incubated,the arm segments of two aptamers'are bound with the loop sequence of FAM-labelled?donor?DNA by Watson-Crick and Hoogsteen base pairings,respectively.As a result,the D–4A FRET probe was formed since the distance between the donor and accepter was shortened,leading to the occurrence of FRET from one FAM molecules to four BHQ-1 molecules and the fluorescence of the donor?FAM?gets efficiently quenched.The as-prepared D–4A FRET probe has stable triple-helix structure in the absence of target Hepatitis C virus?HCV?DNA and very low fluorescence emission,keeping the background signals very low.With the addition of HCV DNA,however,disintegration of the triple-helix DNA structure of D–4A FRET probe occurs since the hybridization of the aptamer sequence with the target,releasing the FAM-labelled DNA and thus the green fluorescence of FAM recovered owing to the separation of the donor and acceptor.With that principle,a fluorescence signal turn-on method of HCV thus sensitively developed.The limit of determination?LOD?was24.57 nM?3??.?2?An enzyme-Free one-donor-multi-donor FRET nucleic acid probe based amplified assay is constructed for detection of microRNA-21 by using aptamer as the recognition element.Compared with the traditional methods,this strategy provides an affordable,sensitive,accurate and rapid detection for target,because the probe developing process is mild,simple,and enzyme-free.This design maintained a D-mA FRET nucleic acid probe,which not only improved the sensitivity but also ensured the accuracy of microRNA-21 detection.The CHA system contained a hairpin structure H1 and the formed probe.After microRNA-21 was added,the aptamer of formed probe was opened by hybridization reaction,exposing the concealed sequence of probe,which was complementary to H1,which could release the target microRNA-21 due to the kinetic mechanisms.The free target microRNA-21 would catalyze the next round of hybridization between probe and H1 to available nonenzymatic signal amplification.As a result,microRNA-21 could be detected sensitively by the change of fluorescence signal.Besides,the selectivity was improved greatly and limit of detection was much lower than that without CHA.At the same time,the current strategy avoided the use of enzyme or complicated equipments,which made it more simple and cost-effective,showing that the proposed strategy can provide a sensitive and selective platform for microRNA-21 detection.In summary,we successfully developed a novel D–mA FRET method for the detection of biomarkers in real sample since it can offer very low background signal and be sensitive to the target.At the same time the proposed method can find wide applications since the strategy can be used to design as a universal analysis platform by changing the aptamer sequence and then to achieve different target detection.Thus we believe that the method we proposed will have great potential as a tool for the detection of biomarkers. |
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