Application Of DNA Strand Displacement In The Construction Of A New Fluorescent Biosensor

With the economic development and social progress,people pay more and more attention to their health.The occurrence of many diseases is accompanied by the expression of the corresponding disease markers.Therefore,the detection of low concentration of these biomarkers is of great significance to nutrition health analysis,disease prevention and early diagnosis.Fluorescence biosensors are devices that specifically recognize biomolecules and convert the identification events into fluorescent signals.Due to the advantages of their ease of assembly,rapid detection,high sensitivity,and low cost,fluorescence biosensors have achieved rapid growth in the clinical and other fields in recent decades.DNA strand displacement is a kinetic-driven nanotechnology developed from the self-assembly of DNA.Its self-initiating,high-accuracy,and high sensitivity has drawn great interest in the construction of biosensors.This paper focuses on the construction of a new fluorescence biosensor based on DNA strand displacement to achieve the detection of different target molecules and to obtain a series of research results.We will summol/Larize the specific research work as follows:Part 1.Steric hindrance inhibition of strand displacement for homogeneous and signal-on fluorescent detection of human serum antibodiesIn this work,based on a new steric hindrance inhibition of DNA strand displacement strategy,we describe the development of a robust fluorescent signal-on method for homogeneous and sensitive detection of antibodies in human serums.The affinity binding of the target antibody with the small molecule moiety on a partial DNA duplex,which contains a unfolded molecular beacon(MB),prevents a protein-conjugated intruding ss DNA from approaching the toehold region of the duplex due to steric hindrance-induced by the bound antibody.As a result,strand displacement of the hybridized MB by the intruding strand is inhibited and fluorescence is retained.Such steric hindrance effect increases with increasing concentration of antibody,leading to elevated fluorescent signals for sensitive detection of the target with a detection limit of 5.6 nmol/L.Moreover,the sensing approach is selective against other control proteins and can be employed to detect the target antibodies in human serum samples.With the advantages of probe immol/Lobilization-free,homogeneous and sensitive signal-on detection mode,the demonstrated antibody-sensing approach thus holds great potential for the detection of different proteins for clinical diagnosis purposes.Part 2.Target-driven allosteric structure switching of aptamer beacon for catalytic and dual amplified fluorescent detection of ATPAbnormal concentrations of ATP are associated with many diseases and cancers and quantitative detection of ATP is thus of great importance for disease diagnosis and prognosis.In the present work,we report a new dual amplified strategy by coupling target recycling with catalytic hairpin assembly(CHA)to achieve high sensitivity for fluorescent ATP detection.The association of the target ATP with the aptamer beacons causes the allosteric structure switching of the aptamer beacons to liberate the pre-locked toehold regions,which hybridize with and unfold the fluorescently quenched hairpin signal probes(HP1)to recycle the target ATP and to trigger CHA between HP1 and the secondary hairpin probes(HP2)to form HP1/HP2 duplexes.Due to the recycling of ATP and CHA,the presence of ATP leads to the formation of many HP1/HP2 duplexes,generating dramatically amplified fluorescent signals for sensitive detection of ATP with a low detection limit of 8.2 nmol/L.Furthermore,the sensing approach shows a high selectivity and can be successfully used to detect the target ATP in human serums to realize its application for real samples.With the distinct advantage of significant signal amplification without the involvement of any nanomaterial and enzyme,the developed method thus holds great potential to be extensively applied for simple and sensitive detection of different small molecules and proteins.Part 2.Dual-input molecular logic circuits for sensitive and simultaneous sensing of multiple micro RNAs from tumor cells.In this work,by using two different mi RNAs as the inputs,we report on the design of molecular logic circuits and its application for sensitive and simultaneous detection of mi RNA-21 and mi RNA-155 from cancer cells.he coexistence of the two mi RNA targets leads to the unfolding of the fluorescently quenched double loop-stem signal probes,and the target mi RNAs are further cyclically reused with the assistance of the duplex fuel probes to generate significantly amplified fluorescent signals for simultaneous detection of the two mi RNA targets at very low levels.With the integration of the enzyme-free target recycling amplification,simple and highly sensitive simultaneous detection of mi RNA-21 and mi RNA-155 at femtomolar level can be achieved.By engineering the design of the signal probes,a broad spectrum of target analytes can be potentially used as inputs to achieve multiplexed detection of the targets for actuate early diagnosis of different diseases.