DNA Nanotechnology-based Biosensor Construction Strategy Study

DNA not only can be used as the code of life,but also has the characteristics of high programmability,automatic synthesis,easy modification and so on,which makes the DNA nanotechnology with DNA molecule as the basic material develop rapidly in the past decades.With the development of DNA nanotechnology,nucleic acid biosensors play an increasingly important role in environmental monitoring,food safety,drug analysis,disease detection and other fields.With the continuous advancement of in vitro screening technologies for functional nucleic acid,more and more nucleic acid molecules with excellent recognition functions have been developed,which further promotes the application range of nucleic acid biosensors.However,in some resource-limited or complex analytical system,due to some inherent defects of nucleic acid molecules and imperfections in the construction strategy of nucleic acid biosensors,the constructed nucleic acid biosensors often fail to perform well under these dynamic and complex analysis systems.Therefore,it is still a huge challenge for researchers to further expand the application of nucleic acid biosensors and improve their performance in complex analytical systems.In order to solve these problems,this thesis focuses on how to reduce the application cost of nucleic acid biosensors and improve the performance of nucleic acid biosensors through reasonable DNA molecular engineering,so that t he designed nucleic acid biosensors can be used in the resource-limited or dynamic complex analysis system.The specific contents are as follows:?1?DNA G4/Hemin can be used as a good peroxidase nanozyme,and it can catalyze the oxidation of tetramethylbenzidine?TMB?in the presence of hydrogen peroxide,resulting in a significant change in the UV-Vis absorption spectrum of the TMB-H₂O₂ system.Therefore,based on this principle,many colorimetric nucleic acid biosensors based on DNA G4/Hemin have been constructed.However,in order to obtain quantitative output results,people often need the help of expensive optical instruments,which greatly limits the wide application of nucleic acid biosensors in resource-limited areas.In this chapter,we found that,DNA G4/Hemin can catalyze the oxidation of TMB-H₂O₂ system,The oxidation system not only has obvious changes in the UV-Vis absorption spectrum,but also has good photo-thermal conversion efficiency after the oxidation of TMB,which can make the temperatu re of the system increase obviously under the irradiation of near-infrared light,and the change of temperature does not need the help of large optical instruments,but can be effectively read by portable thermometers.Thus,this suggests that DNA G4/Hemin may be used to construct nucleic acid photothermal sensors that are more easily available for large-scale applications in resource-limited regions.To achieve this,we constructed a first-generation nucleic acid photothermal biosensor using the DNA G4 double-end blocking strategy.With this biosensor,we can quantify the target with a portable thermometer.This work provides a new design idea for the construction of nucleic acid photothermal biosensors.?2?To further extend the range of applications based on DNA G4/Hemin photothermal biosensors,in this chapter we are committed to transducing more complex target information into readable,quantifiable temperature output signal through advanced DNA engineering.In order to achieve this,we try to reasonably couple different DNA molecular circuits with the DNA photothermal biosensor developed in the previous chapter to build a more advanced nucleic acid biosensor to achieve more complex functions.As a conceptual study,we first coupled the entropy-driven nucleic acid-free enzymatic amplification molecular circuit with the DNA transducer developed in the previous chapter.We successfully realized the detection of ultra-low concentration targets through a portable thermometer.Compared with the simple DNA photothermal sensor in the previous chapter,it has improved by nearly two orders of magnitude.At the same time,in order to realize the detection of complex multi-bit input information,we also realized the coupling of different DNA logic circuits?AND logic circuits,OR logic circuits?with the DNA transducer developed in the previous chapter,and achieved successful logic processing and conversion of multi-bits nucleic acids interactions.Therefore,in this chapter,we demonstrated that integrating dynamic c omplex DNA molecular circuits is a general strategy to construct more advanced DNA photothermal biosensor.We believe that this design concept will provide excellent guidance for the development of the next generation of advanced nucleic acid photothermal sensors.?3?In view of the poor cell penetration ability of traditional nucleic acid biosensors and the prone to false positive signals in complex intracellular environments,in Chapter 4,we constructed a novel DNA tetrahedral biosensor based on DNA nanotechnology.The new tetrahedral biosensor shows good cellular uptake and biostability.At the same time,by combining the fluorescence resonance energy transfer technology,by designing the target-induced fluorescence resonance energy transfer"off"to"on"optical switch,the nucleic acid biosensor almost entirely avoids false-positive signals due to intrinsic interferences,such as nuclease digestion,protein binding and thermodynamic fluctuations in complex biological matrices.The nucleic acid biosensor can achieve accurate imaging of target mRNA in living cells.More importantly,DNA three-dimensional nanostructure construction technology and fluorescence resonance energy transfer"off"to"on"signal output mode combination strategy,provides a new idea for the development of more accurate imaging of nucleic acid biosensors in cells.?4?Since many important analyte concentrations in cells may be at a lower level,some disease markers may further decrease with the occurrence of the disease,while traditional nucleic acid biosensors and targets are mainly based on 1:1 signals.The conversion ratio thus shows a relatively low signal gain.In order to obtain a higher gain signal output,this requires us to design a biosensor that can perform efficient signal amplification on a limited input signal.Therefore,in Chapter 5,we constructed an entropy-driven three-dimensional DNA nano-amplifier by molecular engineering.The amplifier consists of two DNA tetrahedral modules.When there is no target,the two modules do not interact with each other.The signal is always in a quenching state.When a target molecule is present,the target molecule catalyzes the interaction of the two modules,and finally the fluorescently labeled nucleic acid strand is continuously separated from its partially complementary quenching strand chain,so that the fluorescent signal is continuously enhanced.The DNA nanoamplifier realizes ultrasensitive detection of tumor-associated mRNA in living cells,opens new ideas for further application of DNA nanotechnology in cells,and also provides a potential molecular tool for the discovery of intracellular low abundance disease markers.