Application Of DNA Nanotechnology In Biosensors And Intelligent Hydrogels

The revelation of the DNA double-helix structure has revolutionized modern bioscience,and the subsequent discoveries of the supermolecular nanostructures formed by self-assembly of single-stranded DNA and the metal-ion-assisted cooperative stabilized DNA duplexes have further stimulated the researchers’interest in characterization the DNA biopolymers.In addition,DNA nanotechnology has flourished in recent decades and has been widely used in biosensor,DNA hydrogel,functional enzyme,DNA machine,nanostructures,molecular transport,computing and other fields due to the unique properties of DNA,including precise programming ability between complementary strands,biological function and biocompatibility etc.Nucleic acids,proteins and biological small molecules in organisms play important roles in complex biological processes.Their expression levels and content levels often reflect a series of physiological and pathological processes.Therefore,they are widely used as biomarkers of various human diseases,such as malignant tumors,cardiovascular and cerebrovascular diseases and so on.Furthermore,the steady state of physical and chemical properties of biological environment is the primary condition to maintain the free and independent survival of organisms.Therefore,it is of great significance to construct biosensing platforms for disease-related biomarkers or to develop biocompatible materials that are responsive to the physicochemical properties(such as pH,osmotic pressure,temperature)of the internal environment.In this dissertation,several sensitive,rapid and versatile biosensor platforms are developed by the combination of DNA nanotechnology and noble metal nanoparticles/nanoclusters.In addition,we have developed three pH responsive intelligent hydrogels by introducing the triplex structure of DNA.The major content and innovation of this thesis were in the following aspects:1.Hybrid chain reaction(HCR)possesses excellent signal amplification performance,and the multiple cascades of enzymes can effectively improve the catalytic performance of enzyme cascade reaction,thus improving the detection sensitivity.Here,we designed a dual amplification platform based on hybrid chain reaction and enzyme cascade reaction.Two cascade enzymes(GOx,HRP)were modified on two hairpin DNA strands(H1,H2)to form the enzyme functionalized hairpin DNA strands(GOx-H1,HRP-H2).The presence of nucleic acid target(T)would trigger the HCR of GOx-H1 and HRP-H2,accompanied by the formation of GOx/HRP enzyme pairs with a multiple enzymatic cascade,which greatly improves the catalytic performance of enzyme cascades and realize the highly sensitive detection of T.In addition,the platform can be used for the analysis of proteins or biomolecules by introducing aptamers of them,such as ATP.Our detection platform has good sensitivity and the detection limits of T and ATP are 5.2 fM and 0.8 pM,respectively.It provides a general method for the ultra-sensitive detection of nucleic acids and targets with aptamers.2.The high specific binding of the DNA aptamer to the target has an unparalleled advantage in the biosensor,and gold nanoparticles(AuNPs)has emerged as an important fluorescence quencher as well as colorimetric reporter because of its potential fluorescence resonanceenergy transfer(FRET)with various fluorescent substances,high extinction coefficients and unique distance-dependent optical properties.We have developed a fluorometric and colorimetric dual-mode assay for the determination of adenosinetriphosphate(ATP)based on the specific recognition ability of fluorescent labeled aptamer(FAM-Apt)for target(ATP)and cDNAs modified gold nanoparticles(cDNAs-AuNPs).The detection limits and linear ranges could compare with other ATP detection platforms that need expensive equipment or complex operations favorably.Above all,our design may provide a general method to detect various other biologically small molecules,metal ions and proteins based ontheir suitable aptamer.3.We successfully constructed a fluorescence biosensor of detecting microRNA(miR-21,as a model).In addition to the reasonable design of single-stranded DNA probe(including a C-rich sequence as the synthetic template of DNA/silver nanoclusters(DNA/AgNCs),a complementary(Com)sequence to hybridize with the miR-21,and a G-rich sequence to form a complex of G-quadruplex/hemin).It also includes the rational utilization of the distance-dependent property of photoinduced electron transfer(PET)between the preformed DNA/AgNCs(electron donor)and G-quadruplex/hemin complex(electron acceptor).Preferential to other previous PET-based detection methods,we construct the biosensor by utilizing the distance dependent property for the first time and only need to adjust the sequences of Com in different miRNAs assays.4.We systematically investigate and rationally design a classical tMB to construct a miRNAs detection platform.We employ the complementary sequence of miRNAs as the loop and the sequences of protonated cytosine-guanine-cytosine(C-G·C+)and thymine-adenine-thymine(T-A·T)as the triplex stem.It is demonstrated for the first time that the presence of miRNAs would only break the Hoogsteen base-pairing in the stem and hybridize with the tMB to form the rigid heterozygous hybrid duplex structure.These would hinder the fluorescence resonance energy transfer(FRET)between the fluorophore(FAM)and quencher(BHQ1)labeled at the ends of the oligonucleotide,and the fluorescence of FAM will be "turned on".Furthermore,we have extended the application of molecular beacons by replacing the fluorophore FAM with the fluorescent DNA/silver nanoclusters or introducing the complementary sequence of vascular endothelial growth factor(VEGF)aptamer into the loop of tMB.We believe that the tMB would be a universal strategy for the detection of miRNAs and biomarkers with a suitable aptamer,which holds great potential for the early clinical diagnostics of diseases.5.DNA that can be controlled by manipulating pH levels has been successfully developed and is increasingly being improved such that it has been applied in the construction of various DNA-based structures.To further explore the possibilities of pH-activated pure DNA hydrogels,we proposed a new modular strategy for the construction of pH-activated reversible pure DNA hydrogels by introducing two triplex structures which were based on protonated cytosine-guanine-cytosine(C-G·C+)and thymine-adenine-thymine(T-A·T).The C-G·C+ bridges are formed at pH 5.0 and disassemble at pH 7.0,whereas the T-A-T bridges are formed at pH 7.0 and disassemble at pH 10.0.Based on this,the reversible self-assembly of pure DNA hydrogel controlled by pH was realized.More significantly,the introduction of both a fluorophore(FAM)and a quencher(BHQ1)to the hydrogels provides an innovative method for monitoring the self-assembly and disassembly processes through a fluorescence technology.