The Construction Of Biosensor And Drug Delivery System Based On DNA Nano Materials

Early diagnosis and treatment of cancer is of great significance in improving patient survival rates and reducing their psychological and physiological suffering.However,traditional chemotherapy and radiation therapy can cause irreversible damage to normal cells.Therefore,early diagnosis and targeted therapy of cancer are important.DNA nanomaterials,with their addressability and programmability,have advantages such as low environmental pollution,high sensitivity,and high specificity when used as the main component to construct biosensors.Additionally,due to the low toxicity and high biocompatibility of DNA nanomaterials,drug targeting delivery systems constructed using them can reduce the occurrence of rejection reactions.This paper aims to construct a biosensor for tumor markers and a targeted drug delivery system based on DNA nanomaterials.The specific contents are as follows:（1）A fluorescence biosensor for the specific detection of DNA adenine methyltransferase was designed based on DNA rolling circle amplification（RCA）and DNAzyme.When the target is present in the test sample,it undergoes an enzyme-coupled reaction with Dpn I,resulting in the methylation and cleavage of DNA hairpin,generating RCA primers.Simultaneously,using T4 DNA ligase,EXO I,and EXO III,the DNA template strands are joined together,resulting in a circular DNA template.The primers and circular DNA template undergo rolling circle amplification mediated by phi29 DNA polymerase,yielding single-stranded DNA with DNAzyme.The DNAzyme cleaves a molecular beacon,separating the fluorescence group and quench group,resulting in a significant increasement of fluorescence signal.Thus,the concentration of the target is correlated with the fluorescence signal.The dynamic range of this fluorescence biosensor is from 1×10^-3 U·mL^-1 to 200 U·mL^-1,with a LOD of 3.09×10^-4 U·mL^-1.（2）A high-throughput Kras DNA biosensor array chip was constructed using a graphene field-effect transistor as the detection platform.First,a hundred-channel gold/chromium electrode was fabricated on a Si/Si O₂ substrate using bilayer photolithography and electron beam evaporation coating.High-quality graphene films obtained by chemical vapor deposition were transferred onto the electrode surface.Micrometer-scale graphene channels were patterned using bilayer photolithography,and then cDNA was modified onto the graphene surface to form the GFET biosensor array chip.In the presence of Kras DNA,it hybridizes with the cDNA,changing the charge carrier density on the graphene surface and thus altering the Dirac point voltage.The biosensor array chip is very sensitive to the mutation of Kras DNA,and be able to obtain signal within 10 min.The dynamic range is from 1 f mol·L^-1 to 1μmol·L^-1,with a detection limit of 12.03 fmol·L^-1.（3）An electrochemical biosensor for the simultaneous detection of dual ct DNA was constructed based on functionalized DNA hexagonal-nanostructure.First,a DNA hexahedron structure based on DNA self-assembly was designed and bound to the gold electrode surface through the polyadenine structure at the bottom.After introducing Kras DNA,Braf DNA,and two auxiliary probes,the DNA hexahedron structure became complete and formed G-quadruplex-hemin complexes.In the presence of aniline and hydrogen peroxide,the aniline is catalyzed to deposit on the DNA backbones,generating a large current signal.By utilizing the detection principle of an“OR”logic gate,a single concentration of ct DNA can be detected.For Kras DNA,the dynamic range is from 100fmol·L^-1 to 1μmol·L^-1,and the detection limit is 48.73 fmol·L^-1.For Braf DNA,the dynamic range is from 100 fmol·L^-1 to 100 nmol·L^-1,and the detection limit is 44.10fmol·L^-1.（4）A drug delivery system targeting non-small cell lung cancer cell was established based on DNA tetrahedral nanostructures（TDN）.Initially,the TDN modified with different functional componts were synthesized in vitro,and loaded with daunorubicin hydrochloride（DAU-HCl）to form composite nano-drugs for targeted treatment of non-small cell lung cancer cells.The s6 aptamer at the top of the TDN enables it to accumulate around target cells,and with the assistance of the p28 cell-penetrating peptide modified at the other end,it enters the cells through endocytosis.After entering the cells,the siRNA douplex released and bind to protein mRNA,inhibiting the ERBB3protein transcription.At the same time,the released DAU-HCl is embedded in the intracellular DNA double-strands,inhibiting DNA replication and thus inhibiting tumor cell growth.In vitro experiments showed that the cell viratity of target cells was lower to 37.84%after treated with the nano-drugs,which exhibit a strong cell cytotoxicity and a high targeting ability.In this thesis,three biosensors based on DNA nanomaterials were constructed,demonstrating high sensitivity and specificity for different tumor markers,as well as strong anti-interference capability when exposed in human serum samples.These results demonstrate the broad clinical application potential of the constructed biosensors.Furthermore,a targeted therapeutic composite drug based on TDN was developed,exhibiting excellent targeting and cytotoxicity.It can precisely act on target cells,minimizing damage to healthy cells.By combining the biosensors with targeted drug delivery systems,there is a great potential to establish a complete integrated detection-treatment system.Based on the tumor biomarker detection results,appropriate targeted treatment strategies can be selected,enabling accurate delivery drugs to tumor cells,thereby minimizing adverse effects on normal cells.This integrated system provides a feasible option for the detection and treatment of cancer.