Biosensing Research Based On DNA Tetrahedral Nanostructure Probes

DNA sensors are a kind of special biosensors,which have been developed rapidly in recent decades because of their high sensitivity,high speed,low cost and low energy consumption.The development of highly sensitive DNA sensors still faces many challenges.At the interface between electrode and solution,the reduction of mass transfer rate and the increase of crowding effect greatly affect the recognition and hybridization of DNA.We design nanostructured DNA tetrahedron probe to improve the water solubility,chemical stability and biocompatibility of nano-sensor interface.In this thesis,DNA tetrahedron nanostructures are used to construct multivariate and cooperative nanosensor interfaces,and the target molecules can be captured and recognized with high specificity by regulating the nanoscale interfaces.The main contents are as follows:1.A“sandwich-like”biosensor was developed based on the magnetic bead platform for sensitive detection of breast cancer 1?BRCA1?DNA.In the present study,a tetrahedron-structured reporter probe?TSRP?was designed,in which 3vertices of the tetrahedron were labelled with digoxin?Dig?and the other one was labelled with a detection probe.TSRP here provided accurate enzyme loading and well-organized spatial arrangement for optimized signal amplification.The detection limit of this biosensor was as low as 10 fM,which is at least 4 orders of magnitude lower than the single DNA probe?100 pM?,and the signal gain was 2 times higher than the analysis using three one-dimensional?1D?reporter probes.We could distinguish DNA sequences with only 1 base mismatch,and the performance of our TSRP biosensor was proved to be equally good in both PCR products and real fetal calf serum?FCS?sample as in buffer.We believe this work provided a novel avenue for the development of signal amplification strategies.2.We have developed a novel Pb²⁺electrochemical biosensor using specific DNAzyme reaction on a DNA tetrahedron probe,in the presence of Pb²⁺,the substrate strand was cleavaged into two parts and released a“G-rich”oligo which subsequently formed a G-quadruplex/hemin complex,generating a detectable catalysis current signal with the assistant of H₂O₂.The 3D DNA tetrahedron regulated the density and orientation of the probe and thus improved the DNAzyme reaction,and facilitate the complex“two-step”DNA conformational change in the confined space of the interface on the gold electrode,Finally,the LOD of our biosensor was proved to be0.008 nM?3??,which is 9000 times lower than the safety limit of EPA?15?g/L or 72nM?,and nearly 6000 times lower than that of IARC?10?g/L or 48.26 nM?,and more importantly,the specificity and reproducibility of the proposed biosensor was well demonstrated.3.We explored the space charge transfer of tetrahedron based on gold electrode surface initially.It was initially found that the signal value of the overhang probe was limited by its length.Moreover,when the inversion probe is single-stranded,the signal value is large,and when it is double-stranded,the signal value is small.It provides a new idea for E-DNA.