Construction And Application Of The Biosensing Platform Based On Functional Nucleic Acid And Signal Amplification Technology

As an important part of modern analytical technology,biological analysis mainly studies the content,structure and function of bimolecular.Among them,biosensor is a powerful tool for biological analysis and a typical multidisciplinary growth point,involving many disciplines and technologies of physics,chemistry,life science and information science.Based on the characteristics of accuracy,rapidity,easy operation and recycle,biosensors have broad application prospects in analytical chemistry,life science research,clinical diagnosis and analysis,environmental quality testing,and food science.This thesis firstly summarizes the concept,principle and classification of biosensors,then,introduces the basic concept and classification of functional nucleic acid and signal amplification technology,and explores their research potential and application prospects in the field of biosensing analysis.Finally,in order to improve the performance of biosensors,we integrate functional nucleic acid and signal amplification technology with dual-polarization interference and electrochemical technology platforms to construct the highly sensitive,accurate and multifunctional novel biosensor,the specific works are as follows:1.A cadmium ion(Cd2+)aptamer sensor was constructed by combining the nucleic acid aptamer of Cd2+with dual polarization interferometry(DPI).We established a two-layer sensing platform by the layer-by-layer assemble method to explore the interaction mechanism between Cd2+ and aptamer,and quantitatively detect Cd2+.By combining with other spectroscopic characterization methods(UV-vis spectroscopy,Circular dichroism spectroscopy,Fourier transform infrared spectroscopy),we revealed the dependence of the interaction mechanism between Cd2+ and aptamer on Cd2+ concentration,and the real-time conformational changes of aptamer.Based on the real-time mass binding curves,we quantitatively detected Cd2+and calculated the kinetic constant of their interaction.The results showed that the sensor exhibited high sensitivity,specificity,and good reproducibility for Cd2+detection.The novel sensing platform had the advantages of simplicity,sensitivity,reproducibility,multi-channel simultaneous detection,and simultaneous acquisition of interaction kinetic information.2.The transduction hairpin(THP)was introduced into the toehold-mediated strand displacement reaction(TSDR)to form the three-leg DNAzyme walker,and a label-free and sensitive electrochemical sensor was constructed for the first time to detect target nucleic acid.First,electrochemical and DPI techniques verified the feasibility of this biosensing strategy in detail.This sensing strategy utilized the cyclic amplification ability of TSDR and the three-leg DNAzyme walker with efficient catalytic ability to amplify the input signal,which significantly improved the sensitivity of the sensor with a detection limit as low as 0.27 fM for the target miRNA-155.The precise design and introduction of THP made this method with highly specific and versatile.For the detection of different nucleic acid sequences,it is not necessary to change the main circuit of the sensor,but only to redesign the loop sequence of THP.This study provided a simple and effective method for the sensitive and specific detection of nucleic acid biomarkers.3.By combining the signal amplification techniques of enzyme-free catalytic hairpin self-assembly(CHA),hybrid chain reaction(HCR),and copper-based metal-organic frameworks(Cu-MOFs),we established a novel,label-free and highly sensitive electrochemical biosensors.The sensing system mainly included the CHA process in the homogeneous solution and the HCR process on electrode surface.Among them,AuNPs-modified Cu-MOFs were coupled with DNA to introduce Cu-MOFs into HCR process,and finally a large number of Cu-MOFs were immobilized on the electrode surface to generate the strong electrochemical signals.We cleverly combined nucleic acid-based enzyme-free signal amplification technology and nanomaterial-based signal amplification technology,which not only achieved the cascade amplification of input signal,but also amplified the output signal through Cu-MOFs.The sensor accurately and sensitively detected miRNA-21 with the detection limit as low as 0.02 fM,and showed the potential in practical applications.4.Combining the enzyme-free kinetic DNA self-assembly(DDSA)signal amplification techniques(CHA and HCR)with functional nucleic acids(metal ion-dependent DNAzymes,G-quadruplexes,DNA tetrahedral nanostructures),we constructed a novel electrochemical/fluorescence dual-mode biosensor for sensitive and accurate detection of target nucleic acids.The sensing system included the Mg2+-dependent DNAzyme(Mg2+-DNAzyme)-involved CHA-HCR cascade amplification circuit and the electrochemical/fluorescence dual signal mechanism,besides,the sensing system using DNA tetrahedral nanostructures(DTN)as the electrochemical capture probe further improved the sensitivity and accuracy of the electrochemical detection.In the presence of target nucleic acid,the Mg2+-DNAzyme-involved CHA-HCR cascade amplification circuit was activated,and finally generated numerous methylene blue labeled ssDNA and thioflavine T/G-quadruplex to produce the electrochemical and fluorescent signals,simultaneously.Based on the Mg2+-DNAzyme-involved CHA-HCR cascade cyclic amplification process,DTN-modified electrodes,and the specific combination of thioflavine T with G-quadruplex,the sensitivity and specificity of the biosensor were significantly improved.Both modes showed the excellent performance in DNA detection.Two independent signal readout systems ensured the accuracy of the biosensor and provided a new idea for the construction of dual-mode sensors with good analytical performance.