Study On Nucleic Acid Biosensor Based On DNA Nanomachine Signal Amplification System

DNA,which is a biological macromolecule composed of deoxynucleotides,can form different high-level structures with different target molecules through hydrogen bond,Van Der Waals force,hydrophobic effect,electrostatic effect and other non-covalent interactions.At the same time,DNA is also a biomaterial with excellent performance,which has many unique advantages in the construction of biosensors,such as simple structure,predictable design result,easy chemical modification and synthesis,etc.As a new nano technology,DNA nanomachine can respond to external signal stimulation and generate motion similar to the machine and realize signal amplification,which has been widely used in the design of biosensors.In recent years,researchers have taken advantage of the unique properties of DNA to build DNA nanomachines which is similar functional components,such as DNA walking machines and DNA nano-tweezers.Cancer is one of the most common fatal diseases in medicine.Early detection is of great importance to the diagnosis of cancer.Scientific studies have shown that microRNA is closely related to the occurrence of cancer,so it can be used as a diagnostic substance for early cancer.Therefore,it is of practical value to construct a method for detecting microRNA with high sensitivity and low detection limit in clinical diagnosis.In this paper,several different biosensors were constructed by DNA molecular machine and applied to detect microRNA-21,mainly including the following aspects:1.Label-free electrochemical nucleic acid biosensors for the detection of microRNA-21In this work,we constructed an unlabeled biosensor for the detection of microRNA-21 based on G-quadruplex/hemin structure with peroxidase catalytic performance.First of all,the G-rich detection sequence G-DNA and its partially paired switching sequence P-DNA formed a partially complementary double-stranded structure after heating at 95?to slowly cool down.Due to the steric hindration,the detection sequence G-DNA could not be folded into a G-quadruplex/hemin structure.After G-DNA forms the hairpin structure,the DNA enzyme sequence which can cut off the switch sequence P-DNA loses its catalytic activity.Those sequences self-assembled to the gold electrode surface though gold-thiol covalence.When microRNA-21 is present in the solution,the microRNA-21 is partially paired with the startup sequence to release the DNAzyme.After binding to Pb²⁺,the enzyme activity is restored and the switch sequence is cut off.P-DNA,so that the detection sequence G-DNA does not have steric hindrance but can be folded into a stable tetramer structure,combined with hemin and potassium ions have peroxidase activity,catalyze the reduction of H₂O₂,generating electrochemical signals.The stability,recovery and specificity of the biosensor were also investigated.In this work,a microRNA-21molecule can activate a startup sequence DNA enzyme,and then cut off many P-DNA sequence and prompte many rich G sequences can be folded into G-quadruplex/hemin structure,achieving the first level of signal amplification.G-DNA has horseradish peroxidase catalytic activity after combined with hemin and potassium ions for the second signal amplification.This method implements the microRNA-21 ultrasensitive detection,the detection limit is 0.06 fmol/L,it has important significance for microRNA-21 detection.2.Research on ultraviolet-visible spectrum nucleic acid biosensor based on gold nanoparticlesBased on the DNA molecular machine constructed in the previous part of this work,a biosensor for the detection of microRNA-21 was constructed with DNA functionalized gold nanoparticles as the carrier.When microRNA-21 is present,the nanomachine is activated,and G-quadruplex/hemin structure with horseradish peroxidase catalytic activity is formed on the surface of gold nanoparticles,which catalyze the color change of TMB to produce UV-Vis absorption.The stability,recovery and specificity of the biosensor were also investigated.This method use UV-Vis absorption spectrum to detect microRNA-21 in homogeneous solution,which is simple to operate and has good stability.Meanwhile,it has high specificity for detecting microRNA-21,and the detection limit is 15 fmol/L.Furthermore,it can achieve visual detection,which has great application prospect in clinical diagnosis.3.Fluorescence resonance energy transfer nucleic acid biosensor based on chain reaction amplification signalIn this work,a biosensor for detecting microRNA-21 was constructed based on the chain reaction.We designed two different DNA containing DNAzyme(DNA1,DNA2),DNAzyme contained in DNA1 sequence can cut DNA1 and the DNAzyme contained in the DNA2 sequence can cut DNA1.Those sequences can form stable hairpin structure and shielding DNAzyme activity.At the same time,those DNA were respectively modified with fluorescent Cy5 and quencher Dabcyl DNA form hairpin structure make Cy5 close to Dabcyl and causes fluorescence quenching.In present of microRNA-21,those two sequence hairclip structures open and release DNAzyme,which combines with metal ions to restore enzyme activity and cut each other,thus triggering a cascade of enzyme digestion reaction.Then,Cy5 move away from Dabcyl and fluorescence recovery.The content of microRNA-21 is indirectly determined by measuring fluorescence intensity.A small amount of microRNA-21can trigger the DNA cascade cleavage reaction and make the signal amplified,which is beneficial to improve the reaction sensitivity.This method can detect microRNA-21in homogeneous solution with simple operation.This method has linear relationship between 0.01 nmol/L-100 nmol/L and limit detection is 17 pmol/L.