Design Of Nucleic Acid Sensor With Multiple Enzyme-assisted Signal Amplification And Highly Sensitive Detection Of Tumor Markers

Since ancient times,the threat of malignant tumor diseases to human health has been very serious.The K-ras gene,as a tumor proto-oncogene,is a gene necessary for maintaining the normal life activities of biological organisms.Abnormal expression of K-ras gene in cells can cause a variety of cancers such as lung cancer,pancreatic cancer and colorectal cancer.Acute lymphocytic leukemia is a tumor disease of the human blood system.The early onset of acute leukemia is not obvious,and the mortality rate in the later period is high.In the United States,about 3,000 to 4,000people are diagnosed with acute lymphoblastic leukemia every year,and two-thirds of them are children 2 to 5 years old.However,with the development of science and technology,scientists have gradually discovered that the formation of malignant tumors is based on events at the molecular level.And the formation of malignant tumors is caused by changes in genetic information.Therefore,it is extremely important to study the pathogenesis of malignant tumor diseases at the nucleic acid molecular level and to detect and screen malignant tumors at an early stage.In this work,we have successfully developed three novel DNA biosensors based on novel probes to detect nucleic acid biomarkers for malignant tumors.The experimental results show that the designed DNA biosensor shows great potential in gene detection and has broad application prospects in actual detection.Firstly,based on a novel palindrome molecular beacon（PMB）,a highly sensitive biosensor for detecting K-ras gene was constructed.The sensor incorporates a polymerase-mediated signal amplification technology that circulates the target.The PMB in this sensing scheme can form intermolecular dimers.In the presence of polymerase,the molecular beacon probe can be extended using itself as a template.And the target gene is replaced,so that it opens the next molecular beacon.The strand displacement amplification reaction is triggered.The fluorescence signal is greatly enhanced,which enables highly sensitive detection of the K-ras gene with a detection limit as low as 10 pM.In addition,the sensor system has demonstrated excellent detection performance in the detection of complex biological environments and actual samples.Secondly,based on the functionalized hairpin probe（HP）containing the G-quadruplex region and the exonucleaseⅢ（ExoⅢ）cleavage region,a label-free DNA sensor was designed for the detection of Pax-5a gene,which is a biomarker for acute B-lymphocytic leukemia.The non-single-stranded fragments such as stems and G-quadruplexes in the hairpin structure can be digested with a certain concentration of ExoⅢ.The UV signal in the presence or absence of the target can change differently,thereby achieving ultra-sensitive detection of the Pax-5a gene.The sensor has a good ability to discriminate against non-target genes,and its detection ability in a complex biological environment containing serum is also excellent.Finally,we used a multi-enzyme-assisted signal amplification strategy to construct a label-free electrochemical sensor system with higher sensitivity for the detection of Pax-5a gene.In this sensing system,the Pax-5a gene can circulate open the hairpin probe under the action of restriction endonuclease and polymerase.This process generates a large number of G-quadruplex sequences.The ultra-sensitive detection of Pax-5a can be achieved by the G-quadruplex/hemin complex catalyzing the reduction of H₂O₂ on the electrode surface.The detection limit of the sensor system is as low as 4.6 fM.The enzymatic amplification reaction process in the sensor system is performed outside the electrode,which reduces the complexity of operation and avoids the adsorption interference on the electrode surface.At the same time,it provides new ideas for the construction of future sensors.In addition,the biosensor has good specificity and stability,and also has excellent detection capabilities in complex substrate environments.