Electrochemiluminescence Detection Of Tumor Marker MiRNA And Construction Of A Novel Colorimetric Sensor For Glucose

At present,cancer is a kind of disease that human beings are facing.Every year,the number of people who die from cancer is staggering.Most cancers can be measured and treated at an early stage based on detection of tumor markers in the body.However,the concentrations of these tumor markers are extremely low in the early stages of cancer.Therefore,it is particularly important to develop highly sensitive and selective analytical detection methods.Diabetes is also a disease that troubles the health of modern people.The complications of diabetes seriously damage to human tissues and organs such as heart,brain,eyes,kidneys and feet.Therefore,it is of great significance to construct a simple,sensitive and accurate sensor for detecting glucose content in vivo.Electrochemiluminescence combines electrochemistry and optics,and has the advantages of both,such as fast response,high sensitivity and low background signal.The combination of electrochemiluminescence with DNA recognition and signal amplification strategy can be used to construct highly sensitive and highly selective biosensors for the analysis and detection of tumor markers such as miRNA,circulating tumor DNA and carcinoembryonic antigen.Nanozyme is a new generation of artificial enzyme,which has both the unique performance of nanomaterials and the catalytic performance similar to biological enzyme.The introduction of nanozymes into the preparation of biosensors can avoid the weakness of bio-enzyme that are vulnerable to environmental factors,while reducing the cost of analysis.Therefore,the application prospects of biosensors based on nanozyme are very broad.In this thesis,a signal-off electrochemiluminescence biosensor with high sensitivity for miRNA-21 detection was constructed based on the quenching effect of ferrocene on the Ru（bpy）₃²⁺/TPA electrochemiluminescence system and enzymatic-digestion cycle signal amplification strategy.Furthermore,a novel glucose colorimetric biosensor was constructed based on Fe-MOF nanozyme with peroxidase-like activity.The main content of this thesis includes the following:1.Construction of miRNA-21 electrochemiluminescence sensor based on enzyme-digestion cycle amplification strategy.Micro RNAs（miRNAs）are a class of endogenous non-coding RNAs with regulatory functions found in eukaryotes.Studies have shown that the expression of miRNAs is associated with a variety of cancers,indicating that miRNAs play an important role in tumorigenesis.The content measurement for intracellular miRNAs is of great significance for the prevention and early diagnosis of tumors.We constructed a highly sensitive"signal-off"electrochemiluminescence biosensor for the detection of miRNA-21 based on the enzyme-digestion cycle amplification strategy induced by target miRNA-21 and the electrochemiluminescence quenching effect of ferrocene（Fc）on Ru（bpy）₃²⁺/TPA system.The amino-modified double-stranded DNA（ds DNA）was immobilized on the surface of glassy carbon electrode modified with chitosan and glutaraldehyde through the cross-linking effect of glutaraldehyde.In the present work,ferrocene（Fc）-labeled hairpin DNA（Fc-HP）contains bases sequence complementary to target miRNA-21.Therefore,in the presence of miRNA-21,the hairpin structure of Fc-HP is opened,a part of which hybridizes with miRNA-21 to form a double-stranded structure,and the other part is exposed.After adding exonuclease III（Exo III）,the substrate strand of the double-stranded is digested into single nucleotides,resulting in the production of Fc-labeled short-chain DNA（Fc-p DNA）.At the same time,the target miRNA-21 are also released to continue the next hybridization and enzymatic digestion cycle.After such n cycles,a large amount of Fc-p DNA are obtained.The Fc-p DNA obtained from the above reaction are dropped on the surface of the electrode modified with ds DNA,and the Fc-p DNA is introduced onto the surface of the electrode through a strand displacement reaction.Based on the quenching effect of Fc on the electrochemiluminescence of Ru（bpy）₃²⁺/TPA system,the detection of miRNA-21 can be realized.Under the optimal experimental conditions,the detection linear range for miRNA-21 is form 1 p M to 100 n M,and the detection limit Is 0.33 p M（S/N=3）.The constructed sensor exhibits high selectivity,sensitivity and stability for the detection of miRNA-21.2.Construction of a novel glucose colorimetric sensor based on the peroxidase-like property of Fe-MOF.In this work,the metal-organic framework material Fe-MOF was prepared.Study indicated that Fe-MOF exhibited peroxidase-like property and showed strong catalytic capacity toward the oxidation of 3,3’,5,5’-tetramethylbenzidine（TMB）with H₂O₂.Based on the property,we constructed a sensitive colorimetric biosensor for glucose detection.Glucose oxidase（GOx）was immobilized on the surface of Fe-MOF by covalent bonding in the presence of NHS and EDC to form GOx-Fe-MOF.In the presence of glucose,glucose was oxidized into gluconic acid and oxygen was reduced into H₂O₂with GOx as catalyst.The in-situ produced H₂O₂can oxidize TMB.In the room temperature,the reaction between H₂O₂and TMB is very slow.Meaningfully,Fe-MOF can catalyze the oxidation of TMB by H₂O₂.Therefore,the glucose can be measured according to the colour change of TMB before and after being oxidized.The advantages of this work are listed in the following:（1）Fe-MOF is not only the carrier fixing GOx,but also the catalyst between H₂O₂ and TMB reaction;（2）H₂O₂ is generated in situ through the reaction between glucose and O₂,so the interference of background signal can be reduced;（3）Using Fe-MOF as nanozyme instead of natural enzyme,not only avoids the disadvantage of natural enzyme,but also reduces the cost of analysis.Therefore,the glucose sensor has great application potential.