Research On Electrochemical MicroRNA Sensor Based On Catalytic Hairpin Assembly Reaction

Cancer is currently one of the leading causes of death,early diagnosis and prompt treatment can significantly reduce cancer mortality.Studies have shown that the dysregulation of microRNA-21（miRNA-21）is associated with the tumorigenesis,metastasis,and invasion.Therefore,miRNA-21 is considered to be a biomarker for breast cancer tumors.In order to improve the sensitivity and accuracy of miRNA-21detection,a series of electrochemical miRNA-21 sensors have been constructed based on the catalytic hairpin assembly reaction triggered by miRNA-21.We combined the signal amplification strategies such as the electrocatalytic effect of nanomaterials on signaling molecules,the loading effect of nanomaterials on signaling molecules,a novel intercalated signaling molecule,the reaction between signal molecules and electrolytes in electrochemical tests,and novel signal amplification methods to further enhance the sensitivity of the electrochemical biosensor.The content mainly includes the following four parts:（1）The electrocatalytic activity of ZIF-8-derived Fe-N-C nanomaterials towards thionine was investigated for the first time,and a label-free and immobilization-free electrochemical platform based on Fe-N-C-thionine/Fe₃O₄@AuNPs was constructed for the detection miRNA-21.Magnetic Fe-N-C-thionine/Fe₃O₄@AuNPs composites were immobilized on the surface of magnetic glassy carbon electrodes.Fe-N-C and Fe₃O₄ nanoparticles could synergistically catalyze the electroreduction of thionine,and a strong electrochemical reduction signal of thionine was obtained in differential pulse voltammetry test.In the presence of miRNA-21,the catalytic hairpin assembly reaction is triggered and the poorly conductive SiO₂ was then modified on the electrode surface,which slows down the electron transfer rate on the electrode surface.In the range of 1fM-10 nM,the reduction peak current of decreasing thionine was linearly related to the logarithm of miRNA-21 concentration,the detection limit was as low as 0.63 fM.The detection of human serum samples showed that the constructed electrochemical miRNA sensor had good specificity and reproducibility,proving that the platform has broad application prospects in the early diagnosis of tumors.（2）The reaction between the intercalated electrical signal molecules and the electrolyte was studied for the first time,and a double signal amplification e lectrochemical miRNA-21 sensor based on sodium alginate-polypyrrole/AuNPs was constructed.A self-made copper complex with a planar structure was used as an electrical signal molecule and inserted into the double-stranded DNA formed by the catalytic hairpin assembly reaction triggered by miRNA-21.In the square wave voltammetry test,it can trigger the electrochemical reaction on the electrode surface and the chemical reaction between iron and copper was triggered to enhance the electrical signal when the electrolyte contains iron ions.In the range of 1 fM-1 nM,there was a good linear relationship between the peak current and the logarithm of the miRNA-21 concentration,with a detection limit as low as 0.34 fM.This novel dual-signal amplification strategy based on DNA intercalated molecules also has good stability and repeatability in the test of human serum samples.It can be more widely used in the detection of nucleic acid markers.（3）The signal amplification strategies of the first two parts are further combined and optimized.For the first time,the electrocatalytic oxidation activity of magnetic p-Co-BDC to methylene blue was investigated,and an electrochemical miRNA detection platform based on multiple signal amplification of AuNPs-modified Co-terephthalic acid nanosheets was constructed.The two-dimensional p-Co-BDC does not have the adsorption properties of the ZIF-8-derived Fe-N-C in the first part,and it can be modified on the surface of the magnetic glassy carbon electrode to avoid more non-specific adsorption.In the presence of miRNA-21,the catalytic hairpin assembly reaction could be triggered and form double-stranded DNA for the insertion of more methylene blue molecules,enhancing the sensitivity of the detection.In the electrolyte of ascorbic acid,the electrochemical reaction on the electrode surface and the chemical reaction between methylene blue and ascorbic acid can be triggered,which further enhances the electro-oxidative signal of methylene blue in the differential pulse voltammetry test.In the range of 100 a M-10 nM,there was a good linear relationship between the peak current and the logarithm of the miRNA-21 concentration,the detection limit was as low as 86 a M.The detection of miRNA-21 in human serum samples also showed good selectivity,reproducibility and good recovery.The proposed multiplexed signal amplification strategy can realize the ultrasensitive detection of miRNA,which has certain application potential.（4）The first three parts mainly focus on the signal amplification of double-stranded DNA formed by miRNA-21-triggered catalytic hairpin assembly reaction.In the last part,the catalytic hairpin assembly reaction is modified by silver ion.For the first time,silver ions modified catalytic hairpin assembly（CHA）reaction and oxygen reduction reaction（ORR）were combined to amplify the signal for the electrochemical detection of miRNA-21.In the presence of miRNA-21,catalytic hairpin reaction was triggered and the C-Ag⁺-C structural probe formed by hairpin DNA1（H1）and Ag⁺can be opened.Then,Fe₃O₄@PDA@AuNPs modified with capture probe（C1）can immobilize all H1,and Ag NPs as the typical electrocatalyst of oxygen reduction reaction can be electrodeposited in situ in I-T experiments.In addition,with the aid of CdSe quantum dots,the Ag⁺released in solution can also be detected by fluorescence and colorimetry.Ag⁺can change the fluorescence intensity and emission wavelength of CdSe quantum dots,which can reflect the concentration of miRNA-21.The limit of detection（LOD）was 323 a M.Furthermore,the constructed biosensor exhibits good performance in real samples,indicating its potential for practical application.This novel dual-mode detection strategy makes the detection results of miRNA-21 more accurate and reliable.