An Electrochemical Biosensor For MicroRNA Detection Based On Mesoporous Silica Nanochannels And Nucleic Acid Signal Amplification

MicroRNAs(miRNAs),a class of small non-coding RNAs with approximately 18-25 nucleotides in length,plays an important role in regulating cell activity.miRNA was involved in the development of a variety of human diseases that its expression can be applicated in the diagnosis of some specificdiseases including cancer.It is of great significance to develop sensitive and effective methods in miRNA determination.Solid nanopores materials showed similar pore structure to biological pores,which has been extensively applicated in the fields of catalysis,drug delivery,separation,analysis,sensing,and so on.Mesoporous silica nanochannels(MSNs)were a kind of well-developed solid nanopore materials,which displayed the advantages of high poriness,high specific surface area,rich silicon hydroxyl groups on its surface for modification.Once MSNs modified onto electrode surface,electrochemical signal molecules could be aggregated in the pores of MSNs and generate the highly effective electron transmission,which provided a possibility of electrochemical sensing.For the purpose of highly sensitive detection of miRNA,this research constructed two electrochemical biosensors combined MSNs with nucleic acid signal amplication.The major contents were shown as follows:1.An Electrochemical Biosensor for miRNA-21 detection based on Mesoporous Silica Nanochannels and the Amplification of Toeholdmediated Strand Displacement ReactionIn this work,an electrochemical biosensor for miRNA-21 detection was constructed based on mesoporous silica nanochannels(MSNs)with high efficiency of electron transmission and the amplification of toehold-mediated strand displacement reaction(TSDR).The MSNs were grown on the surface of indium tin oxide(ITO)electrode,which was then blocked by the complex of anchor DNA,complementary DNA and trigger DNA as gate molecule through an amide reaction.In the absence of target miRNA-21,the electrochemical signal was very weak that the electroactive molecule[Fe(CN)6]3-/4-with negative charge could not approach the ITO surface due to the electrostatic repulsion with negative charged DNA gate.In the presence of target,miRNA21 hybridized with the cDNA and exposed the toehold,which could then be displaced by Fuel DNA.The complex of Fuel DNA and cDNA were then liberated from MSNs and opened the pores,which led to the approach of[Fe(CN)6]3-/4-to electrode surface with electrochemical response.In addition,due to the TSDR assisted target recycling,significant electrochemical response with highly sensitive detection of miRNA-21 down to 13 fM could be achieved with a linear calibration range from 0.02 to 0.2 pM.This biosensor also applied in complex samples with good performance.2.An Electrochemical Biosensor for miRNA-155 detection based on Mesoporous Silica Nanochannels and DNAzyme-mediated signal AmplificationAn electrochemical sensing strategy for miRNA-155 detection was proposed based on mesoporous silica nanochannels(MSNs)and DNAzyme mediated signal amplification.A hairpin-structured substrate DNA which could be recongnized and hydrolyzed specifically by DNAzyme was designed as gate molecule.This COOH group modified substrate DNA could be linked onto amino group riched MSNs surface through amide reaction.In the absence of target miRNA-155,a Locker DNA hybridized with DNAzyme to block its cleavage site that DNAzyme could not react with substrate DNA.The electroactive molecule[Fe(CN)6]3-/4-with negative charge could not approach the ITO surface,which led to the weak electrochemical signal.In the presence of target,miRNA-155 hybridized with the Locker DNA and released the DNAzyme that substrate DNA was then hydrolyzed with the assistance of Mn2+.The electroactive molecule[Fe(CN)6]3-/4-then approach electrode surface with electrochemical response.At the same time,the released DNAzyme could then hydrolyze the next substrate DNA,generating stronger electrochemical signal.This electrochemical biosensor could achieve the detection limit of miRNA-155 to be 40 pM with a range from 0.2 to 10 nM and very fast response.This biosensor also provided new approach for sensitive miRNA detection and good application potential in clinic diagnosis.