Single Molecule Enzymatic Reaction Detection In Nanopore Confined Space

Nanopore as a single-molecule sensing method has received an increasing attention.It has the unique advantages of label-free,low sample consumption,simple and fast in detection of enzymatic reactions at the single-molecule level.Recently,the applications of nanopore or nanochannel to detect enzymatic reactions are mainly focused on two detection methods: one is mainly detecting the interaction of enzyme molecules or corresponding products with biological nanopore.Another is to detect enzymatic reactions by detecting the change of the rectifying efficiency of the conical nanochannel before and after the enzyme reaction.Although,the enzymatic reactions can be detected by using bio-nanopore,it has limitations of detection due to the poor stability,the low tolerance of solution conditions,and the inability to adjust the size of biological nanopore.In addition,there are high requirements for the enzyme molecules by using conical nanochannel,and the complicated chemical modification makes it difficult to be widely applied.Therefore,it is significantly important to develop an efficient and stable nanopore platform to detect enzymatic reaction at single-molecule level.We detected the single-molecule enzymatic reaction by using silicon nitride nanopores and developed a platform for the detection of single molecule enzymatic reaction in nanopore confined space assisted with DNA nanostructures.This can provide a new approach for single molecule detection,and provides a new idea for clinical medical diagnosis of corresponding enzyme reactions.The specific research content and results are as follows:(1)We proposed a new method for detecting the reaction of horseradish peroxidase based on solid-state nanopores.First,we fabricated a nanopore with diameter of about 50 nm on the100 nm thick free-standing silicon nitride membrane by using focused ion beam(FIB),and then the inner surface of nanopore was modified with horseradish peroxidase(HRP)by employing carbodiimide coupling chemistry.The immobilization of enzyme molecules was characterized by scanning electron microscope(SEM)and current-voltage curves(I-V).The immobilized HRPs remained active in redox reaction that occurred inside a single nanopore channel in the presence of hydrogen peroxide(H2O2)and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS),The ABTS·+ produced in redox reaction would aggregate and interact with the inner surface of nanopore,then the aggregated ABTS·+ passed through nanopore,and translocation events can be detected.We found that the current blockage(?I)trends showed linear dependence on applied biased voltages.The relationship between the dwell time(?t)versus applied biased voltage was the exponentially decaying(?t ~ e-v/v0).Moreover,the real-time single aggregated ABTS·+ molecule translocation events were monitored and investigated.It can be detected that the catalytic reaction of immobilized horseradish peroxidase in a real time through the detection and statistical analysis of translocation events.(2)On the basis of the above research,we proposed a method that assembled enzymes in a nanopore assisted with DNA nanostructure and detected the enzymatic reaction.Due to the detection method above can only obtain a large number of enzymatic reaction in a statistical sense,and there are also inactive enzymes.In order to overcome the limitations of the above research,we proposed a method that assembled enzymes in a nanopore assisted with DNA nanostructure.Firstly,we designed and synthesized DNA nanostructures(DNA tetrahedron,DNA polyhedral)with different sizes and shapes.We used the DNA tetrahedron as a carrier due to the good uniformity of the size.The edge length of DNA tetrahedron structure is about 9 nm that carried four single stranded DNA with design sequences.The inner surface of the solidstate nanopore(~ 40 nm in diameter)was decorated with DNA probes using carbodiimide coupling chemistry.The DNA tetrahedron that carried single HRP enzyme was synthesized,and then hybridized with decorated DNA probes.Due to the limited space of the nanopore,a small amount of horseradish peroxidase molecules can be assembled in the confined space of the silicon nitride nanopore through this method.The assembly of enzyme molecules are characterized by scanning electron microscope(SEM)and current-voltage curves(I-V).When reaction substrates hydrogen peroxide(H2O2)and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)were added into this modified solid-state nanopore channel,the assembled HRP enzyme can induce enzymatic reactions inside the single nanopore.The reaction product ABTS·+ molecule then passed through the solid-state nanopore under applied voltages within the pore,thereby resulting in the changes of open-pore ionic current.According to the analysis of the current blockage(?I)and dwell time(?t)of the aggregated ABTS·+ translocation events,as well as the study of the conductance changes(?G),the catalytic reaction of a very small number of horseradish peroxidases can be detected in a real time.In this chapter,we combined the nanopore sensor with the DNA nanostructure for the first time.Due to the designability of the DNA nanostructure,a fixed number of biomolecules can be assembled in the solid-state nanopore based on this method,which provides a new approach and idea for single molecule detection and the detection of molecular conformation.(3)Based on the DNA tetrahedrons,in this chapter,we proposed a novel sensing approach of GR-5 DNAzyme cleaving specific substrate reactions using relatively larger size solid-state nanopores.Because GR-5 DNAzyme is 30 nt long single-stranded DNA,it is impossible to directly detect it with relatively larger size solid-state nanopores due to detection limitations.We designed and synthesized DNA tetrahedrons with edge length about 9 nm,and then,we combined them with GR-5 DNAzymes to construct complexes(TBD).On this basis,we first use silicon nitride nanopore with a diameter of about 40 nm to detect the TBD,and then added activation ions(Pb2+)to catalyze the cleavage of GR-5,and detect the cleaved products by using the same nanopore.The DNA tetrahedron-binding DNAzyme(TBD)complex provides an improved signal-to-noise ratio because of an increase in excluded volume,induced by the particularity of the event because of its distinctive spatial structure and carried charge.On this basis,different translocation events that are produced by the TBD complex and its cleavage products have been monitored.Additionally,we also used ~ 60 nm diameter silicon nitride nanopores to test their sensing ability.Thus,using DNA tetrahedrons-based nanopores to detect DNAzyme cleavage reactions can enlarge the application range of conventional solid-state nanopore sensors,providing it is not necessary to fabricate ultrasmall diameter nanopores for sensing small molecules.(4)In order to assemble a single enzyme in confined space of nanopore,we try to realize it by assisting pore-controlling DNA nanoplate.We firstly synthesized the DNA nanoplate structure.The outer dimension of the structure is 60×54 nm,the thickness is ~ 4.4 nm(doublelayer DNA double helix),and there is a rectangular hole with a size of 15×14 nm in the center.In order to combine the DNA nanoplate to assemble a single biomolecule in nanopore,we carried out experiments by using silicon nitride nanopores with different diameters(30 nm,23 nm,17 nm).We verified the stability of the current baseline and the voltage withstand capability after the DNA nanoplates docked on the nanopores.We found that the DNA nanoplate would deform and pass through the nanopore after it docked on the 30 nm and 23 nm nanopore when the voltage exceeded 200 m V.We analyzed the translocation behavior of DNA nanoplates through the nanopores.However,deformation and translocation behavior of the nanoplate would not occur until the voltage reached 700 m V when the DNA nanoplate docked on the 17 nm diameter nanopore.Based on this,we bound a short DNA strand(Poly T40)in the center of nanoplate structure by designing the staple strand.Therefore,we assembled a ss DNA in the confined space of the nanopore by docking the DNA nanoplate on the nanopore.After we applied voltages,we obtained new type of current change events,and we performed statistical analysis for the new events.However,due to the large current baseline fluctuations and noise produced by DNA nanoplate docked on the nanopore,and the current leakage between the DNA nanoplate and nanopore,we cannot confirm that the new type of events is completely caused by the conformational change of Poly T40.Finally,we analyzed the potential changes before and after the DNA nanoplates docked on the different diameter nanopores by finite element simulation.We found that it would produced a larger negative potential after the DNA nanoplates docked on the nanopores.In order to test the influce of negative potential on molecule's translocation through nanopore,we detected M13mp18 translocating through ~27 nm nanopore that docked a DNA nanoplate.We found that the negative potential can decelerate M13mp18 translocation around one order of magnitude.Therefore,the approach can provide a new way to slow down analytes translocation in nanopore single molecule detection.