Functional Modification Of Nitride Nanopores For Sensor

Sensing with chemically-modified nanopores is an emerging field that is expected to have major impact on bioanalysis and fundamental understanding of nanoscale chemical interactions down to the single-molecule level.At present,the application of solid-state nanopores is becoming the focus of attention.Compared with biological nanopores,they offer greater flexibility,and such as shape,size and surface properties.The unmodified solid-state nanopores cannot interact with analyses owing to lack specific recognition sites,which is limited in application.Nanopores can expand the scope of analyse by chemical surface modification or modify specific groups.This technique provides a means for label-free,real-time kinetic analysis of biomolecular interactions at the single molecule level including protein-protein,protein-DNA and receptor-ligand interactions,which give new features and function to nanopores and has unique advantages in terms of improving selectivity and sensitivity.In this doctoral thesis,the silicon nitride nanopores were fabricated by FIB and the surface was activated by 3-APTES and optimized the amino silane modified silicon nitride nanopores time for control of the size.The amino silane modified effectively was characterized by different technologies.Change charge amine-functionalized solid-state nanopores was studied by pH tuning,which can overcome the component of the drag force arising from the EOF owing to a lower surface charge density and lower electrophoretic force for polystyrene microspheres(?100 nm).The interaction between the nanopore wall of the chemical modification and translation of PS microspheres was analysed.The blockage current/biased voltage and the duration time/biased voltage function relations when PS microspheres through the nanopore of the chemical modification were discussed.On the basis of silane activated,amino DNA and horseradish peroxidase molecules was covalent coupling by chemical coupling bifunctional reagent for target identification sequences of DNA sensors and hydrogen peroxide sensors.Identify the dynamics of complementary and non-complementary sequences were researched at the nanoscale.The blockage current/biased voltage and the duration time/biased voltage function relations were discussed.For the first time,single horseradish peroxidase molecule was detected based on voltage feedback control.The hydrogen peroxide detection limit and linear relationship was investigated.A real-time monitored single enzyme molecular biochemical reaction and a translocation of the product of enzyme catalysis substrates were explored.So as to make the nanopore into a high specificity,low cost,multi-purpose,integrated/microarray and miniaturization biological sensing and testing platform.The contents of this thesis including:1.Amino silane functional silicon nitride nanoporeWe successfully fabricated Si3N4 nanopore with different size using FIB and the surface was activated by 3-APTES and optimized the amino silane modified silicon nitride nanopores time for control of the size.The amino silane modified effectively was characterized by different technologies.FESEM imaging was introduced to verify the change of the shape and size of before and after the functionalization.The results show that it is possible to control and tune the functionalization efficiency and shrink the nanopore to the chosen size.In addition,EDS was used to characterize the atomic percentage(at.%)before and after the functionalization of nanopores by chemically modified nanopores with the 3-APTES.After the functionalization,there was 24.42%atomic increase in nitrogen around the pore.A new element C after functionalization of the nanopore was observed and O(at.%)also increased from 1.41 to 1.65.In order to further characterize successful immobilization of the 3-APTES on the inner channel wall.?-? curves of before and after the chemical modification nanopores were measured to calculated diameter of modified.This method is easy to operate,good repeatability and stability.Conventional laboratory can complete and change the surface properties as well as provide good reactive for the subsequent biological molecules fixed.2.pH tuning 3-APTES functionalization nanopores translocation of particlesPrior to translocation experiment,we analysed zeta-potential and size measurement of?100 nm PS microspheres in 0.02 M KCl at pH 5.4,7.0,and 10.0 using a Malvern-Zetasizer Nano series,which was used to evaluate surface charge and to demonstrate that these conditions did not promote PS microspheres aggregation.Sequentially,functionalized nanopores were characterized by analysis of Field Emission Scanning Electron Microscopy(FESEM),energy dispersive X-ray spectroscopy(EDS)and electrical measurements.Compared with the image of the pore region before the functionalization,the functionalization layer appeared as a grey shadow in the pore region.After the functionalization,there was 24.42%atomic increase in nitrogen around the pore compared to that before the functionalization.In addition,we observed a new element C after functionalization of the nanopore,and O(at.%)also increased from 1.41 to 1.65.In order to further characterize successful immobilization of the 3-APTES on the inner channel wall.'Diameter of modified nanopore' could be calculated as?139 nm by equation.A linear dependence was found between current drop and biased voltage.Compared with previous studied small nanoparticles,the electrophoretic translocation of negatively charged polystyrene(PS)nanoparticles(diameter?100 nm)were investigated in solution using the Coulter counter principle in which the time-dependent nanopore current was recorded as the nanoparticles were driven across nanopore.Translocation of PS microspheres(?100 nm)needed the threshold voltage of-600 mV.Translocation behaviours were discussed at biased voltages from-500 to-900 mV.An exponentially decaying function(td?e-v/v0)was found between the duration time and biased voltage.The interaction between amine-functionalized nanopore wall and PS microspheres were discussed while the translation of PS microspheres.We explored also translocations of PS microsphere through amine-functionalized solid-state nanopores by varying the solution pH(5.4,7.0,and 10.0)with 0.02 M KC1.All the results suggested that chemical modified nanopores detected not only nanoparticles but also provided an effective platform for the rapid analysis of nanoparticle in solution.3.DNA-functionalized nanopores for sequence-specific recognition of DNA biosensorThree-step approach functionalized silicon nitride nanopores was developed.On the basis of the silanization,a specific DNA probe was introduced on the surface by chemical covalent binding.FESEM imaging,EDS,and electrical measurements were used to confirm the successful immobilization of the DNA probe on the inner channel wall.The DNA to selectively detect complementary target sequences was researched by using 42-mer oligonucleotides as probe molecules.Voltage versus current blockage and event duration was discussed and the results indicate that the current amplitude linearly increases with the voltages.An exponentially decaying function was employed to fit the dwell time dependent on the voltage.In addition,at smaller voltages chain solution for a long time,under the voltage of 150 mV chain solution time is about 3.6 ms,with the increase of voltage,the speed up the melting time,until under the voltage of 400 mV,platform disappear.Three typical current traces was analysed.Event(I)is basically event of DNA specific recognition,which presents under low voltages.The biosensor displayed a good selectivity and specificity to a DNA strand with a sequence complementary to the attached DNA probe.This method can selectivity detect DNA complementary sequence by chemical coupling biological probe,being adaptable to different probe molecules to identification of an unknown DNA oligomer by using specific DNA sequences in the future.4.Horseradish peroxidase functional nanopores hydrogen peroxide sensorSingle enzyme molecules(HRP)was detected for the first time in solution using nanopore based on voltage feedback control.The zeta-potential(?HRP)and hydraulic radius of the HRPs were analysed via Malvern-Zetasizer Nano series in different pH and salt concentration.The results show that ?HRP is positive for pH<4,then drops rapidly becomes negative for pH>5.Hence,the pI value is 4.3 ± 0.2.They did not aggregate in pH 6?7.Salt conditions(0.1?1M)did not promote HRPs aggregation.But they aggregated in 1.5 M?2M KCl.We analysed and discussed single HRP molecular translocation events through solid state nanopore(?28nm).A linear dependence has been found between current blockades versus biased voltage.An exponentially decaying function(td?e-v/v0)has been found between the duration time versus biased voltage.A comparison of the translocation events with?28 nm and?88 nm pores were investigated by analysing histograms of ?G.Peaks were obtained by Gaussian fitting.We observed ?G value almost the same at three biased voltage(?G value 0.06357 ±0.00092 nS at 900 mV,0.06485 ± 0.00080 nS at 700 mV,0.06822 ± 0.00047 nS at 500 mV).?G is foreign to the biased voltage in theory.This similarity ?G value proved the dependability of the results.The ?G value is 0.08898 ± 0.000615nS with nanopore(diameter?28 nm)and 0.11408 ±0.00269 nS with nanopore(diameter?88 nm),which could be easily understand since the same particle would make a bigger influence in a more confined room.The linear range and detection limit was investigated:5?15 nM and 10 pM,respectively.Linear equation:Y=-183.43197X + 3580.72629,the correlation coefficient:0.98623.Real-time monitor a single enzyme molecular biochemical reaction and enzyme catalytic substrate translocation were explored.New translocation events were observed having residence times and amplitudes that differ from those of the enzyme molecule.The product of enzyme catalysis substrates and the enzyme molecule can be effectively distinguished by the nanopore system.This approach offers the potential for further development as studying gene expression,enzyme dynamics at the single-molecule level and quantitative test of small molecules.