Fabrication Of Ultrasensitive Nanopores Sensors And Its Applications

Synthetic nanopores are attracting more and more attention from both industry and academia due to their superior stability,robustness and easily customizable features in comparison with their protein based counterparts,so finding a simple and convenient high mechanical and chemical stability of solid-state nanopores has become the primary problem.With their unique charge transport properties including ion permselectivity,ion concentration polarization,and ionic current rectification?ICR?,synthetic nanopores have been applied into biomimetic ion channels,sensors,ion permselective membranes and ion pumps.In ICR phenomenon,an asymmetric diode-like current-voltage behavior in which the ionic current varies with the polarity and amplitude of the applied voltage can be observed.The nanopore analytical applications based on ICR can be realized by monitoring ionic current change when the analyte molecule recognition event causes the conformational and/or surface charge variation inside nanopore.Meanwhile,to achieve sensitive and selective response to specific molecules,nanopore functionalization is highly significant.However,most of these recognition processes are based on ligand-receptor type conjugation in which analytes are recognized and bound to the inner surface of the nanopore to induce conformational and/or surface charge transitions,while nanopore-based detection of enzyme catalysis is seldom reported.Another key challenge for ICR based nanopore sensing is the receptor regeneration.This issue is critical for sensing devices if online monitoring or repeated determination needs to be performed with one sensor.Though some types of nanopore regeneration have been reported,they are quite limited in specific targets such as Cu²⁺,Ca²⁺,Co²⁺,Cr³⁺,and concanavalin A.However,most of the regeneration processes require multiple steps or relatively harsh conditions such as high temperature or extreme pH to dissociate ligand-receptor conjugation.Therefore,it is also essential to enable facile nanopore regeneration for sensing applications.To target those concerns,this thesis conducted following works:1.Preparation of glass conical nanopores and surface silanization modificationGlass conical nanopores have a simple and convenient preparation method and do not rely on large instruments compared with thin-film solid-state nanopores.Moreover,glass conical nanopores have high mechanical and chemical stability because of their strong geometric structure.By djusting the equipment parameters of the microelectrode pull?P-2000?,glass conical nanopores with suitable pore diameters and tapers were prepared.The morphology and pore diameter of glass conical nanopores were accurately characterized by optical microscopy and field emission scanning electron microscopy?FESEM?,the results show that under the condition of program 6 drawing,the pore diameter of 90 nm can be obtained stably.At the same time,the pore diameter of glass conical nanopore was estimated by electrochemical method.The nanopore diameters are in good agreement with the FESEM characterization.By the reaction of hydroxyl silicon and silicon alkylation reagents,functionalized groups can be introduced on the glass conical nanopore surface,to improve the charge characteristics of glass surface,which provide site for covalent fixed biological probe.This work use absolute ethanol as a solvent,3-aminopropyltriethoxysilane?APTES?,?3-Glycidyloxypropyl?trimethoxysilane?GPTMS?,trimethoxy-[3-?2-methoxyethoxy?propyl]silane as silicon alkylation reagents for glass conical nanopore silanization processing,through surface hydrophilicity of slides,the introduction of active group and the actual application results in the nanopores analyze,confirmed the best silicon alkylation reagents for APTES.The analysis of the introduction amount of amino groups on the inner surface of the nanopores confirmed that the optimal reaction condition was 1%APTES and the reaction time was 2 h.Therefore,the glass conical nanopores used in the subsequent experiments were all silanized under this condition,which laid a foundation for the functional modification of the subsequent nanopores.2.Detection of alkaline phosphatase activity with a functionalized nanopipetteIn this study,a label-free sensing method based on a functionalized nanopipette is proposed for detection of alkaline phosphatase?ALP?activity.This method is based on the unique charge transport properties of nanopores in which the ionic current can be modulated by variation in the charge on the inner surface of the nanopipette.A phosphorylated peptide,O-phospho-L-tyrosine?p-Tyr?,was used as a model substrate and to functionalize the nanopipette.The covalent functionalization procedures were characterized by measuring the current-voltage?I-V?curves and extracting the ionic current rectification status.On ALP catalysis,the removal of the p-Tyr phosphate group induces a decrease in the negative surface charge,subsequently leading to a sensitive response in the ionic current change.An ANOVA test confirmed a linear relationship between the normalized ionic current change and ALP activity(?I/I₀=0.344 C_ALP+0.022,R²=0.914).The functionalized nanopipettes demonstrated a directly measured detection limit of 0.1 mU/mL,and excellent selectivity against four common interfering proteins.Furthermore,this nanopore-based method has potential for use in characterizing or sensing the activities of other enzymes with a similar biochemical process to ALP.3.Ultrasensitive and regenerable nanopore sensing based on target induced aptamer dissociationFor ionic current rectification?ICR?based sensing,nanopore functionalizations are mostly designed for directly binding target molecules to generate detectable signals from surface charge variation.However,this strategy is highly dependent on the charge difference between the captured molecules and surface functionalization layers,which will increase the nanopore design difficulty and subsequently limit the nanopore applicability.Another key challenge for ICR based sensing is the nanopore regenerability that is critical if online monitoring or repeated determination needs to be performed with one sensor.Though some types of nanopore regeneration have been realized on some specific targets or with harsh conditions,it is still highly favored to develop a regenerability using mild conditions for various targets.To address these two challenges,we developed a novel and universal sensing strategy for aptamer-functionalized nanopore that can be easily regenerated after each usage without any harsh conditions and independent of target molecule charge or size for ICR based nanopore sensing.Ochratoxin A?OTA?was used as a model analyte and its corresponding aptamer partially hybridized with the pre-immobilized complementary DNA?cDNA:5'-CHO-TGT CCG ATG CTC CCT TTA-3'?)onto the nanopore inner surface.We demonstrated that the recognition and conjugation of OTA with its aptamer resulted in rectified ionic current variations due to the dissociation between the OTA aptamer and its partially paired c DNA.The performance of this nanopore sensor including sensitivity,selectivity,regenerability,and applicability was characterized using rectified ionic current.This nanopore sensing strategy will provide a promising platform for extensive targets and online sensing applications.