Ultrasensitive DNA Sensor Based On Glass Conical Nanopore

Biological ion channels are biotic nanopores formed by self-assembly of proteins in cell membranes.By regulating the transmembrane transport of molecules and ions,these channels play an important role in complex life processes.However,these biological nanopores are inherently unstable,brittle and sensitive to external stimuli,which limits their practical application to some extent.Therefore,solid-state nanopores have been developed as a substitute for biological nanopores,which can not only simulate the transport function of biological nanopores,but also have better mechanical properties.These are applied to biomimetic ion channels,biosensors and molecular ion detectors.The present research work aims to combine the properties of ion rectification and the enzyme digestion in order to construct a label-free ultrasensitive DNA sensor device,based on a single glass conical nanopore.The dissertaton mainly consists of three chapters:Chapter#1 includes comprehensive introduction of the recent advancements and literature regarding nanopores.It further comprises of the classification of nanopores,based on different materials.Moreover,the latest applications of solid-state nanopores,regarding the fabrication of biomimetic ion channels,molecular ion detectors and biosensors are also summarized here.Apart from this,the principle of ion rectification in nanopore signal analysis and detection system is also introduced in this portion.Finally,the research ideas of the thesis are laid down in this part of dissertation.Chapter#2 consists of the detailed information about the steps and processes involved in the preparation and characterization of the single glass conical nanopore.This part also describes the modem trends in pore size measurement of nanopores.Furthermore,the utilization of scanning electron microscopy for carefully observing the conical tip of the platinum wire and microscopic imaging of single glass nanochannels is also highlighted here.Additionally,the characterization of bright field images of a single glass conical nanopore channel by inverted fluorescence microscopy also makes its way in this chapter.Chapter#3 introduces the DNA detection,approach based on enzyme digestion and ionic current rectification in single glass conical nanopore.Here,we propose a simple sensor,based on single glass conical nanopore,in order to achieve label-free and sequence-specific detection of DNA.Attributed to the unique structural properties,the mentioned nanopore exhibits high sensitivity towards inner surface charge,which can be facilely characterized by ion current rectification.In this research work,enzyme digestion is employed to design the biosensor on the bases of the fact that ribonuclease H holds the potential to specifically hydrolyze RNA strands that are complementary to DNA.Since DNA cannot hybridize with RNA,the negative charged RNA strand was adsorbed electrostatically by the positive charged PAH-modified nanopore and hence,the negative charge on the inner surface of nanopore was increased showing negative ion rectification.However,when the RNA in the DNA-RNA hybrid strand(hybridization of complementary DNA with RNA)was hydrolyzed into a single base by the enzyme,the negative charge on the inner surface was reduced and the polarity of the charge on the inner surface of the nanopore became positive,showing positive ion rectification.The reversal of the ion rectification direction from positive to negative amplifies the change of ion rectification ratio.we have put forward the development of nanopore-based biosensor for DNA with a limit of detection down to 0.1 fM.It is also worth mentioning here that the given sensor exhibits excellent reversibility.We believe that the presented approach would open a new window of opportunities for detecting DNA of unknown sequences.