Construction Of Nanopore Sensing System And Its Applications Of Single Molecular Detection

Since it was first reported in 1996, nanopore sensing has been proven to a powerful technique for high-throughput, lable-free single molecule diction. Due to the potential for distinguishing four kinds of nucleic acids, one promising application of nanopores is high-throughput label-free DNA sequencing, which may cut down the cost of sequencing to 100 dollars. So far, besides the biopolymers like DNA and protein, nanopore has been wildly used for the characterization of size and surface properties of individual nanoparticles and virus. All these results make the scientists believe that nanopore sensing as single molecule detection technique will be developed to be a low-cost DNA sequencing tool and sensitive bio-sensor.In nanopore sequencing, due to the random folded and coil configurations of DNA which influence the corresponding current signal, there have not been a valid theory to account for the dynamics of coiled DNA translocation through nanopore, and it is hard to identify bases of DNA and give a high accuracy by single current blockade. This project focuses on the accuracy of nanopore measurement, and studies the characteristics of the current signals in the translocations of DNA, nanoparticles and virus. It was proved that the random folded and coil configurations did affect the current signals. For a better understanding and analysis of the dynamics of DNA translocation, start from construction unit, we investigated the translocation characteristics and mechanisms of λ-DNA, AuNPs and TMV and revealed the role of anantyes shape, size, charge, rigidity on translocation dynamics, trying to find the major factors that affect the accuracy of nanopore sensing. This project provides guidance for nanopore sequencing and the measurements of biopolymers, nano-materials, and virus in the future.1. The Construction of Nanopore Sensing SystemIn this project, we construct a nanopore sensing system on the base of patch clamp amplifier and the corresponding digitizer, which are used to amplify and record the current respectively. The connections of each component are studied, and we find a way to avoid the electromagnetic noise. Additionally, we designed and made various fiuidic setups according to acquirements of different experiments, which simplified the experiments, reduced the addition of samples, and avoided the most electromagnetic noise.2. Study of the translocation of X-DNA through solid-state nanoporeHere, a 30-60 (tip-base) nm conical nanopore fabricated in 100 nm thick silicon nitride (Si3N4) membrane by focused ion beam (FIB) has been employed for the analysis of X.-DNA translocations at different voltage biases from 200 to 450 mV. The distributions of translocation time and current blockage, as well as the events frequencies as a function of voltage are investigated. The presence and configurations of X-DNA molecules are well characterized. Also, we find that greater applied voltages markedly increase the events rate, and stretch the coiled λ-DNA molecules into linear form. However, compared to 6-30 nm ultrathin solid-state nanopores, a threshold voltage of 181 mV is found to be necessary to drive DNA molecules through the nanopore due to conical shape and length of the pore. The speed is slowed down-5 times, while the capture radius is-2 fold larger. The results show that the large nanopore in thick membrane with an improved stability and throughput also has the ability to detect the molecules at a single molecular level. Additionally, due to random folded configurations, we find multi-level current blockades, and the velocity of DNA shows a nonlinear relation with applied voltage. For a better understanding of the dynamics of DNA translocation, as DNA can be model by short rods connected with small balls, we next performed the translocation of simpler AuNPs and rod-shaped TMV where the dynamics of the translocation were fully studied.3. Study of the translocation of AuNPs and AuNPs-DNA conjugates through solid-state nanoporeIn this study, we present the first observations of the intriguing biphasic (low slat) and asymmetrical (0.5 M KC1) events in the translations of DNA modified gold nanoparticles through-60 nm nanopores. An electric field dependent conductance change and quadratic nonlinear electrophoresis were observed as well. The induced charges and the change of the DNA structure are used to account for the dynamics of the biphasic events and asymmetric events. Then, we develop an approximation of the conductance change of nanopore based on induced-charge electrophoresis (ICEP). The effect of salt concentration, the applied voltage, and particle radius on the conductance change are studied. The results shows solid-state nanopores may not only be used for single nanoparticle scanning, but also a much easier and powerful platform for direct observation and study of nonlinear electrophoresis of single nanoparticle. This study will provide valuable suggestions when using AuNPs-analytes probing system integrated nanopore sensor for a target probe /analytes detection.4. Study of the translocation of TMV through solid-state nanoporeHere, we present the first translocation of rigid rod-shaped Tobacco mosaic virus (TMV) through various solid-state nanopores. Interestingly, due to the high rigidity of TMV, three types of events, and a strong current fluctuation during the translocation of TMV are observed. A kinetic model is then proposed to address the dynamics of the translocation, followed with corresponding dynamics simulations. Based on the model we are able to reveal the behaviors of TMV during its translocation through nanopore by the characteristics of the event. Then, we study The conductance change due to the trapping of TMV near the pore entrance, the vertical translocation, and the rotation of TMV during the translocation are then studied, indicating a pore geometry-dependent conductance changes and the fluctuations. At the end, we investigate the rotation frequency of TMV during the translocation, and the results indicate TMV orientation tends to be parallel to the electric field during the translocation through nanopore. This study gives a fundamental understanding of the dynamics of rod-shaped particles translocating through nanopore and how the current responds to it. It opens a new possible way to characterize the rigidity of analytes by nanopores.In summary, we proved that the random folded and coil configurations would affect the current signals. By studying the translocation characteristics and mechanisms of AuNPs and TMV, the influences of the surface charge, configuration change, interactions with pore wall, rotation during translocation of analytes were understood. It provides guidance for nanopore sequencing and the measurements of biopolymers, nano-materials, and virus in the future.