Research On Gene Sequencing Technology Based On Solid State Nanopores

Gene sequencing technology can be used as a methord to measure genetic information in biological research, it also plays an important role in gene diagnosis and gene therapy application. Gene sequencing technology has made great progress since the first-generation sequencing technology was invented in 1977. Different with the first-generation and second-generation sequencing technology, the third generation sequencing technology characterized as single-base sequencing feature. It can reduce the cost of sequencing to one thousand dollar or less and is faster and more accurate than the first-generation and second-generation sequencing technology. This will lead to gene sequencing technology oriented towards common people and induce a dramatic revolution in the medical field.In this dissertation, the first-generation and second-generation sequencing technologies are reviewed first and the advantages of the third generation sequencing technology are discussed. Then the state of the art for the third generation gene sequencing techology is introduced briefly. Following the introduction chapter, fabricating 5nm diameter nanopore is discussed. A platform for biomolecular detection experiments is set up in this dissertation. DN A molecules are detected in this plateform:(1) In our work, three type material nanopores are fabricated, which include silicon material based nanopore, graphene nanopore and Polydimethylsiloxane (PDMS) nanochannel. To fabricate the silicon material based nanopore, a silicon chip with an etched window is fabricated first with the microfabrication process. Then, we used FIB and TEM to etch a nanopore with two steps sequentially, the first of which is to reduce the thickness of the SiN membrane with a focused ion beam operated with in a FIB system (Helios 600i NanoLab, FEI Company, Hillsboro, USA). Fllowing the milling process, we used a high energy electron beam in a high resolution transmission microscopy(HRTEM) to irradiate a nanopore in the center of the reduced area on the silicon chip. With this methord, we can fabricate nanopore with diameter smaller than 5nm. To prepare the graphene nanopore, we first fabricate a silicon chip with an etched window. The top layer for the etched window is a thin SiN film with thickness of 1 OOnm. We can use the FIB to drill the SiN film with a through pore. The diameter of the through pore is controlled in 500 nm. Then a graphene film with thickness of single layer or a few layer is transferred to cover the 500 nm diameter through pore. Fllowing the transfer process, a nanopore is irradiated on the graphene film with the TEM operated at 300KeV. The diameter of the graphene nanopore can be controlled under 2 nm. Except nanopore fabrication, we also do a lot of study on nanochannel fabrication. We have fabricated a seriers of PDMS nanochannels with diameter ranging from 10 nm to 100 nm and length ranging from 100 nm to several micrometers through the process of embedding Polyethylene oxide (PEO) nanofibers.(2) In the world, we first investigated the electrolyte conductance of the nanopore from low to high concentrations. We found that the conductivity was governed by the surface charge density of the nanopore at low concentration while at high concentration, the conductivity of the nanopore will be lower than the bulk value when the nanopore’s size is smaller than 4.5nm. To further explain this phenomenon, we simulated the ions mobility in nanopore using GROMAS and propose a new model to describe how the ions move in nanoscale channel. We also modified the surface charge density of the nanopore to study how the surface charge density influences the conductivity of the nanopore. Based on that we also study the conductance properties of the PDMS nanochannel with different salt concentrations, we found that the conductance in nanochannel will decrease as the concentration increased especially at high concentration. In nanochannel, the viscosity of the solution will increase as the concentration increases, resulting the decrease of the mobility and conductivity of ions.. In order to study the rectification of conical nanopore, we compared the conductance of the conical nanopore with hydrophobic and hydrophilic surface and found after hydrophilic treatment., the rectification disappeared. This result can effectively improve the DNA molecular capture rate in nanopore detection experiments.(3) One challenge of nanopore sequencing is the electric noise. In order to decrease the noise and improve the signal to noise ratio, we investigated three methords to improve thte signal to noise ratio, including coating the nanopore with PDMS, hydrophilic treatment on the nanopore surface and deposition Al2O3 membrane on the nanopore surface. All of these methords can effectively improve the signal.(4) In order to study the translocation of DNA molecular through the nanopore, we designed biomolecules detection experiment platform and integrated it with nanopore together. Using our own fabricated Si3N4 nanopore, we can distinguish the posture of 48 kb λ DNA moleculars when it transports through nanopore according to the shape of the signal. We did further theoretical analysis about the amplitude and the duration time of the block signal of DNA transportation. We found when the nanopore is smaller than 7 nm,48 kb λ DNA moleculars will stick on the surface of the nanopore, it will slow down the transpotation speed of smaller DNA moleculars just as the surface has been modified. Using 2.5 nm size nanopore, it can distinguish the differences between 3 nt "G" homopolymer and 3 nt "T" homopolemer. During the experiment of graphene nanopore, we detected 48kb λ DNA from 100 mV to 1000 mV and figured out the linear relationship between the applied voltage and the duration time. In order to distinguish the differences between 3 nt "C" homopolymer and 3 nt "G" homopolymer, we designed a new methord by means of gradient salt concentration. Due to the different surface charge of these two kinds of homopolymers, the amplitude of the block signal is a little different.