| As the messenger of genetic information, DNA is one of the most important materials in the evolution process of life. The rapid and accurate detection of DNA sequence can not only help people prevent and treat diabetes, hemophilia, cancer and some other genetic diseases, but also unravel the mystery of life origin. Since the 70’s of last century, three generations of DNA sequencing technology have been developed. Based on Sanger sequencing method, it took about 15 years and $3 billions for the first-generation DNA sequencing technology to complete the Human Genome Project. By using the second-generation DNA sequencing with the characteristic of high throughput, the cost of the individual human genome sequencing could be reduced to less than 1 week and $1 million. In recent years, the third-generation sequencing based on nanopore has become a hot research topic. When the DNA molecule passes through a nanopore, a characteristic blockade ionic current can be detected to determine the information of DNA molecule. With the advantages of rapidity, accuracy and low-cost, this technology is expected to complete an individual human genome sequencing within $1000 and 24 h which was the target set by the National Institutes of Health (NIH). Nevertheless, there are still some key issues need to be resolved. For example, the high translocation speed of DNA makes it difficult to detect the blockade ionic current accurately. It was reported that increasing the friction between DNA and nanopore can to some extent reduce the translocation speed of DNA.However, lots of previous studies on DNA were mainly focused on surface self assembly, molecular morphology and mechanical stability of DNA molecule, as well as the research on the specific functional devices based on DNA. There was few research focused on the friction behavior of DNA. Therefore, in order to make a comprehensive, in-depth understanding on the friction mechanism between DNA and nanopore materials, it is very essential to carry out the research on the friction behavior of DNA. The related research results can not only promote the development and application of the third-generation sequencing based on nanopore, but also enrich and improve the basic theory of bio-nanotribology.In this thesis, the effects of nanopore materials, NaCl concentration, solution pH and chain length of DNA on the friction behavior of DNA against nanopore materials were investigated by an atomic force microscopy (AFM). Firstly, DNA molecules were successfully assembled on the spherical SiO2 tip. Based on the friction tests of DNA against various nanopore materials in liquid environment, the optimization suggestion of nanopore material for the nanopore sequencing was proposed. After that, DNA films were successfully prepared on the mica surface. By using a Si3N4 tip, the effects of NaCl concentration, solution pH and chain length of DNA on the friction behavior of DNA aganist Si3N4 nanopore material were systematically investigated. Moreover, in order to verify the optimization results of solution pH in this thesis, translocation situation of DNA molecules when passing through a silicon nitride nanopore in different pH solution was tested with the help of reseaech group of Professor Chen Yunfei at the Southeast University. The main conclusions can be summarized as following:(1) Based on the successful preparation of the SiO2 tip assembled with DNA molecues, the effect of nanopore materials on the friction behavior of DNA was revealed.After the modification of SiO2 tip by 3-aminopropyltriethoxy, the SiO2 tip assembled with DNA molecues (DNA-SiO2 tip) was successfully prepared. Then, the friction behavior of DNA against five typical nanopore materials (APS-silica, silicon nitride, silica, graphene, graphite) was studied. It was indicated that under the same load, the friction force between DNA and five nanopore materials was ordered as follows:APS-silica>silicon nitride> silica> graphene> graphite. Due to the special layered nanostructure of graphene and graphite, the friction force of DNA-SiO2 tip on them was significantly less than on the other three nanopore materials. Further analysis indicated that the hydrophobic surface can to some extent increase the friction force between DNA and nanopore materials. Due to the electrostatic attraction between the positively charged APS-silica surface and DNA, the friction force between DNA-SiO2 tip and substrate was larger. The research offers the optimization suggestion of nanopre materials, and also provides a reference for the selection of tip material in the following research on friction behavior of DNA.(2) The effect of NaCl concentration on the conformation and friction behavior of DNA was clarified.The DNA films were successfully prepared on the mica surface. By using the Si3N4 AFM tip, effect of NaCl concentration on the conformation and friction behavior of DNA was studied. It was found that with the increase of Na+ ion concentration in solution, the relatively extended DNA molecule can be turned into a coiled conformation due to the screening of the negative charged phosphate group outside of the DNA backbone. When the NaCl concentration of solution was low, the friction force between Si3N4 tip and DNA was larger due to the larger bending rigidity of DNA. In the DNA nanopore sequencing process, lower NaCl concentration can make DNA molecules more extended and pass through the nanopore in an ideal posture. Moreover, the larger friction of DNA in low NaCl concentration can reduce the translocation speed of DNA molecues. Therefore, lower NaCl concentration was more conducive to the third-generation sequencing based on nanopore.(3) The effect of sulotion pH on the conformation and friction behavior of DNA was clarified.By using the Si3N4 tip, the force curve and friction were tested on the DNA films in sulotions with different pH. The influence mechanism of solution pH on the conformation and friction of DNA was analysed. It was found that the variation of solution pH would affect both the force between bases and the electrostatic interaction between DNA and Si3N4 tip. When the solution pH was low, there was an electrostatic attraction between the Si3N4 tip and DNA. Such attraction would increase the interaction between them and further lead to the increase of friction. When the solution pH was high, there was an electrostatic repulsion between the Si3N4 tip and DNA films. Such repulsion would decrease the interaction between them and further lead to the decrease of friction. Moreover, the conformation variation of DNA molecule in different pH solution could also affect the friction of DNA. In low pH, DNA was mainly in a "train-like" conformation, the tip would be subjected to a greater resistance from DNA molecule during the shearing process. However, in high pH solution, the DNA molecules joined in loops and in an open coil conformation in solution. Such conformation would decrease the resistance during the shearing process of tip. The "train-like" conformation in low pH sulotion could make DNA molecules reach into and pass through the nanopore in an ideal posture. Moreover, the high friction of DNA in low pH solution can reduce the translocation speed of DNA molecues. The experimental results of translocation situation of DNA in different pH solution also verified that low pH value can decrease the translocation speed of DNA. Therefore, low pH solution was more conducive to the third-generation sequencing based on nanopore.(4) The study showed that chain length had insignificant effect on the friction behavior of DNA, and the read length of nanopore sequencing will not be limited by firction.The DNA films with different chain lengths were prepeared on the mica surface. By using a Si3N4 tip, the influence of chain length on the conformation and friction behavior of DNA was studied. It was indicated that when adsorbed on APS-modified mica as films in solution, the conformation of DNA molecules would be a combination of loops and "train-like". With the increase of the chain length, the DNA molecule is more extended. However, under low normal load, the chain length had insignificant effect on the friction force of DNA. Such results indicated that the DNA translocation speed was affected insignificantly by chain length. Therefore, from the perspective of tribology, the read length of nanopore sequesing will not be limited by friction.In summary, this thesis systematically studies the effect of nanopore material, NaCl concentration, solution pH and chain length of DNA on the friction behavior of DNA. The research resaults can not only enrich the basic theory of bio-nanotribology, but also provide the theoretical basis for the optimization of the third-generation sequencing based on nanopore. |
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