Preparation Of Quasi-Interpenetrating Network/Nanoparticle Composite Matrix And Its Performance For DNA Sequencing

Separation and sequencing of DNA are vital to reveal genetic code.Capillary electrophoresis(CE) is one of the most significant techniques for the analysis of charged biomacromolecules(e.g.DNA and proteins,etc.).During the separation and sequencing analysis of DNA by CE,the sieving matrices are very important because they determine the migration behavior and the resolution of DNA.At present, non-gel sieving matrices(i.e.,noncross-linking polymer solutions) have been employed widely in CE,which usually include linear homopolymers,copolymers, mixtures,etc.Among them,linear polyacrylamide(LPA) with high molecular weight (MW) possesses high sequencing ability and long read length.However,high-MW LPA solution is very viscous and has no self-coating ability.On the contrary,poly(N, N-dimethylacrylamide)(PDMA) shows excellent self-coating ability but offers relatively poor sieving performance.Although a lot of polymer solutions have been developed and tested as sieving matrices for CE,no single existing homopolymer solution can fully meet all expectations.Therefore,searching for the sieving matrices with low viscosity,high-sieving ability and self-coating ability is still an important issue for high-throughput DNA analysis.However,testing and developing a new polymer matrix usually takes a lot of time and effort.Recently,addition of certain additives(such as montmorillonite clay,gold nanoparticles,polymer nanoparticles, bacterial cellulose fibrils,carbon nanotubes) into low viscous polymer solutions has been proved to be a very efficient and simple method to overcome the difficulty of filling capillaries and improve DNA separation performance due to their unique properties.Although various additives used to separate dsDNA have been investigated in the past several years,the study on additives for ssDNA sequencing is very deficient at present.So we aim at improvement of the performances of ssDNA sequencing by incorporating additives into polymer to form composite matrices.In this dissertation,based on the synthesis and characterization of noncross-linking quasi-interpenetrating network(quasi-IPN),we prepared three composite matrices and studied their DNA sequencing performances by CE.1.Preparation and characterization of quasi-interpenetrating networkThree LPA samples with viscosity-average molecular weight of 1.5,3.3,and 6.5 MDa were synthesized by using inverse emulsion polymerization and then noncross-linking quasi-IPN were prepared by the polymerization of N, N-dimethylacrylamide(DMA) monomer in LPA aqueous solution.These matrices combine high sequencing ability of LPA and excellent self-coating ability of PDMA. LPA and quasi-IPN were characterized by Ubbelohde viscometer and ~1H NMR and the results demonstrate the preparation of LPA and quasi-IPN.2.Preparation of quasi-interpenetrating network/gold nanoparticle composite matrix and its performance for DNA sequencingGold nanoparticles(GNPs) with particle sizes of about 20,40,and 60 nm were prepared and added into noncross-linking quasi-IPN composed of LPA with different viscosity-average MW of 1.5,3.3 and 6.5 MDa and PDMA to form polymer/metal composite matrices,respectively.The effects of the parameters in relation to quasi-IPN/GNPs matrices,such as GNP contents,GNP particle sizes,LPA MW, solution concentrations,and temperature,on ssDNA sequencing performances were studied.Without complete optimization(such as base calling software), quasi-IPN3/GNPs40-1 yielded a readlength of 766 bases at 98%accuracy in about 57 min by using the ABI 310 Genetic Analyzer at 50℃and 150 V/cm.Resolutions of quasi-IPN/GNPs were higher than those of quasi-IPN without GNPs and approximated those of quasi-IPN with higher-MW LPA without GNPs in the bare fused-silica capillaries.So the use of quasi-IPN/GNPs with low-MW LPA could avoid the problems in relation to LPA with high MW such as difficult preparation, very high viscosity,and easy degradation,and thus help for full automation.The sequencing time of quasi-IPN/GNPs was shorter than that of quasi-IPN under the same sequencing conditions.Furthermore,the separation reproducibility of quasi-IPN/GNPs solution was excellent and shelf life was more than one year.The results showed that these novel matrices could improve ssDNA sequencing performances due to the interactions between GNPs and polymer chains and the formation of physical cross-linking points as demonstrated by intrinsic viscosities and glass transition temperatures,which prevented the polymer chains from sliding away from each other and thus could form relatively more stable "pore" sizes(more robust sieving matrix networks) and increase the apparent MW of the matrices and their sieving properties.3.Preparation of quasi-interpenetrating network/functionalized gold nanoparticle composite matrix and its performance for DNA sequencingA new matrix additive,PDMA-functionalized gold nanoparticle(GNP-PDMA), was prepared by "grafting-to" approach to avoid GNP aggregation at high concentration of salt in buffer solution,and then incorporated into quasi-IPN composed of LPA(3.3 MDa) and PDMA to form novel polymer/metal composite sieving matrix(quasi-IPN/GNP-PDMA) for DNA sequencing by CE.Without complete optimization(such as base calling software),quasi-IPN/GNP-PDMA-2 yielded a readlength of 801 bases at 98%accuracy in about 64 min by using the ABI 310 Genetic Analyzer at 50℃and 150 V/cm.Compared with previous quasi-IPN/GNPs,quasi-IPN/GNP-PDMA can further improve DNA sequencing performances.This is because the presence of GNP-PDMA can improve the compatibility of GNPs with the whole sequencing system,enhance the entanglement degree of networks,and increase the GNP concentration in system,which consequently lead to higher restriction and stability,higher apparent MW,and smaller pore size of the total sieving networks.Furthermore,the composite matrix was also compared with quasi-IPN containing higher-MW LPA and commercial POP-6.The results indicate that the composite matrices are potential ones for DNA sequencing to achieve full automation.4.Preparation of quasi-interpenetrating network/functionalized multi-walled carbon nanotube double-network composite matrix and its performance for DNA sequencingPDMA-functionalized multi-walled carbon nanotubes(MWNT-PDMA) were prepared via atom transfer radical polymerization(ATRP),and then added into quasi-IPN composed of LPA(3.3 MDa) and PDMA to form polymer/nanotube double-network composite sieving matrices for DNA sequencing by CE.The effects of MWNT-PDMA concentration in matrices and MW of PDMA side chains in MWNT-PDMA on ssDNA sequencing performances were studied in detail.Without complete optimization(such as base calling software),quasi-IPN/MWNT-PDMA2-Ⅱyielded a readlength of 792 bases at 98%accuracy in about 62 min by using the ABI 310 Genetic Analyzer at 50℃and 150 V/cm.The CE results show that,compared with quasi-IPN,the novel composite matrices can improve ssDNA sequencing performances due to the formation of a double-network consisting of a flexible quasi-IPN polymer network and a rigid MWNT network based on unique tubular structure,which makes the total sieving networks more restricted and stable and increases the apparent MW of the matrices.Furthermore,the PDMA side chains on MWNT-PDMA may entangle with homo LPA or PDMA in quasi-IPN to further stabilize the matrix network.Therefore,more restricted,stable,and smaller nanopore structure in quasi-IPN/MWNT-PDMA matrix results in more excellent properties. Furthermore,these double-network composite matrices were also compared with other matrices and the results indicate that they are promising ones for DNA sequencing.In conclusion,in the presence of nanoparticles(GNPs,GNP-PDMA,or MWNT-PDMA),the separation had the advantages of high resolution,speediness, excellent reproducibility,long shelf life,and easy automation.The composite matrices seem able to combine optimal sieving ability and dynamic coating ability with moderate viscosity.Therefore,less viscous composite matrix solutions(with nanoparticles) due to lower solution concentration and lower-MW LPA could be used to replace more viscous solutions(without nanoparticles) due to higher solution concentration or higher-MW LPA to separate DNA,while the sieving performances were approximate even higher,which helped to achieve full automation especial for capillary array electrophoresis(CAE) and microchip electrophoresis(MCE).