准互穿聚合物网络/纳米粒子复合介质的制备及其用于DNA测序性能的研究
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摘要
DNA的分离和测序分析是揭开遗传密码的关键。毛细管电泳(CE)是分析带电生物大分子(如DNA和蛋白质等)最重要的技术之一。由于筛分介质在DNA的分离和测序分析过程中影响DNA的迁移特性和分离度,因此对筛分介质的研究显得非常重要。目前,无胶筛分介质(即非交联的高分子溶液)广泛应用于毛细管电泳中,通常包括线形均聚物、共聚物、混合物等。其中,高分子量线形聚丙烯酰胺(LPA)具有筛分能力强、读出长度长等优点。但是,高分子量LPA溶液的粘度很高而且没有自涂覆能力。与之不同的是,聚N,N-二甲基丙烯酰胺(PDMA)具有极好的自涂覆能力,但筛分能力较弱。虽然已经研制和测试了大量高分子溶液用作毛细管电泳筛分介质,但没有一种单一均聚物溶液能满足所有的应用要求。因此,寻求同时具有高筛分能力和自涂覆能力的低粘度高分子溶液仍是DNA高效分析的一个重要课题。然而,研制一种新型聚合物介质通常需要花费大量时间和精力。近年来的研究表明,在低粘度的高分子溶液中加入某种添加剂(如多羟基化合物、蒙脱土、金纳米粒子、聚合物纳米粒子、细菌纤维素原纤维、碳纳米管等)是一种克服毛细管填充困难及改善DNA分离性能的非常有效且简单的方法。尽管在过去的数年时间里各种用于双链DNA(dsDNA)分离的添加剂已引起了广泛的关注,但有关用于单链DNA(ssDNA)测序的添加剂的研究还很少。因此,本论文的目的是通过将添加剂加入到聚合物中制成复合介质来提高ssDNA测序性能。首先制备并表征了准互穿聚合物网络(quasi-IPN),在此基础上制备了三种用于毛细管电泳DNA测序的复合介质,并研究了它们用作DNA测序介质的性能。
1.准互穿聚合物网络的制备及表征
通过反相乳液聚合法制备了粘均分子量分别为1.5、3.3和6.5 MDa的LPA,接着在LPA水溶液中引发N,N-二甲基丙烯酰胺(DMA)单体聚合生成非交联的quasi-IPN,该介质结合了LPA的高筛分能力和PDMA的自涂覆能力。利用乌氏粘度计和~1H NMR光谱仪对LPA和quasi-IPN进行了表征,结果证明了LPA和quasi-IPN的生成。
2.准互穿聚合物网络/金纳米粒子复合介质的制备及其用于DNA测序性能的研究
制备了粒径约为20、40和60 nm的金纳米粒子(GNPs),将其分别加入由LPA(1.5、3.3和6.5 MDa)和PDMA所组成的quasi-IPN中生成了聚合物/金属复合介质(quasi-IPN/GNPs)。详细研究了各种参数(如GNPs含量、GNPs粒径、LPA分子量、溶液浓度和温度)对ssDNA测序性能的影响。在测序条件(如碱基读出软件)未完全优化的情况下,使用quasi-IPN3/GNPs40-1作为介质,利用ABI 310遗传分析仪在50℃和150 V/cm下得到的读出长度(98%准确度)为766碱基,耗时约57 min。quasi-IPN/GNPs的分离度高于不含GNPs的quasi-IPN,接近于含较高分子量LPA但不含GNPs的quasi-IPN。所以使用含低分子量LPA的quasi-IPN/GNPs可以克服使用高分子量LPA所带来的问题(如难以制备、测序浓度下粘度高及容易降解等),从而有助于实现全自动化。在相同测序条件下,quasi-IPN/GNPs的测序时间要少于quasi-IPN。此外,quasi-IPN/GNPs的分离重现性非常好,而且寿命大于一年。结果表明,GNPs与高分子链之间有相互作用并且生成了物理交联点,它们避免了高分子链彼此之间的滑移,从而可以形成相对更稳定的孔径(更稳固的筛分网络)并增加介质的表观分子量及它们的筛分性能,所以这些复合介质可以显著改善DNA的测序性能。
3.准互穿聚合物网络/官能化的金纳米粒子复合介质的制备及其用于DNA测序性能的研究
为了解决GNPs在高盐浓度下易附聚的问题,由“grafting-to”法制备了一种新型介质添加剂,即PDMA官能化的GNPs(GNP-PDMA),并将其加入到由LPA(3.3 MDa)和PDMA所组成的quasi-IPN中得到了用于毛细管电泳DNA测序的聚合物/金属复合筛分介质(quasi-IPN/GNP-PDMA)。在测序条件(如碱基读出软件)未完全优化的情况下,使用quasi-IPN/GNP-PDMA-2作为分离介质,利用ABI 310遗传分析仪在50℃和150 V/cm下得到的读出长度(98%准确度)为801碱基,耗时约64 min。由于GNP-PDMA的存在可以改善GNPs与整个测序体系的相容性、增大网络的缠结程度并增加GNPs在体系中的含量,从而导致整个筛分网络更加受限和稳固、介质的表观分子量更高并且孔径更小,因此与之前的quasi-IPN/GNPs相比,quasi-IPN/GNP-PDMA可以进一步增强ssDNA的测序性能。此外,与其它商用介质的比较表明,该复合介质是一种非常有潜力的全自动化DNA测序(特别是微流动体系)介质。
4.准互穿聚合物网络/官能化的多壁碳纳米管双网络复合介质的制备及其用于DNA测序性能的研究
通过原子转移自由基聚合法(ATRP)制备了PDMA官能化的多壁碳纳米管(MWNT-PDMA)添加剂,并加入到由LPA(3.3 MDa)和PDMA所组成的quasi-IPN中,得到了用于毛细管电泳DNA测序的聚合物/纳米管双网络复合筛分介质(quasi-IPN/MWNT-PDMA)。详细研究了MWNT-PDMA含量和MWNT-PDMA上PDMA侧链分子量对ssDNA测序性能的影响。在测序条件(如碱基读出软件)未完全优化的情况下,使用quasi-IPN/MWNT-PDMA2-Ⅱ作为介质,利用ABI 310遗传分析仪在50℃和150 V/cm下得到的读出长度(98%准确度)为792碱基,耗时约62 min。CE结果表明,与quasi-IPN相比,该复合介质可以显著改善DNA测序性能,这是因为在复合筛分介质中形成了双网络,它包括一个柔性的quasi-IPN聚合物网络和一个刚性的MWNTs(具有独特的管状结构)网络。这两种不同类型的网络可以共存于介质溶液中并相互作用,从而避免聚合物之间彼此滑移、稳固和制约总的筛分网络、增加介质的表观分子量并减小介质的孔径。此外,MWNT-PDMA上的PDMA侧链可以与quasi-IPN中的LPA或PDMA发生缠结,从而进一步稳固介质网络。因此,quasi-IPN/MWNT-PDMA介质中的这种更受限、更稳固且孔径更小的纳米结构使得测序性能更优良。复合介质与其它介质的比较结果进一步表明,它们非常有希望用于DNA测序。
总之,在纳米粒子(GNPs、GNP-PDMA或MWNT-PDMA)存在条件下,DNA的测序表现出分离度高、重现性好且测序介质寿命长等特点,因此易于实现快速、高效分离DNA的目的。这些新型复合介质同时具备了优良的筛分能力、自涂覆能力和适宜的粘度等特点。因此,含纳米粒子的低粘度介质溶液(低溶液浓度、低分子量LPA)可用于取代不含纳米粒子的高粘度介质溶液(高溶液浓度、高分子量LPA),而二者的筛分性能相当甚至前者更高。这非常有利于实现DNA微流动体系分离的全自动化,特别是毛细管阵列电泳(CAE)和微芯片电泳(MCE)的全自动化。
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 network
Three 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 sequencing
Gold 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 sequencing
A 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 sequencing
PDMA-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).
1.准互穿聚合物网络的制备及表征
通过反相乳液聚合法制备了粘均分子量分别为1.5、3.3和6.5 MDa的LPA,接着在LPA水溶液中引发N,N-二甲基丙烯酰胺(DMA)单体聚合生成非交联的quasi-IPN,该介质结合了LPA的高筛分能力和PDMA的自涂覆能力。利用乌氏粘度计和~1H NMR光谱仪对LPA和quasi-IPN进行了表征,结果证明了LPA和quasi-IPN的生成。
2.准互穿聚合物网络/金纳米粒子复合介质的制备及其用于DNA测序性能的研究
制备了粒径约为20、40和60 nm的金纳米粒子(GNPs),将其分别加入由LPA(1.5、3.3和6.5 MDa)和PDMA所组成的quasi-IPN中生成了聚合物/金属复合介质(quasi-IPN/GNPs)。详细研究了各种参数(如GNPs含量、GNPs粒径、LPA分子量、溶液浓度和温度)对ssDNA测序性能的影响。在测序条件(如碱基读出软件)未完全优化的情况下,使用quasi-IPN3/GNPs40-1作为介质,利用ABI 310遗传分析仪在50℃和150 V/cm下得到的读出长度(98%准确度)为766碱基,耗时约57 min。quasi-IPN/GNPs的分离度高于不含GNPs的quasi-IPN,接近于含较高分子量LPA但不含GNPs的quasi-IPN。所以使用含低分子量LPA的quasi-IPN/GNPs可以克服使用高分子量LPA所带来的问题(如难以制备、测序浓度下粘度高及容易降解等),从而有助于实现全自动化。在相同测序条件下,quasi-IPN/GNPs的测序时间要少于quasi-IPN。此外,quasi-IPN/GNPs的分离重现性非常好,而且寿命大于一年。结果表明,GNPs与高分子链之间有相互作用并且生成了物理交联点,它们避免了高分子链彼此之间的滑移,从而可以形成相对更稳定的孔径(更稳固的筛分网络)并增加介质的表观分子量及它们的筛分性能,所以这些复合介质可以显著改善DNA的测序性能。
3.准互穿聚合物网络/官能化的金纳米粒子复合介质的制备及其用于DNA测序性能的研究
为了解决GNPs在高盐浓度下易附聚的问题,由“grafting-to”法制备了一种新型介质添加剂,即PDMA官能化的GNPs(GNP-PDMA),并将其加入到由LPA(3.3 MDa)和PDMA所组成的quasi-IPN中得到了用于毛细管电泳DNA测序的聚合物/金属复合筛分介质(quasi-IPN/GNP-PDMA)。在测序条件(如碱基读出软件)未完全优化的情况下,使用quasi-IPN/GNP-PDMA-2作为分离介质,利用ABI 310遗传分析仪在50℃和150 V/cm下得到的读出长度(98%准确度)为801碱基,耗时约64 min。由于GNP-PDMA的存在可以改善GNPs与整个测序体系的相容性、增大网络的缠结程度并增加GNPs在体系中的含量,从而导致整个筛分网络更加受限和稳固、介质的表观分子量更高并且孔径更小,因此与之前的quasi-IPN/GNPs相比,quasi-IPN/GNP-PDMA可以进一步增强ssDNA的测序性能。此外,与其它商用介质的比较表明,该复合介质是一种非常有潜力的全自动化DNA测序(特别是微流动体系)介质。
4.准互穿聚合物网络/官能化的多壁碳纳米管双网络复合介质的制备及其用于DNA测序性能的研究
通过原子转移自由基聚合法(ATRP)制备了PDMA官能化的多壁碳纳米管(MWNT-PDMA)添加剂,并加入到由LPA(3.3 MDa)和PDMA所组成的quasi-IPN中,得到了用于毛细管电泳DNA测序的聚合物/纳米管双网络复合筛分介质(quasi-IPN/MWNT-PDMA)。详细研究了MWNT-PDMA含量和MWNT-PDMA上PDMA侧链分子量对ssDNA测序性能的影响。在测序条件(如碱基读出软件)未完全优化的情况下,使用quasi-IPN/MWNT-PDMA2-Ⅱ作为介质,利用ABI 310遗传分析仪在50℃和150 V/cm下得到的读出长度(98%准确度)为792碱基,耗时约62 min。CE结果表明,与quasi-IPN相比,该复合介质可以显著改善DNA测序性能,这是因为在复合筛分介质中形成了双网络,它包括一个柔性的quasi-IPN聚合物网络和一个刚性的MWNTs(具有独特的管状结构)网络。这两种不同类型的网络可以共存于介质溶液中并相互作用,从而避免聚合物之间彼此滑移、稳固和制约总的筛分网络、增加介质的表观分子量并减小介质的孔径。此外,MWNT-PDMA上的PDMA侧链可以与quasi-IPN中的LPA或PDMA发生缠结,从而进一步稳固介质网络。因此,quasi-IPN/MWNT-PDMA介质中的这种更受限、更稳固且孔径更小的纳米结构使得测序性能更优良。复合介质与其它介质的比较结果进一步表明,它们非常有希望用于DNA测序。
总之,在纳米粒子(GNPs、GNP-PDMA或MWNT-PDMA)存在条件下,DNA的测序表现出分离度高、重现性好且测序介质寿命长等特点,因此易于实现快速、高效分离DNA的目的。这些新型复合介质同时具备了优良的筛分能力、自涂覆能力和适宜的粘度等特点。因此,含纳米粒子的低粘度介质溶液(低溶液浓度、低分子量LPA)可用于取代不含纳米粒子的高粘度介质溶液(高溶液浓度、高分子量LPA),而二者的筛分性能相当甚至前者更高。这非常有利于实现DNA微流动体系分离的全自动化,特别是毛细管阵列电泳(CAE)和微芯片电泳(MCE)的全自动化。
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 network
Three 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 sequencing
Gold 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 sequencing
A 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 sequencing
PDMA-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).
引文
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