Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Dro...Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.展开更多
In this study,we compared FeNi alloy magnetic nanoparticles(MNPs) prepared by either combustion or chemical precipitation methods.We found that the FeNi MNPs generated by combustion method have a rather high saturatio...In this study,we compared FeNi alloy magnetic nanoparticles(MNPs) prepared by either combustion or chemical precipitation methods.We found that the FeNi MNPs generated by combustion method have a rather high saturation magnetization Ms of ～180 emu/g and a coercivity field Hc of near zero.However,the alloy nanoparticles are easily aggregated and are not well dispersive such that size distribution of the nanoparticle clusters is wide and clusters are rather big(around 50～700 nm).To prepare a better quality and well dispersed Fe-Ni MNPs,we also developed a thermal reflux chemical precipitation method to synthesize FeNi3 alloy MNPs.The precursor chemicals of Fe(acac)3 and Ni(acac)2 in a molecular ratio of 1:3 reacted in octyl ether solvent at the boiling point of solvent(～300 ℃) by the thermal reflux process.The 1,2-hexadecandiol and tri-n-octylphosphine oxide(TOPO) were used as reducer and surfactant,respectively.The chemically precipitated FeNi3 MNPs are well dispersed and have well-controlled particle sizes around 10～20 nm with a very narrow size distribution(±1.2 nm).The highly monodispersive FeNi3 MNPs present good uniformity in particle shape and crystallinity on particle surfaces.The MNPs exhibit well soft magnetism with saturation magnetization of ～61 emu/g and Hc～0.The biomedically compatible FeNi MNPs which were coated with biocompatible polyethyleneimine(PEI) polymer were also synthesized.We demonstrated that the PEI coated FeNi MNPs can enter the mammalian cells in vitro and can be used as a magnetic resonance imagine(MRI) contrast agent.The results demonstrated that FeNi MNPs potentially could be applied in the biomedical field.The functionalized magnetic beads with biocompatible polymer coated on MNPs are also completed for biomedical applications.展开更多
文摘Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.
文摘In this study,we compared FeNi alloy magnetic nanoparticles(MNPs) prepared by either combustion or chemical precipitation methods.We found that the FeNi MNPs generated by combustion method have a rather high saturation magnetization Ms of ～180 emu/g and a coercivity field Hc of near zero.However,the alloy nanoparticles are easily aggregated and are not well dispersive such that size distribution of the nanoparticle clusters is wide and clusters are rather big(around 50～700 nm).To prepare a better quality and well dispersed Fe-Ni MNPs,we also developed a thermal reflux chemical precipitation method to synthesize FeNi3 alloy MNPs.The precursor chemicals of Fe(acac)3 and Ni(acac)2 in a molecular ratio of 1:3 reacted in octyl ether solvent at the boiling point of solvent(～300 ℃) by the thermal reflux process.The 1,2-hexadecandiol and tri-n-octylphosphine oxide(TOPO) were used as reducer and surfactant,respectively.The chemically precipitated FeNi3 MNPs are well dispersed and have well-controlled particle sizes around 10～20 nm with a very narrow size distribution(±1.2 nm).The highly monodispersive FeNi3 MNPs present good uniformity in particle shape and crystallinity on particle surfaces.The MNPs exhibit well soft magnetism with saturation magnetization of ～61 emu/g and Hc～0.The biomedically compatible FeNi MNPs which were coated with biocompatible polyethyleneimine(PEI) polymer were also synthesized.We demonstrated that the PEI coated FeNi MNPs can enter the mammalian cells in vitro and can be used as a magnetic resonance imagine(MRI) contrast agent.The results demonstrated that FeNi MNPs potentially could be applied in the biomedical field.The functionalized magnetic beads with biocompatible polymer coated on MNPs are also completed for biomedical applications.
