Preparation of a Hyper‐cross‐linked Polymer Monolithic Column and Its Application to the Sensitive Determination of Genomic DNA Methylation

Abstract

A hyper-cross-linked polymer monolithic column, poly(methacrylatoethyl trimethyl ammonium-co-vinylbenzene chloride-co-divinylbenzene) (MATE-co-VBC-co-DVB) with phenyl and quaternary ammonium groups was successfully prepared in the current study. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The poly(MATE-co-VBC-co-DVB) monolithic column was demonstrated to have strong anion exchange/reversed-phase (SAX/RP) mixed-mode retention for analytes on capillary liquid chromatography (cLC). By using this monolithic column, we developed a rapid and sensitive method for the detection of DNA methylation. Our results showed that six nucleobases (adenine, guanine, cytosine, thymine, uracil, and 5-methylcytosine (5-mC)) can be baseline separated within 15 min by electrostatic repulsion and hydrophobic interactions between nucleobases and the monolithic stationary phase. The limit of detection (LOD, signal/noise = 3) of 5-mC is 0.014 pmol and endogenous 5-mC can be distinctly detected by using only 10 ng genomic DNA, which is comparable to that obtained by mass spectrometry analysis. Furthermore, by using the method developed here, we found that DNA methylation inhibitor 5-azacytidine (5-aza-C) and 5-aza-2'-deoxycytidine (5-aza-CdR) could induce a significant decrease of genome-wide DNA methylation in human lung carcinoma cells (A549) and cervical carcinoma cells (HeLa).

Assessing Gibberellins Oxidase Activity by Anion Exchange/Hydrophobic Polymer Monolithic Capillary Liquid Chromatography-Mass Spectrometry

“…In addition, the continuous monolithic matrix was obtained and attached well to the inner wall of the monolithic column (Figure 2B) with through-pores of approximate 1.5 µm (Figure 2C). The distribution of mesopore in the poly(META- co -DVB- co -EDMA) monolith ranges from 3.3 to 4.0 nm, and the specific surface area is 426 m 2 /g (Figure 2D), which is comparable to the hypercrosslinked monolithic column [38], [39], [40].…”

Preparation and characterization of poly-(methacrylatoethyl trimethylammonium chloride-co-vinylbenzyl chloride-co-ethylene dimethacrylate) monolith

“…Binary porogen consist of 1-propanol and 1,4-butanediol was selected since they could perfectly homogenize polymer solution of MATE, VBC, and EDMA. The ratio of MATE : VBC was adopted from Chen and coworkers [33]. Each variation of the polymerization condition has significant effect on the structure of monolith.…”

“…Poly-(MATE-co-VBC-co-EDMA) monolith was prepared by one-step thermally initiated in situ polymerization method, as described by Chen et al [33] with minor modification as shown in Fig 1. Firstly, porogen 1-propanol was mixed thoroughly with 1,4-butanediol. Subsequently, all monomers and cross-linker (MATE, VBC and EDMA) were added into porogenic solvents and mixed properly to form polymerization solution, with composition as indicated in Table 1.…”

Preparation and characterization of poly-(methacrylatoethyl trimethylammonium chloride-co-vinylbenzyl chloride-co-ethylene dimethacrylate) monolith

“…Poly-(MATE-co-VBC-co-EDMA) monolith was prepared by one-step thermally initiated in situ polymerization method, as described by Chen et al [33] with minor modification as shown in Fig 1. Firstly, porogen 1-propanol was mixed thoroughly with 1,4butanediol. Subsequently, all monomers and cross-linker (MATE, VBC and EDMA) were added into porogenic solvents and mixed properly to form polymerization solution, with composition as indicated in Table 1.…”