Development of an epoxy-based monolith used for the affinity capturing of Eschericha coli bacteria

Abstract

An epoxy-based monolith has been developed for use as hydrophilic support in bioseparation. This monolith is produced by self-polymerization of polyglycerol-3-glycidyl ether in organic solvents as porogens at room temperature within 1 h. One receives a highly cross-linked structure that provides useful mechanical properties. The porosity and pore diameter can be controlled by varying the composition of the porogen. In this work, an epoxy-based monolith with a high porosity (79%) and large pore size (22 μm) is prepared and used in affinity capturing of bacterial cells. These features allow the passage of bacterial cells through the column. As affinity ligand polymyxin B is used, which allows the binding of gram-negative bacteria. The efficiency of the monolithic affinity column is studied with Escherichia coli spiked in water. Bacterial cells are concentrated on the column at pH 4 and eluted with a recovery of 97 ± 3% in 200 μL by changing the pH value without impairing viability of bacteria. The dynamic capacity for the monolithic column is nearly independent of the flow rate (4 × 10⁹ cells/column). Thereby, it is possible to separate and enrich gram-negative bacterial cells, such as E. coli, with high flow rates (10 mL/min) and low back pressure (<1 bar) in a volume as low as 200 μL compatible for real-time polymerase chain reaction, microarray formats, and biosensors.

Introduction

Monoliths have become an interesting alternative to columns packed with beads since their introduction as a separation material in the late 1980s [1], [2]. They overcome the limitations of chromatography beads and have been successfully employed for separation involving large biological molecules such as proteins, plasmid DNA, and viruses [3], [4], [5]. Conventional particle-based supports consist of few micrometer-sized porous particles. Because the pores within the particles are nearly closed, the liquid inside them is stagnant. Therefore, the molecules to be separated are transported to the active sites inside the narrow pores and back to the mobile phase mainly by diffusion. Since diffusion itself is a rather slow process, especially in the case of large molecules with a low mobility, longer contact times of the molecules with the resin surface are required. Polymeric monoliths consist of a solid continuous phase permeated by a continuous network of through-pores and can be distinguished depending on their shape, backbone material, porosity or pore size. Despite their differences they all are characterized by low mass transfer. Since the flow of the liquid within the channels is driven by the pressure difference, the molecules to be separated are transported to the active sites located on the surface of the channels by convection. Therefore, it is possible to perform an efficient separation of large molecules within high flow rates at low back pressure and with less loss in column efficiency, resulting in fast separation [6]. The rigid monoliths can be used in reversed-phase, ion-exchange, hydrophobic interaction, and affinity chromatography [7], [8], [9].

There are several different types of monolithic supports currently available (see Table 1). They are basically synthesized from different chemical compounds to form silica [10], acrylamide [11], styrene [12], agarose [13], and methacrylate [14] monoliths. Silica monoliths were prepared as single blocks with through-pores (1–2 μm) and mesopores (5–25 nm) by a sol–gel process. Because of the high hydrophobicity and strong non-specific adsorption with biomolecules, their application is limited in bioseparation [15]. Organic polymer-based monolithic counterparts have found their use mainly in bioseparation [7]. Polyacrylamide and polymethacrylate are the most frequently used organic polymers for casting of monoliths for separation of biomolecules. Cryogels were synthesized by cross-linking water-soluble polyacrylamide at sub-zero temperatures [16]. The resulting polymer bed is hydrophilic, spongy and elastic, and possesses macropores that are typically 10–100 μm in diameter. Cryogels have been used to separate a variety of different cells, including T-cells and B-cells, to separate bacteria and also to process crude feedstock such as Escherichia coli lysate [17], [18], [19]. The elution can be performed by using a mechanical force to compress the separation media [20]. The methacrylate monolith is synthesized by free radical polymerization of methacrylate induced thermally or by radiation in the presence of porogens and initiator. The resulting monolith contains macropores (1–2 μm) and epoxy groups that can be used directly for ligand immobilization. In addition, the diol groups that can be generated on this material tend to give a support with low non-specific binding for many biological agents, as it has been noted with other affinity supports [21].

However, the majority of the monolith research has been focused in free radical polymerization of vinyl polymers, and only a few attempts have been made to investigate other polymer systems. Therefore, the objective of the present study was to prepare a monolithic support with large pores by a very simple, fast and gentle polyaddition reaction and to evaluate its potential as carrier for the chromatography of large biomolecules, e.g. bacterial cells. The capture of bacterial cells was reported in previous works by using silica beads [22], [23]. It was considered to use the monolithic column as selective pre-enrichment column for bioanalytical detection methods, e.g. real-time polymerase chain reaction (RT-PCR), microarray formats, and biosensors [24], [25], [26].