Macroporous Monolithic Layers as Efficient 3-D Microarrays for Quantitative Detection of Virus-like Particles

Abstract

The main objective of the present paper was to test the recently developed new type of 3-D protein microarray system based on glycidyl methacrylate-co-ethylene glycol dimethacrylate (GMA-EDMA) monolithic material for efficient and fast virus detection. The large-size synthetic particles bearing adsorption-responsible biomolecules on their surface were used as a virus model. Two affinity pairs were chosen for present study. Model virus-like particles, close to the dimensions of human viruses, were developed by means of protein (one of affinity partners) covalent binding to the outer carboxylated surface of polymer latexes (polystyrene based, 80-nm diameter). Recently, it was shown that the adsorption of similar synthetic particles was defined by a protein covering the particle surface. The corresponding complement was immobilized on the surface of prepared by photoinitiated polymerization GMA-EDMA macroporous layers. The detection of a formed biocomplementary complex between protein-bearing latex particle and immobilized affinity partner was carried out by two different methods: (1) similar to an ELISA approach using horse radish peroxidase conjugated with monoclonal antibodies and (2) direct method using two markers. In parallel, the pairing of native proteins was also evaluated. The adsorption behavior of studied particles has been additionally investigated by affinity adsorption at static and dynamic (frontal elution) conditions using the same GMA-EDMA material shaped as a short monolithic column (CIM Disk, BIA Separations, Ljubljana, Slovenia). The results obtained for these virus-mimicking supramolecular structures can be further used for the construction of a rapid, highly sensitive, and highly specific test intended for precise diagnostics of some respiratory tract infection viruses.

Polymethacrylate monolithic columns for capillary liquid chromatography

“…Kalashnikova et al [229] tested a new type of threedimensional (3-D) GMA-EDMA micro-arrays for the detection and quantitative evaluation of affinity characteristics of specially designed large-size virus-like model particles. A micro-array represents an analytical device that employs the bio-recognition capability of bioactive compounds (such as enzymes, antibodies, nucleic acids, micro-organisms, and cells) immobilized on a solid surface.…”

Affinity monolith chromatography: a review of principles and recent analytical applications

“…One advantage to using GMA/EDMA monoliths in AMC is that the epoxy groups, as provided by GMA, can be directly used for the immobilization of various binding agents (e.g., proteins or other amine-containing ligands). This approach, known as the “epoxy method”, is relatively simple and fast to perform [9, 10, 39, 40]. However, the epoxy groups are susceptible to hydrolysis, which can result in lower amounts of immobilized ligand than other available amine-coupling methods [9].…”

Porous polymer monoliths for extraction: Diverse applications and platforms

“…One of the most exciting recent developments in CIM has been described by Kalashnikova et al [134,135] in the application of these supports for isolation of the influenza virus. This has been accomplished using both 3-D microarrays for quantitative detection [134] as well as in the disk format for fine purification [135].…”

“…This has been accomplished using both 3-D microarrays for quantitative detection [134] as well as in the disk format for fine purification [135]. A range of suitable affinity ligands were immobilized on poly(GMA-co-EDMA) monoliths and the interaction with both virusmimicking synthetic particles and influenza A virus was investigated.…”