Investigation of polyacrylamide hydrogel-based molecularly imprinted polymers using protein gel electrophoresis.

Hazim F. EL Sharif,Subrayal M. Reddy,Flavio Giosia

doi:10.1002/jmr.2942

Abstract

In conjunction with polyacrylamide gel electrophoresis (PAGE), molecular imprinting methods have been applied to produce a multilayer mini-slab in order to evaluate how selectively and specifically a hydrogel-based molecularly imprinted polymer (MIP) binds bovine haemoglobin (BHb, ~64.5 kDa). A three-layer mini-slab comprising an upper and lower layer and a MIP, or a non-imprinted control polymer dispersion middle layer has been investigated. The discriminating MIP layer, also based on polyacrylamide, was able to specifically bind BHb molecules in preference to a protein similar in molecular weight such as bovine serum albumin (BSA, ~66 kDa). Protein staining allowed us to visualise the protein retention strength of the MIP layer under the influence of an electric field. This method could be applied to other proteins with implications in effective protein capture, disease diagnostics, and protein analysis.

Highlights

Molecular imprinting is a means of introducing sites of specific molecular arrangement into an otherwise uniform polymeric matrix.[1,2,3] These techniques have found applications in biomedical engineering, as chiral stationary phase in high-performance liquid chromatography, as chiral selector in capillary electrophoresis methods and in the design of new drug delivery systems.[4,5,6] Compared to traditional molecularly imprinted polymers (MIPs) made in organic solvents, aqueous media synthesis of chemically and mechanically stable MIPs has received much traction over the past 20 years and is an interesting challenge in chemistry.[7]
What we present is the first report of hydrogel-based MIPs applied to slab electrophoresis, involving a multilayer resolving system
Hydrogel MIPs and non-imprinted polymer (NIP) (0.1 g) were treated with a 3 mg/mL protein solution prepared in 0.2 mL reverse osmosis (RO) water, and polymer/protein solutions were mixed on a rotary vortex mixer allowed to associate at room temperature ($22C) for 20 minutes followed by centrifugation

Summary

| INTRODUCTION

Molecular imprinting is a means of introducing sites of specific molecular arrangement into an otherwise uniform polymeric matrix.[1,2,3] These techniques have found applications in biomedical engineering, as chiral stationary phase in high-performance liquid chromatography, as chiral selector in capillary electrophoresis methods and in the design of new drug delivery systems.[4,5,6] Compared to traditional molecularly imprinted polymers (MIPs) made in organic solvents, aqueous media synthesis of chemically and mechanically stable MIPs has received much traction over the past 20 years and is an interesting challenge in chemistry.[7]. The resulting hydrogel materials are hydrophilic and highly crosslinked Due to their high-water compatibility, hydrogel-based MIPs have been shown to retain protein stability and provide a robust means for recognition of target analytes over long periods.[7,23,24] For hydrogel protein imprinting, water-soluble monomers such as acrylamide and functionalised acrylamides have been used alongside the cross-linker N,N0-methylenebisacrylamide (MBAm) to produce polyacrylamidebased MIPs.[4,12,13] Polyacrylamide (PAM) is biocompatible and inert to. Ogiso et al[25,26] applied MIP technology to gel electrophoresis to develop a simple and inexpensive DNA detection method. Illustrated is the application of hydrogel-based MIPs in mini-slab gel electrophoresis whereby a MIP dispersion is layered between two control layers. We explore how, during electrophoresis, a protein imprinted polymer can retain its template protein vs a protein, which is analogous in molecular weight

| MATERIALS AND METHODS

| RESULTS AND DISCUSSION

| CONCLUSIONS